Ex vivo effects of low dose delayed release rapamycin on agonist-induced platelet aggregation, activation and procoagulant platelet phenotypes in domestic cats

Ex vivo effects of low dose delayed release rapamycin on agonist-induced platelet aggregation, activation and procoagulant platelet phenotypes in domestic cats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ex vivo effects of low dose delayed release rapamycin on agonist-induced platelet aggregation, activation and procoagulant platelet phenotypes in domestic cats Meg Shaverdian, Nghi Nguyen, Stuart Fitzgerald, Louise Grubb, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7068900/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Cardiogenic arterial thromboembolism (CATE) is a complication of hypertrophic cardiomyopathy (HCM) with a high mortality rate. Despite anti-platelet drugs use, on-treatment recurrence rate remains high indicating a critical need to discover novel therapies. Studies in both humans and cats show that low dose delayed release rapamycin (LDDRR) can reduce the progression of left ventricular hypertrophy. However, its effect on platelets is unclear. In this study we assessed the ex vivo effects of LDDRR on platelet aggregation, alpha-granule secretion indicated by an increase in P-selectin, and procoagulant platelet phenotypes including loss of mitochondrial membrane potential (ΔΨm) and phosphatidylserine (PS) exposure. Cats were treated with 0.3 mg/kg LDDRR orally every 7 days for 4 consecutive weeks. Blood was collected at 3, 24 and 48 hours after the last dose. While LDDRR had no effect on aggregation, it significantly decreased P-selectin expression in thrombin + COL (3 hrs), and ADP (24, 48 hrs) samples. LDDRR had protective effects on ΔΨm in all agonists at all times. PS exposure was reduced at 24 hrs in thrombin ± COL samples. Our study indicated that LDDRR in cats can safely modulate platelet activation, procoagulant phenotypes and tendency in varying degrees making it an effective candidate to prevent CATE. Health sciences/Cardiology Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Biological sciences/Physiology rapamycin procoagulant platelets cardiogenic arterial thromboembolism hypertrophic cardiomyopathy mitochondrial membrane potential phosphatidylserine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hypertrophic cardiomyopathy (HCM) is the most common form of cardiomyopathy in domestic cats affecting approximately 1 in 7 (~ 15%) cats in the general population. 1 , 2 Familial HCM in cats closely resembles human HCM and has been served as a naturally occurring large animal translational model for evaluating the efficacies of novel compounds like aficamten, a cardiac myosin inhibitor, that was recently approved for use in human HCM patients. 3 , 4 Manifestations of feline HCM largely mirror those seen in human HCM such as left ventricular outflow tract obstruction (LVOTO), sudden death, congestive heart failure and thrombosis. 5 In humans, HCM affects about 1 in 500 people. 6 A common clinical sequala of human HCM is atrial fibrillation, which affects about 23% of HCM patients and is commonly associated with thromboembolism. 7 , 8 While AF in human HCM patients is a serious complication associated with stroke and mortality, most cats develop thromboembolic diseases in the absence of AF. 9 In cats, the prevalence of cardiogenic arterial thromboembolism (CATE) secondary to HCM is about 11.3% with a mortality rate of up to 67%. 10 The pathogenesis of CATE and ischemic stroke in human and feline HCM patients in the absence of AF is not well understood. However, given the high morbidity and mortality associated with CATE, research is ongoing to understand the underlying mechanisms of CATE in the hopes of discovering novel therapeutic targets that can benefit humans and cats. Since platelets are the primary responders in hemostasis, many studies, to date, have documented increased platelet adhesion and activation in cats and humans with HCM. Platelets in both humans and cats with HCM have increased P-selectin expression suggesting that platelets may play a significant role in CATE pathogenesis. 11 – 15 Moreover, mean platelet volume is significantly higher in HCM patients. 16 Cats with HCM have also been found to have increased neutrophil extracellular traps in the form of free circulating histones, which may further exacerbate thrombosis by priming platelets and augmenting fibrin formation. 17 Procoagulant platelets are a specialized subset of activated platelets that play a role in thrombin generation and fibrin formation in hemostasis. 18 However, disease processes that favor the over production of procoagulant platelets have been associated with pathological thrombosis. Upon activation with strong agonists like thrombin and collagen, the mitochondrial membrane potential depolarizes causing increased permeability of the mitochondrial membrane and sustained intracellular hypercalcemia. This eventually leads to externalization of the electronegative phospholipid, phosphatidylserine, on the platelet surface, serving as platforms for the assembly of coagulation complexes such as tenase and prothrombase complexes to facilitate thrombin generation. 18 While procoagulant platelets are essential in normal hemostasis, excessive or dysregulated procoagulant platelet activity is associated with thrombotic disorders such as ischemic stroke, coronary arterial disease, and venous thrombosis. 19 – 21 Since procoagulant platelets play a crucial role in thrombin generation and fortification of clot structures, any imbalance in their generation and function can lead to either hemorrhage or thrombosis. Understanding the regulation of procoagulant platelet formation may, therefore, provide therapeutic insights for targeting thrombotic diseases without impairing normal hemostasis. In humans with AF, anticoagulants such as warfarin are considered the standard of care for the primary and secondary prevention of CATE. 22 However, in HCM patients without AF, antiplatelet drugs are recommended for the secondary prevention of ischemic stroke while their clinical benefit as primary prevention remains unclear. Due to the absence of AF in most feline HCM patients, an antiplatelet drug is the first line therapy for primary CATE prevention. 5 , 23 Nevertheless, the on-treatment recurrence rate for CATE in cats remains high at around 49% suggesting that conventional antiplatelet drugs may have limited efficacy in preventing intracardiac thrombosis and CATE. 24 Conventional antiplatelet drugs such as aspirin and clopidogrel target the pathways involved in platelet aggregation, but do not affect the cellular processes that mediate the formation of procoagulant platelets. As a subset of platelets are continuously exposed to high concentrations of potent agents, sustained intracellular hypercalcemia results in procoagulant phenotypes, characterized by the formation of procoagulant membrane and proteolytic downregulation of platelet integrins, which are crucial for platelet aggregation. As a result, even when aggregation is inhibited, procoagulant platelet formation continues with ongoing thrombin generation and fibrin polymerization to stabilize the clot structure. Despite this, no current drugs have been found to target cellular processes specific to procoagulant platelet formation without causing systemic adverse effects. 18 This knowledge gap in current treatment strategies highlights the potential of developing novel therapies to target procoagulant platelets, thereby reducing the risk of ischemic stroke and other thromboembolic events without impairing normal hemostasis. Multiple in vitro studies have investigated potential therapeutic targets to modulate procoagulant platelet formation. Rapamycin, also known as sirolimus, is a macrolide compound that inhibits the mechanistic/mammalian target of rapamycin (mTOR) pathway, a key regulator of cell growth, metabolism, and immune responses. 25 By inhibiting mTOR complex 1 (mTORC1), rapamycin increases autophagy, suppresses protein synthesis, reduces inflammation, and modulates cellular metabolism, making it a promising candidate for a range of clinical conditions. 25 Rapamycin is used to treat various conditions such as preventing organ transplant rejection, and cancer, and has recently been approved by the US Food and Drug Agency for the treatment of feline HCM. Low dose delayed release rapamycin (LDDRR) has been shown to have cardioprotective effects in HCM-affected cats by reducing or halting the progression of left ventricular hypertrophy. 25 , 26 Findings in humans suggest that similar mTOR inhibition may also help reduce the progression of hypertrophy. 27 – 29 Additionally, a multi-omic study found that rapamycin-treated cats with HCM-had downregulations in proteins associated with the complements, inflammation, coagulation cascade and von Willebrand Factors, suggesting that rapamycin may have anti-thrombotic effects. 30 The effects of rapamycin on platelet function remain poorly understood with most studies limited to in vitro models, which yielded conflicting results. In vitro treatment of human platelets with rapamycin increases platelet aggregation and secretion in response to ADP and thrombin in one study while another study showed that collagen response is modulated by reducing platelet aggregation and spreading. 31 – 33 An in vitro study in human platelets demonstrated that rapamycin reduces procoagulant platelet formation by protecting mitochondrial membrane potential (ΔΨm) and limiting phosphatidylserine (PS) externalization to prevent excessive thrombin generation via an mTORC1-independent mechanism. 34 Moreover, the effects of rapamycin on platelet function and procoagulant platelet formation have yet to be demonstrated in an ex vivo large animal model. Given the potential antithrombotic and cardioprotective effects of rapamycin in HCM, further research is crucial to explore its potential as a therapeutic strategy for preventing thrombosis in both cats and humans with HCM. Herein, we conducted the first ex vivo study assessing the pharmacodynamic effects of LDDRR on platelet aggregation, activation and procoagulant platelet tendency in domestic cats. We hypothesized that following a month-long treatment of once weekly rapamycin, platelet activation and formation of procoagulant phenotypes would be safely reduced without compromising platelet aggregation. Furthermore, by utilizing a previously validated scoring system to evaluate procoagulant tendency in cats, we hypothesized that rapamycin would modulate the overall platelet procoagulant tendency at all time points after rapamycin treatment. 35 Results Patient outcome None of the 8 cats studied exhibited any notable adverse effects secondary to low dose delayed-release rapamycin (LDDRR) given once every 7 days for 4 weeks throughout the study period. Table 1 summarizes the selected hematology variables before and after rapamycin treatment. Figure 1 summarizes a modified experimental approach to characterize procoagulant platelet phenotypes in cats. Table 1. Selected hematological findings in 8 cats before and 3, 24, and 48 hours after treatment with delay-release rapamycin. Baseline 3h 24h 48h P value Baseline vs 3h Baseline vs 24h Baseline vs 48h White Blood Cells x 10 9 /L (Ref: 3.50-20.70) 5.20 (4.53-6.69) 4.93 (4.03-5.75) 5.28 (4.89-5.75) 5.22 (4.30-6.52) 0.94 0.46 0.84 Lymphocytes x 10 9 /L (Ref: 0.83-9.10) 2.81 (1.84-4.17) 1.54 (1.22-2.69) 2.08 (1.44-2.99) 2.11 (1.59-2.84) 0.01 0.64 0.46 Monocytes x 10 9 /L (Ref: 0.09-1.21) 0.11 (0.06-0.29) 0.23 (0.18-0.28) 0.24 (0.17-0.28) 0.22 (0.08-0.30) 0.51 0.64 0.61 Neutrophils x 10 9 /L (Ref: 1.63-13.37) 2.51 (±1.05) 2.84 (±0.61) 2.88 (±0.38) 2.75 (±0.67) 0.51 0.37 0.69 Hematocrit, % (Ref: 33.70-55.40) 35.09 (±4.96) 35.21 (±8.21) 36.06 (±3.86) 35.18 (±3.95) 0.96 0.61 0.94 Platelets x 10 9 /L (Ref: 125-618) 248.6 (±115.6) 309.5 (±124.8) 325.5 (±63.56) 291.1 (±61.60) 0.38 0.06 0.46 Mean Platelet Volume, fl (Ref: 8.6-14.9) 9.54 (±0.59) 9.35 (±0.85) 9.47 (±0.62) 9.52 (±0.82) 0.32 0.62 0.95 Neutrophil-to-lymphocyte ratio 0.91 (0.53-1.17) 1.55 (1.01-2.15) 1.36 (0.82-2.26) 1.39 (0.83-1.67) 0.11 0.19 0.31 Platelet-to-lymphocyte ratio 110.7 (±86.66) 188.9 (±100.3) 165.6 (±80.89) 136.9 (±50.00) 0.10 0.15 0.46 Rapamycin did not affect adenosine diphosphate induced platelet aggregation Platelet aggregation in response to adenosine diphosphate (ADP) at 24 and 48 hrs, measured by whole blood impedance platelet aggregometry in heparinized blood, was variable among cats (co-efficient of variance =16.3%, 18.1%, respectively). No differences in any aggregometry variables were noted following treatment with LDDRR at 24 and 48 hrs compared to baseline (area under the curve (AUC) p=0.10, aggregation unit (AU) p =0.19, velocity (AU/min) p =0.08) (Figure 2). Of the 8 cats, 4 (50%) had increased aggregation (AUC), 2 (25%) had decreased aggregation (AUC) and 2 (25%) had no changes in aggregation (AUC) at 24 hrs. No significant difference in aggregation at baseline and 24 hrs after dosing was found (AUC baseline = 111.8 ± 21.02 vs AUC 24 hrs = 125.8 ± 18.24, p=0.11). At 48 hrs, 3 of the 8 cats (37.5%) had increased aggregation but, overall, this difference did not differ from baseline measurements (AUC 48 hrs. : 131.8 ± 25.94, p=0.09) (Figure 2A). Low dose delayed-release rapamycin prevented the loss of platelet mitochondrial membrane potential in the presence of potent agonists Protective effects on platelet mitochondrial membrane potential (ΔΨm) were detected at 3, 24 and 48 hrs after the last dose of rapamycin when ΔΨm was measured as percent change (%) in tetramethylrhodamine methyl ester (TMRM)-positive platelets (Figure 3C, D). Rapamycin prevented the loss of ΔΨm induced by thrombin at 3 hrs (baseline= -15.6%, IQR -23.8 to -0.4 vs 3 hrs = -3.6%, IQR -7.2 to 1.1, p=0.04), 24 hrs (-2.5%, IQR -6.9 to 2.1, p=0.02) and 48 hrs (-1.2%, IQR -5.2 to 0.5, p=0.04) post LDDRR treatment. With the addition of collagen to thrombin-activated cells, ΔΨm was also preserved at 3 hrs (-8.5%, IQR -12.5 to -1.3, p=0.02), 24 hrs (-5.1%, IQR -12.6 to 0.2, p=0.02) and 48 hrs (-6.2%, IQR -16.9 to -2.5, p=0.02) when compared to baseline (-25.3%, IQR -45.7 to -16.5). Similarly, protective effects were found upon stimulation with thrombin and CVX (baseline= -75.6%, IQR -82.4 to -53.4 vs 3 hrs= -28.5%, IQR -61.3 to -17.3, p=0.02) (24 hrs= -34.8%, IQR -53.3 to -15.4, p=0.02) (48 hrs = -44.9%, IQR -60.4 to -20.8, p=0.02) (Figure 3D). When the loss of ΔΨm was measured as fold change (log 10) in TMRM median fluorescence intensity (MFI FC log 10 ), we did not detect any significant differences in ΔΨm in response to thrombin in the presence or absence of collagen or CVX at all time points (Figure 3E). Platelets treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), which disrupts the proton gradient across the mitochondrial inner membrane, leading to a loss of ΔΨm, served as negative controls. Surprisingly, treatment of platelets with CCCP at 3 (MFI FC Log 10 = -0.02 ± 0.1;) and 24 hrs (-0.009 ± 0.1) after the last dose of LDDRR showed significant reductions in the loss of ΔΨm when compared to baseline (-0.1 ± 0.1) (p=0.03, p=0.03, respectively) (Figure 3E). Phosphatidylserine externalization was significantly modulated at 24 hours after rapamycin dosing Following the measurements of ΔΨm, exogenous calcium was added to the remaining aliquots of PRP, and phosphatidylserine (PS) externalization was assessed by detecting the percent change (%) in annexin V-positive platelets over time (Figure 4C, D). At 24 hrs after the last dose of LDDRR, externalization of PS in response to thrombin (Change PS baseline = 39.7% ± 53.0 vs Change PS 24 hrs = -5.8% ± 31.4, p=0.006) and a combination of thrombin and COL (Change PS baseline = 45.4%, IQR 12.8 to 152.8 vs Change PS 24 hrs = 11.4%, IQR -4.4 to 22.1, p=0.02) were significantly decreased (Figure 4D). However, changes in PS on platelets were widely variable among cats at 3 and 48 hrs after LDDRR dosing. Overall, rapamycin had no significant effect on PS externalization when platelets were stimulated with thrombin and CVX (p=0.14) (Figure 4D). Rapamycin modulated alpha granule secretion in response to adenosine diphosphate and thrombin in the presence of collagen Final aliquots of platelets were assessed for P-selectin (CD62P) in response to ADP, and thrombin in the presence or absence of COL or CVX. Response was evaluated based on fold change (log 10) in median fluorescence intensity between baseline and at 3, 24 and 48 hrs after LDDRR treatments. We found that alpha-granule secretion in response to thrombin and COL at 3 hours was modulated compared to baseline (MFI FC baseline = 0.09 ± 0.08 vs MFI FC 3 hrs = -0.02 ± 0.1, p=0.006). Response to ADP was also significantly decreased at 24 (MFI FC 24h = 0.006 ± 0.05) and 48 hrs (MFI FC = 0.004±0.06) compared to baseline (MFI FC baseline = 0.07 ± 0.06, p=0.004, p=0.007, respectively) (Figure 5 C, D). No significant changes in P-selectin expression were noted in platelets stimulated with thrombin in the presence or absence of CVX (p=0.17, p=0.21, respectively). Ex vivo effects of rapamycin on the tendency of procoagulant platelet formation in individual cats Figure 6 summarizes the distribution of platelet procoagulant tendency scores in the 8 cats using the previously established scoring system. 35 Significant reductions in procoagulant tendency scores among individual cats were recorded over time when platelets were stimulated with thrombin or thrombin + COL (p=0.04 and p=0.004, respectively) in comparison to baseline. Most reductions in procoagulant platelet tendency scores were noted at 24 hrs post dose compared to baseline especially when thrombin with or without COL were used as agonists. None of the cats had scores greater than 2 in the thrombin group at all time points. Only 1 out of 8 cats (12.5%) had a tendency score ≥ 2 at 24 hrs in the thrombin and COL group compared to 5/8 (62.5%) at baseline. Cats in the thrombin + CVX group had slight decreases in their tendency scores at 3 and 24 hrs after LDDDR treatment. Discussion Here, we conducted the first ex vivo study evaluating the effects of LDDRR on platelet aggregation, activation and procoagulant phenotypes in domestic cats. We found that LDDRR treatment in cats was well tolerated and the aggregation response to ADP remained unchanged after treatment. However, ADP-mediated platelet activation, measured by P-selectin upregulation, was modulated. One of the procoagulant platelet markers, ΔΨm, was protected at all time points after LDDRR, following ex vivo treatments of platelets with strong agonists. Additionally, the other procoagulant phenotype, PS, was reduced at 24h after LDDRR. Lastly, P-selectin as a marker of procoagulant platelets also was significantly decreased in the presence of thrombin and COL shortly after rapamycin treatment. Using a novel scoring system by factoring in all 3 procoagulant markers, we found that LDDRR was most efficacious at reducing the platelet procoagulant tendency scores at 24 hrs and to a lesser extent. At 48 hrs post dose. Our complete blood count (CBC) showed an increasing trend in platelet count was noticed at 3 and 24 hrs compared to the baseline. Since feline platelets are highly subjected to in vitro activation and aggregation, which can lead to falsely low platelet counts on automated analyzers, the modulation in platelet activation by rapamycin might have decreased in vitro aggregate, thereby increasing the platelet count on CBC. Another possibility is that rapamycin might have induced apoptosis, triggering platelet generation or turnover. However, research regarding rapamycin’s effect on apoptosis seems to be cell dependent. For instance, while rapamycin induces apoptosis in dendritic cells, it inhibits apoptosis in monocytes and macrophages. 36 Its apoptotic effect on platelets remains unexplored. A significant drop in lymphocyte count was observed at 3 hrs, followed by recovery at 24 and 48 hrs. The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are commonly used as non-specific indicators of systemic inflammation and stress. Trends of increased NLR and PLR were noted following rapamycin treatment. Since overall neutrophil count did not increase after LDDRR, the observed increase in NLR and PLR was likely due to decreased lymphocyte counts rather than systemic inflammation or stress. In healthy cats, NLR typically ranges from 1.5 to 2.8, suggesting that the observed increase in NLR is unlikely to be clinically significance. PLR, on the other hand, exceeded the reported reference range (100 to 164) in cats, likely due to both increased platelet counts and decreased lymphocytes. 37 However, the transient decrease in lymphocyte count following rapamycin treatments remains unclear. One possibility is that rapamycin enhances autophagy, and in conjunction with mTORC inhibition, affects specific lymphocytes sub-sets, leading to a transient decrease in lymphocyte count. Because mTOR activity is crucial for T cell proliferation, rapamycin disrupts the cell cycle and reduces the proliferation of T cells, ultimately reducing peripheral lymphocyte numbers. 38 – 40 Further studies in clinical cats with underlying inflammation are needed to delineate the clinical significance of these hematological changes. In agreement with multiple human studies, our study did not observe any changes in ADP-mediated platelet aggregation. 41 – 43 In a study by Pelekanou et al., human platelets pre-treated with rapamycin had similar aggregation response upon stimulation with ADP and collagen when compared to baseline. 41 ​ Additionally, in kidney transplant patients, short-term rapamycin treatment did not affect in vitro aggregation response to SFLLRN, a thrombin-receptor activating peptide (PAR-1) agonist. 42 These findings are clinically significant as they indicate that LDDRR does not negatively impact platelet aggregation. One possible explanation for this observation is that autophagy is not essential for platelet aggregation. 44 A study using Atg7 knockout mice, which lack the key platelet-specific gene critical for autophagy, has shown that their platelets maintain normal aggregation in response to ADP, collagen and thrombin. 44 Another plausible explanation is that low dose, short-term rapamycin treatment used in our study partially inhibits mTORC1 while sparing mTORC2, which regulates pathways involved in the platelet aggregation and cytoskeletal rearrangement. However, evidence regarding rapamycin's effect on platelet aggregation remains conflicting. Several studies have reported that rapamycin enhances platelet aggregation. 31 , 33 These discrepancies may be due to differences in aggregation assays, as light transmission aggregometry may be more sensitive in detecting subtle changes in platelet function. Also, the use of different agonists such as collagen and thrombin could provide a more comprehensive assessment of aggregation. To study procoagulant phenotypes, we utilized a modified approach to evaluate a panel of 3 markers by flow cytometry. 35 , 45 These markers include ΔΨm loss, PS externalization and P-selectin expression. This novel technique of evaluating procoagulant markers sequentially instead of simultaneous detection was developed specifically to study procoagulant platelets in cats due to several reasons. 35 First, feline platelets are more susceptible to in vitro activation due to their sensitivity to subtle changes in ambient temperature, causing cold-induced activation, irreversible shape change and microaggregates. 46 Unlike human and murine platelets, when feline platelets are exposed to low concentrations of exogenous calcium, they can undergo spontaneous aggregation in the absence of strong agonists. The sensitive nature of feline platelets, therefore, prevents the simultaneous detections of PS, P-selectin and ΔΨm. 35 The first procoagulant marker evaluated was ΔΨm after stimulation with strong platelet agonists. We showed that LDDRR prevented the loss of platelet ΔΨm with thrombin, COL or CVX stimulation at all time points. This preservation of ΔΨm is significant since ΔΨm loss is a marker of mitochondrial permeability transition pore (mPTP) opening. Potent agonists like thrombin and COL bind to their respective receptors, initiating a signaling cascade that transiently increases intracellular calcium, which facilitates calcium entry into the mitochondria via an electrochemical gradient across the inner mitochondrial membrane. Located on the inner mitochondrial membrane, the mPTP, which is regulated by cyclophilin D (CypD), opens to allow calcium efflux to the cytosol. This results in sustained cytosolic hypercalcemia, which is crucial to mediate intracellular signaling to promote procoagulant platelet formation. 18 Our data align with findings by Śledź et al., who assessed ΔΨm using the potentiometric dye, JC-1, in healthy human platelets, and found that in vitro rapamycin prevented the loss of ΔΨm in an mTORC1-independent manner. 34 The exact mechanisms underlying these observations in platelets are unclear, but rapamycin has been shown to reduce mitochondrial oxidative stress by reducing reactive oxygen species, promoting mPTP opening and calcium efflux. Rapamycin has also been shown to induce mitophagy, a specific type of autophagy, in other cell types, to facilitate removal of damaged or dysfunctional mitochondria improving mitochondrial function. 47 Clinically, individuals, especially elderly with age-related decline in mitochondrial efficiency, and those with cardiovascular diseases, are prone to mitochondrial dysfunction and loss of platelet ΔΨm. 48 , 49 In these groups, impaired mitochondrial function may exaggerate generation of procoagulant platelets, thereby increasing the risk of thrombotic complications. Thus, therapeutic strategies such as rapamycin, which preserves mitochondrial integrity, may hold promise when used in conjunction with traditional antithrombotic therapies. Following mPTP opening, supramaximal calcium stimulates the activation of TMEM16F, a scramblase enzyme, which promotes the translocation of the negatively charged PS from the inner to the outer side of the plasma membrane. As mentioned previously, the exposed PS facilitates thrombin generation by creating a surface for coagulation enzymes binding. 18 While LDDRR prevented ΔΨm loss induced by potent agonists at all timepoints, PS externalization was reduced only at 24 hrs but reverted to baseline at 48 hrs. Consistent with our findings, Śledź et al., which measured PS externalization in a similar fashion after thrombin and CVX stimulation, found that in vitro rapamycin treatment partially modulated PS externalization in a dose-dependent manner. 34 Since plasma concentration of rapamycin declines after peak concentration at 3 hrs post administration, this decrease in plasma concentration could explain the incomplete inhibition of PS externalization. However, the protective effect of rapamycin on mitochondrial membrane integrity lasted beyond 24 hrs, indicating that, in addition to mitochondria, there were other sources of calcium that could facilitate PS exposure and procoagulant platelet generation. One plausible explanation which could have caused this discrepancy in our results is the methodology used to assess PS flip. Following the measurement of ΔΨm, more exogenous calcium was added to PRP prior to quantifying PS expression. This exogenous calcium could enter the platelet cytosol via a process called store-operated calcium entry (SOCE), which serves to elevate intracellular calcium and, in part, plays a vital role in sustained supramaximal hypercalcemia. 50 Previous activation of platelets with PAR (thrombin) and glycoprotein VI agonists (COL, CVX) could have depleted calcium stores in the dense tubular system via inositol triphosphate-mediated release, activating Orai1 channels on the plasma membrane to promote calcium entry into the cytosol. 50 While mitochondria play a crucial role in providing calcium necessary for the sustained elevation of intracellular calcium required for procoagulant platelet formation, it is important to note that other mechanisms, such as SOCE, also synergistically elevate calcium to achieve supramaximal calcium levels. 50 This influx of extracellular calcium might explain the externalization of PS despite ΔΨm hyperpolarization under rapamycin treatment. Further experiments are needed to test this hypothesis. Nevertheless, rapamycin’s significant effect on reducing the PS exposure is crucial as it contributes to pathogenic or abnormal thrombin generation. P-selectin, a transmembrane protein found on the platelet alpha granules, was utilized, not only as a marker of alpha granule secretion, but also to distinguish between apoptotic and procoagulant platelets, since PS and ΔΨm loss are also features of apoptotic platelets. 35 , 45 However, while procoagulant platelets have surface expression of P-selectin, apoptotic platelets do not undergo alpha-granule release, and is, therefore, recommended as a surface marker by the International Society of Thrombosis and Haemostasis. 45 , 51 , 52 P-selectin, as a marker of procoagulant platelet, was significantly decreased in thrombin and COL-treated platelets 3 hrs after LDDRR treatment. Several studies have investigated the effects of rapamycin on P-selectin expression in platelets with varying results. One study in human platelets demonstrated that rapamycin did not have any immediate effects on thrombin-induced P-selectin expression. 53 Conversely, another study found that rapamycin decreases platelet P-selectin expression in older mice in a venous thrombosis model. 54 These findings suggest that rapamycin's effects on P-selectin expression may be age and species-dependent. The lack of significant modulation in P-selectin expression did not parallel with the decreases in PS externalization and ΔΨm upon stimulation with the respective agonists throughout the study period. Despite not evaluating all procoagulant markers simultaneously, this suggests that LDDRR might shift platelets from a procoagulant phenotype to a classical activation maintaining their response to agonists. Since we observed P-selectin upregulation in response to all procoagulant agonist groups before and after LDDRR treatment, it was unlikely that platelets underwent apoptosis. However, further studies utilizing more specific apoptotic markers will be needed to further rule out apoptosis due to strong agonist stimulations. These findings demonstrate the importance of evaluating multiple markers to accurately define procoagulant phenotypes. While P-selectin upregulation in response to strong agonists was preserved after LDDRR, platelet activation induced by ADP was significantly decreased at 24 and 48 hrs. The variation in rapamycin’s effects on P-selectin expression across different agonists may stem from the downstream signaling pathways initiated by agonists. Intracellular signaling pathways activated by ADP may be more prone to rapamycin modulation than those mediated by thrombin and GPVI agonist. One plausible explanation is that ADP-mediated platelet activation occurs primarily through the receptor, P2Y 12 , which activates the Akt signaling cascade, rather than PKC that thrombin utilizes. 53 , 55 No studies to date have examined the specific effects of mTORC1 inhibition on ADP-mediated activation. However, our results suggest that there are agonist-specific differences in the regulation of mTOR signaling, requiring further investigations. If rapamycin reduces ADP-mediated platelet priming or activation through an mTORC1-dependent mechanism, then rapamycin may elicit synergistic platelet inhibitory effects of P2Y 12 inhibitors like clopidogrel and prasugrel. Like human beings, cats also have reduced responses to P2Y 12 inhibitors secondary to pharmacogenomic variations and other co-morbidities resulting in reduced efficacy and increased on-treatment thrombotic events. As mentioned previously, LDDRR is conditionally approved for the treatment of feline HCM. Given that clopidogrel is the recommended first-line antithrombotic prevention in HCM cats, future studies should focus on evaluating the clinical benefits of dual therapy consisting of rapamycin and P2Y 12 inhibitors. It could enhance the therapeutic efficacy of clopidogrel by further attenuation of the ADP signaling pathway. We further validated our previously established scoring system to assess platelet procoagulant tendency in cats by demonstrating reductions in procoagulant scores at 24 hrs and, to a lower extent, 48 hrs, after LDDRR treatment. These findings suggest that rapamycin may provide short-term therapeutic benefits in preventing thrombosis associated with HCM. To better understand the physiological relevance of these findings, additional assessments such as thrombin generation, thrombus formation under shear stress and viscoelastic evaluations should be performed before and after LDDRR treatments. These assays would provide deeper insights into rapamycin’s effects on clot formation and fibrinolysis. Our data also suggest that the anti-procoagulant effects of rapamycin maybe transient, emphasizing the need to investigate the pharmacodynamics beyond 48 hrs. This would help determine the optimal dosing regimen to maintain sustained suppression of procoagulant platelet formation. Furthermore, additional studies should explore the potential interactions between LDDRR and antithrombotics, which may prolong the suppression of procoagulant platelet formation and enhance overall therapeutic efficacy. This study has several limitations. Rather than characterizing and quantifying procoagulant platelets by assessing all three markers simultaneously, we developed a scoring system that integrates data from each marker individually to evaluate the platelet procoagulant tendency in individual cats. Quantifying procoagulant platelets in cats presents a challenge due to the inherent sensitivity of feline platelets. While a stepwise evaluation of each procoagulant phenotype may be more physiological, it carries notable limitations. For instance, analyzing PS and P-selectin separately may impair our ability to distinguish between apoptotic and procoagulant platelets, as PS exposure occurs in both states. During protocol development, simultaneous detection of PS and P-selectin was attempted, but this led to suboptimal P-selectin antibody binding and in vitro clot formation. The co-detection of P-selectin or other activation markers such as CD40L with PS may better differentiate apoptotic from procoagulant platelets in feline samples in addressing this limitation. Additionally, the small sample size may underpower the detection of notable trends due to Type II error. Platelet function testing was performed in clinically healthy cats, which might not fully reflect the platelet hyperresponsiveness seen in HCM affected cats. Although mitochondrial membrane potential and PS externalization were assessed, rapamycin’s mechanism of action was not explored. Future studies are needed to investigate the mechanistic approach. Conclusion This was the first ex vivo study in a large animal model investigating the anti-platelet effects of rapamycin. Not only was rapamycin safe and well-tolerated, it also was effective in suppressing procoagulant platelet tendency. Considering that rapamycin did not alter platelet aggregation and no cats in our study had any adverse effects of hemorrhage, the modulation in procoagulant tendency by rapamycin suggests that rapamycin may prevent pathologic thrombosis without affecting hemostasis. Given the benefit of LDDRR in reversing HCM, clinical trials evaluating the additional antithrombotic effect of rapamycin in HCM-affected cats and individuals at risk of arterial thrombosis and stroke are warranted. Materials and Methods Animals Eight cats (4 intact males and 4 intact females) from a colony of Maine Coon/outbred mixed domestic cats from the Feline HCM Research Colony at University of California, Davis and North Carolina State University were enrolled for this study. Physical examinations, complete blood count using an automated hematology analyzer (HM5, Zoetis, Parsippany, NJ) and transthoracic echocardiogram were performed by the corresponding author and board-certified cardiologist (JAS) to ensure that all cats were clinically healthy prior to the study. No cats showed any echocardiographic evidence of HCM at the time of the study. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of California, Davis (protocol number: 21895) and carried out in accordance with relevant guidelines and regulations. All methods were reported in compliance with the ARRIVE guidelines. Drug administration Cats were treated with low-dose delayed release rapamycin (LDDRR) 0.3 mg/kg rapamycin orally every 7 days for 4 consecutive weeks according to previously published study and pharmacokinetic data from the manufacturer. 26 At the time of the study, formulation of rapamycin provided by the manufacturer was not commercially available for veterinary use while conditional approval is sought from the FDA Center for Veterinary Medicine. During the study period, all cats were fed the same diet and housed in groups with environmental enrichment. Cats were monitored twice daily for any potential adverse effects such as gastrointestinal signs (vomiting, diarrhea, inappetence) and bleeding diathesis (hematomas, hematuria, hematemesis, petechia and ecchymosis) throughout the study period and up to 1 week after drug administration. Blood collection Four to 6 ml of blood was then collected from either the medial saphenous or jugular vein using a 21- or 23-gauge butterfly needle. Blood was transferred immediately to tubes containing 3.2% trisodium citrate or sodium heparin. Each tube was gently inverted and inspected for any possible clotting. Blood sampling occurred 24 hrs before the first dose of delay-released rapamycin, and at 3, 24 and 48 hrs after the last dose in week 4. Whole blood impedance platelet aggregometry Platelet aggregation was measured by whole blood impedance platelet aggregometry (Multiplate, Roche, Mannheim, Germany) at 24 and 48 hrs after the last dose of rapamycin as previously described. Briefly, heparinized whole blood is diluted with equal volumes of pre-warmed 0.9% NaCl and incubated at 37°C for 3 minutes under physiologic shear generated by Teflon-coated magnetic stir bars at 800-rpm. ADP (6.25 µM, MilliporeSigma, Burlingon, MA) was then added and platelet aggregation, measured as electrical impedance over a 6-minute period, was reported as maximum aggregation, velocity (AU/min) and area under the curve (AUC; AU x minutes). Platelet rich plasma isolation Within 30 minutes of blood collection, citrated whole blood was placed in a 37°C bead bath for 30 minutes to facilitate red blood cell sedimentation. Platelet rich plasma (PRP) was then generated by centrifugation at 300 × g (21°C, no brakes) for 5 minutes. PRP was assessed for “swirling” to ensure that platelets were still in their resting discoid shape. An automated hematology analyzer (HM5, Zoetis, Parsippany, NJ) was used to measure platelet counts in PRP and further confirmed by manual blood smear analysis. Prior to activation, PRP was rested for an additional 30 minutes at 37°C. Procoagulant platelet characterization Mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) externalization and P-selectin expression were analyzed consecutively by flow cytometry to identify and characterize procoagulant platelets as previously described. 35 In brief, platelets were first standardized to 1x10 7 cells/ml using Tyrode HEPES (pH 7.2, 5.5 mM dextrose, 1mM CaCl 2 ) to a final volume of 200 µl. Tetramethylrhodamine methyl ester (TMRM) (320nM, Invitrogen, Eugene, OR) was added to PRP and placed in 21% oxygen at 37°C under gentle rocking for 15 minutes. TMRM-loaded platelets were subsequently stimulated with bovine alpha thrombin (0.001U/ml, 5 minutes at 37°C, Haematologic Technologies, Inc., Essex Junction, VT) prior to further activation with equine type I collagen (COL) (4 µg/ml, CHRONO-LOG Corp., Havertown, PA), or convulxin (CVX) (100 ng/ml, Cayman chemicals, Ann Arbor, MI) for an additional 5 minutes. Unstimulated platelets served as positive control while platelets treated with either 20 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Invitrogen, Eugene, OR) or 10 µM A23187 (Millipore Sigma, Burlington, MA) served as negative controls (Figs. 2A - C). Platelets were then analyzed live by flow cytometry (Beckman-Coulter FC500, Beckman-Coulter Inc., Miami, FL). Shortly after, the remaining aliquots of platelets were loaded with additional 1mM CaCl 2 (final concentration 2mM). To quantify PS externalization, platelets were labelled with annexin V conjugated to fluorescein isothiocyanate (FITC) (1:200, Cat: 556419, BD Pharmingen, San Jose, CA) for 45 minutes at 37°C followed by fixation with 0.1% methanol free paraformaldehyde (Thermo Scientific, Waltham, MA). Unstimulated platelets and platelets treated with A23187 served as the negative and positive controls, respectively. The final marker, P-selectin, was analyzed in a separate aliquot of PRP, standardized to 1x10 7 cells/ml with Tyrode HEPES (pH 7.2, 5mM dextrose, no divalent cations). Resting platelets and adenosine diphosphate (ADP) (20 µM, Sigma-Aldrich, St. Louis, MO) treated platelets served as negative and positive controls, respectively. After platelet activation with the aforementioned agonists, rat anti-mouse monoclonal antibody conjugated to FITC specific to P-selectin (CD62P) (1:200, Clone: RB40.34, BD Biosciences, San Diego, CA) along with mouse anti-human monoclonal antibody conjugated to allophycocyanin against β3-integrin (CD61) (1:1000, Clone:VI-PL2, eBioscience, San Deigo, CA) were added to the platelets. Platelets then were incubated for 45 minutes at 37°C followed by fixation similar to annexin V. Flow Cytometry Analysis Platelets were identified by forward- and side-scatter properties previously established using 0.9 µm and 3 µm calibration beads in the log scale as described and 10,000 events were analyzed for each experimental condition (Figs. 3A, 4 A, 5 A). 12 Gating of TMRM, PS and P-selectin positive platelets (Figs. 3B, 4 B, 5 B). The magnitude in the loss of ΔΨm in response to agonists was calculated using Equations 1 and 2 using values of TMRM median fluorescence intensity (MFI) and the percentage of TMRM-positive cells (Fig. 3E, F). Externalization of PS in response to agonists was evaluated as change in percentage increase using the following Eq. 3. Lastly, agonist-induced upregulation of P-selectin was quantified using Eq. 4. All flow cytometry data were assessed using commercially available software (FlowJo, Tree Str Inc., Ashland, OR). Equation 1: $$\:TMRM\:MFI\:Fold\:Change\:\left(\text{log}10\right)=\left(\text{log}10\:MFI\:activated\right)-\left(\text{log}10\:MFI\:resting\right)$$ Equation 2: $$\:TMRM\:\left(\%\:change\right)=\frac{\left(\%TMRM\:activated\right)-\left(\%TMRM\:resting\right)}{\left(\%TMRM\:resting\right)}\:\times\:100$$ Equation 3: $$\:Annexin\:V\:\left(\%\:change\right)=\frac{\left(\%AV\:activated\right)-\left(\%AV\:resting\right)}{\left(\%AV\:resting\right)}\times\:100$$ Equation 4: $$\:P-selectin\:MFI\:Fold\:Change\:\left(\text{log}10\right)=\left(\text{log}10\:MFI\:activated\right)-\left(\text{log}10\:MFI\:resting\right)$$ Scoring system for identifying the tendency of procoagulant platelet formation in cats The procoagulant tendency of platelets in each cat was assessed using a previously established scoring system. 35 In brief, the distributions of the 3 procoagulant markers were assessed after in vitro treatments with the 3 agonist groups (thrombin, thrombin + COL and thrombin + CVX. Arbitrary cut-offs of each procoagulant marker were assigned based on the medians and interquartile ranges of platelet responses generated in a population of 8 healthy cats. A score of 1 was assigned to the specific phenotype if the response to the agonist group was above or below the 40th percentile of the data generated in the population. Table 2 outlines the proposed scoring system for assessing procoagulant tendency in individual cats. A score of 0 was assigned if P-selectin upregulation did not exceed the anticipated increase as it was categorized as an apoptotic response. A total score of 3 in an individual was considered to have the highest procoagulant tendency while a score of 0 was considered to have apoptotic or the lowest procoagulant tendency. Table 2 Criteria for determining the tendency of procoagulant scores in individual cats. Median fluorescence intensity (MFI), tetramethyl rhodamine methyl ester (TMRM), interquartile range (IQR). Procoagulant marker Median (IQR) Assigned score 1. P-selectin 0.1 (0.005 to 0.13) 1: If MFI FC (log10) ≥ 0.05 from resting 0: If MFI FC (log10) < 0.05 from resting 2. Mitochondrial membrane potential-TMRM -32.4% (-54.2% to -17.9%) 1: If change (%) in ΔΨm ≥ -22.5% from resting 0: If Change (%) in ΔΨm < -22.5% from resting 3. Phosphatidylserine-Annexin V 45.4% (12.7–152.8%) 1: If change (%) in PS ≥ 30% from resting 0: If Change (%) in PS < 30% from resting Statistical analysis Based on preliminary data, sample size calculations showed that a minimum of 6 cats were needed to detect the lowest detectable effect of 30% in platelet activation with 80% power and an alpha-priori of 0.05. Due to the high plausibility of in vitro activation and spontaneous aggregation when processing feline platelets, a total of 8 cats were included. The normality of data was determined by D’Agostino and Pearson test. Data that were normally distributed were presented as mean ± standard deviation while non-parametric data were presented as median and interquartile range (IQR). Paired parametric and non-parametric paired data were compared using t -tests and Wilcoxon signed-rank test, respectively, as appropriate based on normality. Inter-individual variability was assessed by co-efficient of variation, which is calculated by the ratio of standard deviation to mean. Repeated measures ANOVA and Kruskal-Wallis one-way analysis of variance were utilized to analyze the treatment effect over time with post-hoc analyses by Tukey test. Fisher’s exact test was used in categorical data analysis to compare procoagulant tendency scores among cats. Data were considered statistically significant if p < 0.05. Statistical analyses were conducted using commercially available software (Prism 10.0, GraphPad Software, La Jolla, CA). Declarations Competing Interests Funding and disclosures: This study was funded by TriviumVet (Waterford, Ireland); industry sponsor did not have any influence over data analysis, final results or conclusion of this study. Funding for M.S. was provided in part by both University of California, Davis and TriviumVet. Authors S.F. and L.G. are employees of TriviumVet and J.A.S serves as a scientific advisory board member for TriviumVet. The authors, N.N. and R. H. L.L. declare no conflict of interest. Funding and disclosures: This study was funded by TriviumVet (Waterford, Ireland); industry sponsor did not have any influence over data analysis, final results or conclusion of this study. Funding for M.S. was provided in part by both University of California, Davis and TriviumVet. Authors S.F. and L.G. are employees of TriviumVet and J.A.S serves as a scientific advisory board member for TriviumVet. The remaining authors, N.N. and R.H.L.L. declare no conflict of interest. Author Contribution M.S, J.A.S, S.F, L.G and R.H.L.L conceived the research. J.A.S, N.N, M.S. and R.H.L.L selected the study subjects. M.S., N.N, R.H.L.L collected the data. M.S., N.N, R.H.L.L., J.A.S. analyzed the data. M.S and R.H.L.L drafted the manuscript and all authors contributed to the revision of the manuscript and approved of the final version of the manuscript for submission. The remaining authors, N.N. and R.H.L.L. declare no conflict of interest. Acknowledgement The authors would like to thank Carina Gonzalez for her assistance with the data collection for this study. Data Availability The authors declare that all data supporting the findings of this study are available within the article or from the corresponding author upon request. References Payne, J. R. & Brodbelt, D. C. Luis Fuentes, V. Cardiomyopathy prevalence in 780 apparently healthy cats in rehoming centres (the CatScan study). J. Vet. Cardiol. 17 (Suppl 1), 244–257. https://doi.org/10.1016/j.jvc.2015.03.008 (2015). Paige, C. F., Abbott, J. A., Elvinger, F. & Pyle, R. L. Prevalence of cardiomyopathy in apparently healthy cats. J. Am. Vet. Med. Assoc. 234 , 1398–1403. https://doi.org/10.2460/javma.234.11.1398 (2009). Freeman, L. M., Rush, J. E., Stern, J. A., Huggins, G. S. & Maron, M. S. Feline Hypertrophic Cardiomyopathy: A Spontaneous Large Animal Model of Human HCM. Cardiol. Res. 8 , 139–142. https://doi.org/10.14740/cr578w (2017). Sharpe, A. N. et al. Pharmacokinetics of a single dose of Aficamten (CK-274) on cardiac contractility in a A31P MYBPC3 hypertrophic cardiomyopathy cat model. J. Vet. Pharmacol. Ther. 46 , 52–61. https://doi.org/10.1111/jvp.13103 (2023). Luis Fuentes, V. et al. ACVIM consensus statement guidelines for the classification, diagnosis, and management of cardiomyopathies in cats. J. Vet. Intern. Med. 34 , 1062–1077. https://doi.org/10.1111/jvim.15745 (2020). Maron, B. J. et al. Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults. Circulation 92 , 785–789. https://doi.org/10.1161/01.cir.92.4.785 (1995). Olivotto, I. et al. Impact of atrial fibrillation on the clinical course of hypertrophic cardiomyopathy. Circulation 104 , 2517–2524. https://doi.org/10.1161/hc4601.097997 (2001). Siontis, K. C. et al. Atrial fibrillation in hypertrophic cardiomyopathy: prevalence, clinical correlations, and mortality in a large high-risk population. J. Am. Heart Assoc. 3 , e001002. https://doi.org/10.1161/JAHA.114.001002 (2014). Côté, E. et al. Atrial fibrillation in cats: 50 cases (1979–2002). J. Am. Vet. Med. Assoc. 225 , 256–260. https://doi.org/10.2460/javma.2004.225.256 (2004). Hogan, D. F. Feline Cardiogenic Arterial Thromboembolism: Prevention and Therapy. Vet. Clin. North. Am. Small Anim. Pract. 47 , 1065–1082. https://doi.org/10.1016/j.cvsm.2017.05.001 (2017). Tablin, F. et al. Platelet activation in cats with hypertrophic cardiomyopathy. J. Vet. Intern. Med. 28 , 411–418. https://doi.org/10.1111/jvim.12325 (2014). Li, R. H., Stern, J. A., Ho, V., Tablin, F. & Harris, S. P. Platelet Activation and Clopidogrel Effects on ADP-Induced Platelet Activation in Cats with or without the A31P Mutation in MYBPC3. J. Vet. Intern. Med. 30 , 1619–1629. https://doi.org/10.1111/jvim.14568 (2016). Tan, A. W. K. et al. Platelet Priming and Activation in Naturally Occurring Thermal Burn Injuries and Wildfire Smoke Exposure Is Associated With Intracardiac Thrombosis and Spontaneous Echocardiographic Contrast in Feline Survivors. Front. Vet. Sci. 9 , 892377. https://doi.org/10.3389/fvets.2022.892377 (2022). Dimitrow, P. P., Undas, A., Bober, M., Tracz, W. & Dubiel, J. S. Obstructive hypertrophic cardiomyopathy is associated with enhanced thrombin generation and platelet activation. Heart 94 , e21. https://doi.org/10.1136/hrt.2007.126896 (2008). Weikert, U., Kühl, U., Schultheiss, H. P. & Rauch, U. Platelet activation is increased in patients with cardiomyopathy: myocardial inflammation and platelet reactivity. Platelets 13 , 487–491. https://doi.org/10.1080/0953710021000057857 (2002). Icli, A. et al. Increased mean platelet volume in hypertrophic cardiomyopathy. Angiology 65 , 420–424. https://doi.org/10.1177/0003319713483919 (2014). Li, R. H. L. et al. Circulating neutrophil extracellular traps in cats with hypertrophic cardiomyopathy and cardiogenic arterial thromboembolism. J. Vet. Intern. Med. 37 , 490–502. https://doi.org/10.1111/jvim.16676 (2023). Agbani, E. O. & Poole, A. W. Procoagulant platelets: generation, function, and therapeutic targeting in thrombosis. Blood 130 , 2171–2179. https://doi.org/10.1182/blood-2017-05-787259 (2017). Prodan, C. I., Vincent, A. S. & Dale, G. L. Coated-platelet levels are elevated in patients with transient ischemic attack. Transl Res. 158 , 71–75. https://doi.org/10.1016/j.trsl.2011.02.010 (2011). Pasalic, L. et al. Novel assay demonstrates that coronary artery disease patients have heightened procoagulant platelet response. J. Thromb. Haemost . 16 , 1198–1210. https://doi.org/10.1111/jth.14008 (2018). Kaiser, R. et al. Procoagulant platelet activation promotes venous thrombosis. Blood 144 , 2546–2553. https://doi.org/10.1182/blood.2024025476 (2024). Ommen, S. R. et al. AHA/ACC Guideline for the Diagnosis and Treatment of Patients With Hypertrophic Cardiomyopathy: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 142, e558-e631 (2020). (2020). https://doi.org/10.1161/CIR.0000000000000937 Blais, M. C. et al. Consensus on the Rational Use of Antithrombotics in Veterinary Critical Care (CURATIVE): Domain 3-Defining antithrombotic protocols. J. Vet. Emerg. Crit. Care (San Antonio) . 29 , 60–74. https://doi.org/10.1111/vec.12795 (2019). Hogan, D. F. et al. Secondary prevention of cardiogenic arterial thromboembolism in the cat: The double-blind, randomized, positive-controlled feline arterial thromboembolism; clopidogrel vs. aspirin trial (FAT CAT). J. Vet. Cardiol. 17 (Suppl 1), 306–317. https://doi.org/10.1016/j.jvc.2015.10.004 (2015). Sciarretta, S., Forte, M., Frati, G. & Sadoshima, J. New Insights Into the Role of mTOR Signaling in the Cardiovascular System. Circ. Res. 122 , 489–505. https://doi.org/10.1161/CIRCRESAHA.117.311147 (2018). Kaplan, J. L. et al. Delayed-release rapamycin halts progression of left ventricular hypertrophy in subclinical feline hypertrophic cardiomyopathy: results of the RAPACAT trial. J. Am. Vet. Med. Assoc. 261 , 1628–1637. https://doi.org/10.2460/javma.23.04.0187 (2023). Shioi, T. et al. Rapamycin attenuates load-induced cardiac hypertrophy in mice. Circulation 107 , 1664–1670. https://doi.org/10.1161/01.CIR.0000057979.36322.88 (2003). Paoletti, E. et al. Effect of sirolimus on left ventricular hypertrophy in kidney transplant recipients: a 1-year nonrandomized controlled trial. Am. J. Kidney Dis. 52 , 324–330. https://doi.org/10.1053/j.ajkd.2008.04.018 (2008). McMullen, J. R. et al. Inhibition of mTOR signaling with rapamycin regresses established cardiac hypertrophy induced by pressure overload. Circulation 109 , 3050–3055. https://doi.org/10.1161/01.CIR.0000130641.08705.45 (2004). Rivas, V. N. et al. Multi-Omic, Histopathologic, and Clinicopathologic Effects of Once-Weekly Oral Rapamycin in a Naturally Occurring Feline Model of Hypertrophic Cardiomyopathy: A Pilot Study. Anim. (Basel) . 13. https://doi.org/10.3390/ani13203184 (2023). Wu, Q., Huang, K. S., Chen, M. & Huang, D. J. Rapamycin enhances platelet aggregation induced by adenosine diphosphate in vitro. Platelets 20 , 428–431. https://doi.org/10.1080/09537100903114552 (2009). Aslan, J. E., Tormoen, G. W., Loren, C. P., Pang, J. & McCarty, O. J. S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation. Blood 118 , 3129–3136. https://doi.org/10.1182/blood-2011-02-331579 (2011). Babinska, A. et al. Enhancement of human platelet aggregation and secretion induced by rapamycin. Nephrol. Dial Transpl. 13 , 3153–3159. https://doi.org/10.1093/ndt/13.12.3153 (1998). Śledź, K. M. et al. Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity. Biochem. Pharmacol. 177 , 113975. https://doi.org/10.1016/j.bcp.2020.113975 (2020). Shaverdian, M., Nguyen, N. & Li, R. H. L. A novel technique to characterize procoagulant platelet formation and evaluate platelet procoagulant tendency in cats by flow cytometry. Front. Vet. Sci. 11 , 1480756. https://doi.org/10.3389/fvets.2024.1480756 (2024). Castedo, M., Ferri, K. F. & Kroemer, G. Mammalian target of rapamycin (mTOR): pro- and anti-apoptotic. Cell. Death Differ. 9 , 99–100. https://doi.org/10.1038/sj.cdd.4400978 (2002). Donato, G., Pennisi, M. G., Persichetti, M. F., Archer, J. & Masucci, M. A. Retrospective Comparative Evaluation of Selected Blood Cell Ratios, Acute Phase Proteins, and Leukocyte Changes Suggestive of Inflammation in Cats. Anim. (Basel) . 13. https://doi.org/10.3390/ani13162579 (2023). Kay, J. E., Kromwel, L., Doe, S. E. & Denyer, M. Inhibition of T and B lymphocyte proliferation by rapamycin. Immunology 72 , 544–549 (1991). Battaglia, M. et al. Rapamycin promotes expansion of functional CD4 + CD25 + FOXP3 + regulatory T cells of both healthy subjects and type 1 diabetic patients. J. Immunol. 177 , 8338–8347. https://doi.org/10.4049/jimmunol.177.12.8338 (2006). Thomson, A. W., Turnquist, H. R. & Raimondi, G. Immunoregulatory functions of mTOR inhibition. Nat. Rev. Immunol. 9 , 324–337. https://doi.org/10.1038/nri2546 (2009). Pelekanou, V., Trezise, A. E., Moore, A. L. & Kay, J. E. FK 506 and rapamycin do not affect platelet aggregation or mitochondrial function. Transpl. Proc. 23 , 3200–3201 (1991). López, E. et al. Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients. J. Cell. Mol. Med. 17 , 636–647. https://doi.org/10.1111/jcmm.12044 (2013). Hollerbach, A. et al. Platelet Activation by Antiphospholipid Antibodies Depends on Epitope Specificity and is Prevented by mTOR Inhibitors. Thromb. Haemost . 119 , 1147–1153. https://doi.org/10.1055/s-0039-1685453 (2019). Ouseph, M. M. et al. Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis. Blood 126 , 1224–1233. https://doi.org/10.1182/blood-2014-09-598722 (2015). Josefsson, E. C., Ramström, S., Thaler, J. & Lordkipanidzé, M. & group, C. s. Consensus report on markers to distinguish procoagulant platelets from apoptotic platelets: communication from the Scientific and Standardization Committee of the ISTH. J Thromb Haemost 21, 2291–2299 (2023). https://doi.org/10.1016/j.jtha.2023.05.001 Tablin, F., Walker, N. J., Klein, S. D., Field, C. L. & Crowe, J. H. Animal models for studies on cold-induced platelet activation in human beings. J. Lab. Clin. Med. 135 , 339–346. https://doi.org/10.1067/mlc.2000.105619 (2000). Li, Q. et al. Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke. Biochem. Biophys. Res. Commun. 444 , 182–188. https://doi.org/10.1016/j.bbrc.2014.01.032 (2014). Panel, M., Ghaleh, B. & Morin, D. Mitochondria and aging: A role for the mitochondrial transition pore? Aging Cell. 17 , e12793. https://doi.org/10.1111/acel.12793 (2018). Kadenbach, B., Ramzan, R., Moosdorf, R. & Vogt, S. The role of mitochondrial membrane potential in ischemic heart failure. Mitochondrion 11 , 700–706. https://doi.org/10.1016/j.mito.2011.06.001 (2011). Millington-Burgess, S. L. & Harper, M. T. Cytosolic and mitochondrial Ca. Platelets 32 , 855–862. https://doi.org/10.1080/09537104.2021.1881951 (2021). Zhang, H. et al. Bcl-2 family proteins are essential for platelet survival. Cell. Death Differ. 14 , 943–951. https://doi.org/10.1038/sj.cdd.4402081 (2007). Hua, V. M. et al. Necrotic platelets provide a procoagulant surface during thrombosis. Blood 126 , 2852–2862. https://doi.org/10.1182/blood-2015-08-663005 (2015). Moore, S. F., Hunter, R. W. & Hers, I. Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets. J. Thromb. Haemost . 12 , 748–760. https://doi.org/10.1111/jth.12552 (2014). Yang, J. et al. mTORC1 promotes aging-related venous thrombosis in mice via elevation of platelet volume and activation. Blood 128 , 615–624. https://doi.org/10.1182/blood-2015-10-672964 (2016). Woulfe, D., Jiang, H., Mortensen, R., Yang, J. & Brass, L. F. Activation of Rap1B by G(i) family members in platelets. J. Biol. Chem. 277 , 23382–23390. https://doi.org/10.1074/jbc.M202212200 (2002). Additional Declarations Competing interest reported. Funding and disclosures: This study was funded by TriviumVet (Waterford, Ireland); industry sponsor did not have any influence over data analysis, final results or conclusion of this study. Funding for M.S. was provided in part by both University of California, Davis and TriviumVet. Authors S.F. and L.G. are employees of TriviumVet and J.A.S serves as a scientific advisory board member for TriviumVet. The authors, N.N. and R. H. L.L. declare no conflict of interest. Cite Share Download PDF Status: Published Journal Publication published 11 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 06 Feb, 2026 Reviews received at journal 04 Feb, 2026 Reviews received at journal 28 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 05 Jan, 2026 Reviews received at journal 14 Nov, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviewers invited by journal 30 Oct, 2025 Editor assigned by journal 30 Oct, 2025 Editor invited by journal 31 Jul, 2025 Submission checks completed at journal 30 Jul, 2025 First submitted to journal 30 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7068900","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":542871607,"identity":"49d342a3-197b-48b5-92e5-f68976b81509","order_by":0,"name":"Meg Shaverdian","email":"","orcid":"","institution":"University of California","correspondingAuthor":false,"prefix":"","firstName":"Meg","middleName":"","lastName":"Shaverdian","suffix":""},{"id":542871608,"identity":"225262ea-d01d-4bf4-92aa-35a452b56a34","order_by":1,"name":"Nghi Nguyen","email":"","orcid":"","institution":"University of California","correspondingAuthor":false,"prefix":"","firstName":"Nghi","middleName":"","lastName":"Nguyen","suffix":""},{"id":542871609,"identity":"e044a774-5a96-41b5-aae1-c56a6f31fc93","order_by":2,"name":"Stuart Fitzgerald","email":"","orcid":"","institution":"TriviumVet","correspondingAuthor":false,"prefix":"","firstName":"Stuart","middleName":"","lastName":"Fitzgerald","suffix":""},{"id":542871610,"identity":"056725b4-b225-4484-963e-2ade7f66cb83","order_by":3,"name":"Louise Grubb","email":"","orcid":"","institution":"TriviumVet","correspondingAuthor":false,"prefix":"","firstName":"Louise","middleName":"","lastName":"Grubb","suffix":""},{"id":542871611,"identity":"77e7f715-a9a6-42c3-a2c0-a70fa9be958e","order_by":4,"name":"Joshua A. Stern","email":"","orcid":"","institution":"North Carolina State University","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"A.","lastName":"Stern","suffix":""},{"id":542871612,"identity":"72ac14af-eb31-4145-b5af-48c8d65035e5","order_by":5,"name":"Ronald H. L. Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAp0lEQVRIiWNgGAWjYDACCeaGAx8qoBwe4rQwNhyccYZULcy8baRoMZ/d2HiAd94defkZCYwP3rYR1sEgc+dgwwHJbc8MN9xIYDacS4wWCYnEhgOG2w4nGEgksEnzEq0lcc7hBKDD2H8Tr+Vgw+EEhhsJbMxEaznYcOyw4YYzD5sl55wjSkvy4c9/ag7Ly7cnH/zwpowILUiAsYE09aNgFIyCUTAKcAMAzCY6JCB9R9kAAAAASUVORK5CYII=","orcid":"","institution":"North Carolina State University","correspondingAuthor":true,"prefix":"","firstName":"Ronald","middleName":"H. L.","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-07 22:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7068900/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7068900/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-46991-z","type":"published","date":"2026-04-11T15:58:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95618573,"identity":"6d29aadb-8f64-492d-b1f3-0cbb17572e2c","added_by":"auto","created_at":"2025-11-11 09:17:38","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2053411,"visible":true,"origin":"","legend":"","description":"","filename":"ExVivoRapamycinPlateletCats7.30.25.docx","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/6f3e900ff5635849063ea097.docx"},{"id":95618571,"identity":"28543637-b1ac-49a7-87cb-8869c7e6a199","added_by":"auto","created_at":"2025-11-11 09:17:38","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8081,"visible":true,"origin":"","legend":"","description":"","filename":"825e6aa009344f52a21bd339cd33b579.json","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/78f23958ac9b1c530277c4ff.json"},{"id":95656647,"identity":"dc3103f0-4a02-4b89-8bc0-c5886d289eda","added_by":"auto","created_at":"2025-11-11 16:19:14","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":164786,"visible":true,"origin":"","legend":"","description":"","filename":"825e6aa009344f52a21bd339cd33b5791enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/d02323a64bba24121ceca08a.xml"},{"id":95618569,"identity":"5778aa61-6bde-4963-a602-271f19972d6e","added_by":"auto","created_at":"2025-11-11 09:17:38","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134848,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/1f1e794d43d649a0b2eaffcb.jpeg"},{"id":95656710,"identity":"37ff3b25-d2c0-42a6-9bfa-3298950e6b9f","added_by":"auto","created_at":"2025-11-11 16:19:25","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141140,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/bbf6f5ec1cbe5cb741e310ae.jpeg"},{"id":95656646,"identity":"9d67d101-1cd2-42d7-be96-79fc18411425","added_by":"auto","created_at":"2025-11-11 16:19:14","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":809744,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/404ba5937398af52e555fe89.jpeg"},{"id":95618579,"identity":"b86b2823-c213-4a1e-82df-65bdd356cb8d","added_by":"auto","created_at":"2025-11-11 09:17:39","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":264186,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/850b0a0ac9f8bc56ba4c24b0.jpeg"},{"id":95656149,"identity":"4b65431a-32e5-4647-8e70-24e35ac3ce5e","added_by":"auto","created_at":"2025-11-11 16:17:52","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169799,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/70fe886c5af218d8411411e8.png"},{"id":95618588,"identity":"986673cc-6b26-4413-adef-4add4f1ffb31","added_by":"auto","created_at":"2025-11-11 09:17:39","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":336888,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/6f2f662adbe949b5a1150ddf.jpeg"},{"id":95656530,"identity":"e29fc229-36ad-410f-8789-009df4984245","added_by":"auto","created_at":"2025-11-11 16:18:56","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18640,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/48f110027ce6beb4ef84f1a8.png"},{"id":95618575,"identity":"65096a5b-3a6e-4ba0-b0dd-8fe3759bd361","added_by":"auto","created_at":"2025-11-11 09:17:38","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22014,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/50649c98699c5b4ca2bed657.png"},{"id":95618582,"identity":"a364554a-0025-40de-9c4b-2ee28e0228f9","added_by":"auto","created_at":"2025-11-11 09:17:39","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":611252,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/8efbc713d7e2ac6dfe53fe88.png"},{"id":95618586,"identity":"8d01a7e2-9a62-4e0c-b26f-2e3c11775bb1","added_by":"auto","created_at":"2025-11-11 09:17:39","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":36490,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/0f3597e712e5f4cc8e8c0c63.png"},{"id":95618581,"identity":"714be679-eeb7-4270-997c-d7a9c91681ca","added_by":"auto","created_at":"2025-11-11 09:17:39","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48079,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/454d6052a02a9f8f4ffb6b2c.png"},{"id":95618585,"identity":"f8b7b8e1-47bd-47c7-9460-f87f4349fca0","added_by":"auto","created_at":"2025-11-11 09:17:39","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":182189,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/3041f308a018bb35e8ca5c58.png"},{"id":95656485,"identity":"d0de1cad-9fad-4fa0-8656-e73c39d68c00","added_by":"auto","created_at":"2025-11-11 16:18:49","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161949,"visible":true,"origin":"","legend":"","description":"","filename":"825e6aa009344f52a21bd339cd33b5791structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/c0d81578068394c5668b69a2.xml"},{"id":95618584,"identity":"8e6c5b89-7ced-4a89-a669-a258122f1a37","added_by":"auto","created_at":"2025-11-11 09:17:39","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176368,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/8ef4f9349f5adcbc4f754012.html"},{"id":95618566,"identity":"3958af10-4406-4b23-b1be-b498d359ac8a","added_by":"auto","created_at":"2025-11-11 09:17:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA summary of workflow used in this study.\u003c/strong\u003e Eight cats were treated with low dose (0.3 mg/kg) delayed release rapamycin (LDDRR) orally every 7 days for 4consecutive weeks. Blood was collected at 3, 24 and 48 hours following the final dose of rapamycin. Platelet-rich plasma (PRP) was isolated and part of the isolated PRP was used for stepwise assessment of mitochondrial membrane potential and PS exposure and the remaining part was used to assess platelet activation detected by P-selectin.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/0a79ab0c8adb4c6e64e10667.png"},{"id":95618568,"identity":"4584d68a-5ed8-4a8b-9031-4a244536f176","added_by":"auto","created_at":"2025-11-11 09:17:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47355,"visible":true,"origin":"","legend":"\u003cp\u003ePlatelet aggregation, measured as (A) area under the curve (AUC), (B) aggregation unit (AU) and (C) velocity (AU/min), was evaluated by whole blood electrical impedance aggregometry in 8 cats. Diluted whole blood was treated with 6.25µM adenosine diphosphate (ADP) before or at 24 and 48 hours after-LDDRR treatment for 4 weeks. No difference in platelet aggregation was found at 24 and 48 hours when compared to baseline measurements. Dotted lines represent third quartile, median and first quartile. Width of the plots demonstrate frequency at which values occur.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/75c58b239144310936512a21.png"},{"id":95618570,"identity":"d613055b-ad13-49d9-b1e1-153c2134f767","added_by":"auto","created_at":"2025-11-11 09:17:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":173818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEx vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emeasurement of mitochondrial membrane potential in live platelets. \u003c/strong\u003ePlatelets isolated from 8 cats were loaded with tetramethylrhodamine methyl ester (TMRM)before stimulation with thrombin, thrombin/collagen (COL) and thrombin/convulxin (CVX). The extent of mitochondrial membrane potential loss was determined by measuring the percent change (%) in TMRM-positive platelets and fold change (FC) (log10) in TMRM median fluorescence intensity (MFI). \u003cstrong\u003e(A)\u003c/strong\u003e First, platelets were identified on flow cytometry using forward (FS) and side scatter (SS) properties and \u003cstrong\u003e(B)\u003c/strong\u003e gating for TMRM-positive platelets were established in fluorescent-minus-one (FMO) controls in thrombin + COL stimulated platelets. \u003cstrong\u003e(C)\u003c/strong\u003e Histograms demonstrating the loss of TMRM following stimulation with thrombin + CVX (red) compared to the resting (unstimulated) platelets (blue) at baseline. At 24 and 48 hours after rapamycin treatment, a graduate increase in TMRM positive platelets was shown demonstrating mitochondrial protective effect despite stimulation with thrombin and CVX.\u003cstrong\u003e (D)\u003c/strong\u003eViolin plots demonstrating that rapamycin at 3, 24 and 48 hours prevented the loss of mitochondrial membrane potential upon stimulation by all three agonists. \u003cstrong\u003e(E)\u003c/strong\u003e Loss of mitochondria membrane potential was shown to be protected by rapamycin when platelets were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) when the change in TMRM was measured as MFI FC. CCCP-treated platelets served as negative controls. *p \u0026lt; 0.05, **p\u0026lt;0.01. Dotted lines represent third quartile, median and first quartile. Width of the plots demonstrate frequency at which values occur.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/a3e3588d77cd0fdfc8eef264.png"},{"id":95658002,"identity":"de58b471-dfc4-451e-916f-28f3593546f6","added_by":"auto","created_at":"2025-11-11 16:22:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx vivo effect of rapamycin on platelet phosphatidylserine externalization in 3-, 24-, and 48-hours post-treatment. \u003c/strong\u003eMagnitude of phosphatidylserine (PS) flip was measured as percent change (%) based on baseline measurements. PS on platelets was detected by annexin V (AV) by flow cytometry following activation by thrombin, thrombin/ collagen (COL) and thrombin/ convulxin (CVX). \u003cstrong\u003e(A) \u003c/strong\u003eResting or unstimulated platelets had minimal externalized PS. \u003cstrong\u003e(B) \u003c/strong\u003eFluorescent minus one (FMO) control in thrombin + COL treated platelets was used to establish gating to identify AV-positive platelets. \u003cstrong\u003e(C)\u003c/strong\u003e Histograms showing an increase in PS depicted by a shift to the right after stimulation with thrombin + COL (dark purple) sample compared to unstimulated platelets (blue) at baseline and 24, 48 hours post rapamycin dosing. \u003cstrong\u003e(D)\u003c/strong\u003e Violin plots showing that PS flip was prevented at 24 hours post rapamyhcin treatment in thrombin (p=0.006) and thrombin + COL activated (p=0.02) platelets. *p \u0026lt; 0.05. ** p\u0026lt;0.01. Dotted lines represent third quartile, median and first quartile. Width of the plots demonstrate frequency at which values occur.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/0a8d9866bfb6bc0d7b41c22a.png"},{"id":95657418,"identity":"14953d17-1330-45c5-a92f-fd25a9328841","added_by":"auto","created_at":"2025-11-11 16:20:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe magnitude of platelet alpha granule secretion, measured by P-selectin, after stimulation of platelets with ADP, thrombin, thrombin + COL or thrombin + CVX at 3 time points post rapamycin treatment in 8 cats.\u003c/strong\u003e Platelets from 8 cats were activated with adenosine diphosphate (ADP) or thrombin in the presence or absence of collagen (COL) and convulxin (CVX). The magnitude of alpha granule secretion was measured as fold change (log 10) in P-selectin median fluorescence intensity (MFI) based on unstimulated (resting) platelets. \u003cstrong\u003e(A)\u003c/strong\u003e Resting platelets showing minimal P-selectin expression based on \u003cstrong\u003e(B)\u003c/strong\u003e fluorescence-minus-one control (FMO) to establish P-selectin gate. \u003cstrong\u003e(C)\u003c/strong\u003e Histograms showing platelets before (blue) and after ADP stimulation (pink) at baseline along with 24, 48 hours post rapamycin. Modulation in P-selectin upregulation was most profound at 24 hours. \u003cstrong\u003e(D) \u003c/strong\u003eViolin plots showing that\u003cstrong\u003e \u003c/strong\u003erapamycin significantly modulated platelet activation in response to ADP at 24 and 48 hours and thrombin + COL at 3 hours post rapamycin. *p \u0026lt; 0.05. **p\u0026lt;0.005. Dotted lines represent third quartile, median and first quartile. Width of the plots demonstrate frequency at which values occur.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/cb62fde1cbd284b2a397c3c1.png"},{"id":95656283,"identity":"d1256d1d-c96c-4de7-a19d-f0dd3a524b22","added_by":"auto","created_at":"2025-11-11 16:18:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeat maps showing the platelet procoagulant tendency scores in response to thrombin, thrombin/collagen (COL) and thrombin/convulxin (CVX) in 8 cats at 3, 24 and 48 hours after rapamycin treatment.\u003c/strong\u003e A procoagulant tendency score out of 3 was assigned to each cat at different time points based on the agonist groups using procoagulant platelet markers, loss of mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) externalization and P-selectin expression. A score of 3 represents the highest procoagulant tendency in an individual while a score of 0 represents resting or apoptotic platelets. Rapamycin reduced procoagulant tendency at 24 and 48 hours after rapamycin when platelets were stimulated with thrombin in the presence or absence of COL. To a lesser degree, procoagulant propensity scores were reduced at 24 hours after rapamycin treatment when platelets were activated.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/f96a8bb9866206e224fcbe3d.png"},{"id":106809873,"identity":"4ebee9d1-a55d-4143-abc1-7fe831be9c3b","added_by":"auto","created_at":"2026-04-13 16:13:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1663213,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7068900/v1/cab60a4b-90d7-4686-a3e0-adcb7fc519a9.pdf"}],"financialInterests":"Competing interest reported. Funding and disclosures: This study was funded by TriviumVet (Waterford, Ireland); industry sponsor did not have any influence over data analysis, final results or conclusion of this study. Funding for M.S. was provided in part by both University of California, Davis and TriviumVet. Authors S.F. and L.G. are employees of TriviumVet and J.A.S serves as a scientific advisory board member for TriviumVet. The authors, N.N. and R. H. L.L. declare no conflict of interest.","formattedTitle":"Ex vivo effects of low dose delayed release rapamycin on agonist-induced platelet aggregation, activation and procoagulant platelet phenotypes in domestic cats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypertrophic cardiomyopathy (HCM) is the most common form of cardiomyopathy in domestic cats affecting approximately 1 in 7 (~\u0026thinsp;15%) cats in the general population.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Familial HCM in cats closely resembles human HCM and has been served as a naturally occurring large animal translational model for evaluating the efficacies of novel compounds like aficamten, a cardiac myosin inhibitor, that was recently approved for use in human HCM patients.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Manifestations of feline HCM largely mirror those seen in human HCM such as left ventricular outflow tract obstruction (LVOTO), sudden death, congestive heart failure and thrombosis.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e In humans, HCM affects about 1 in 500 people.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e A common clinical sequala of human HCM is atrial fibrillation, which affects about 23% of HCM patients and is commonly associated with thromboembolism.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e While AF in human HCM patients is a serious complication associated with stroke and mortality, most cats develop thromboembolic diseases in the absence of AF.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e In cats, the prevalence of cardiogenic arterial thromboembolism (CATE) secondary to HCM is about 11.3% with a mortality rate of up to 67%.\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe pathogenesis of CATE and ischemic stroke in human and feline HCM patients in the absence of AF is not well understood. However, given the high morbidity and mortality associated with CATE, research is ongoing to understand the underlying mechanisms of CATE in the hopes of discovering novel therapeutic targets that can benefit humans and cats. Since platelets are the primary responders in hemostasis, many studies, to date, have documented increased platelet adhesion and activation in cats and humans with HCM. Platelets in both humans and cats with HCM have increased P-selectin expression suggesting that platelets may play a significant role in CATE pathogenesis.\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Moreover, mean platelet volume is significantly higher in HCM patients.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Cats with HCM have also been found to have increased neutrophil extracellular traps in the form of free circulating histones, which may further exacerbate thrombosis by priming platelets and augmenting fibrin formation.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eProcoagulant platelets are a specialized subset of activated platelets that play a role in thrombin generation and fibrin formation in hemostasis.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, disease processes that favor the over production of procoagulant platelets have been associated with pathological thrombosis. Upon activation with strong agonists like thrombin and collagen, the mitochondrial membrane potential depolarizes causing increased permeability of the mitochondrial membrane and sustained intracellular hypercalcemia. This eventually leads to externalization of the electronegative phospholipid, phosphatidylserine, on the platelet surface, serving as platforms for the assembly of coagulation complexes such as tenase and prothrombase complexes to facilitate thrombin generation.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e While procoagulant platelets are essential in normal hemostasis, excessive or dysregulated procoagulant platelet activity is associated with thrombotic disorders such as ischemic stroke, coronary arterial disease, and venous thrombosis.\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Since procoagulant platelets play a crucial role in thrombin generation and fortification of clot structures, any imbalance in their generation and function can lead to either hemorrhage or thrombosis. Understanding the regulation of procoagulant platelet formation may, therefore, provide therapeutic insights for targeting thrombotic diseases without impairing normal hemostasis.\u003c/p\u003e\u003cp\u003eIn humans with AF, anticoagulants such as warfarin are considered the standard of care for the primary and secondary prevention of CATE.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e However, in HCM patients without AF, antiplatelet drugs are recommended for the secondary prevention of ischemic stroke while their clinical benefit as primary prevention remains unclear. Due to the absence of AF in most feline HCM patients, an antiplatelet drug is the first line therapy for primary CATE prevention.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Nevertheless, the on-treatment recurrence rate for CATE in cats remains high at around 49% suggesting that conventional antiplatelet drugs may have limited efficacy in preventing intracardiac thrombosis and CATE.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eConventional antiplatelet drugs such as aspirin and clopidogrel target the pathways involved in platelet aggregation, but do not affect the cellular processes that mediate the formation of procoagulant platelets. As a subset of platelets are continuously exposed to high concentrations of potent agents, sustained intracellular hypercalcemia results in procoagulant phenotypes, characterized by the formation of procoagulant membrane and proteolytic downregulation of platelet integrins, which are crucial for platelet aggregation. As a result, even when aggregation is inhibited, procoagulant platelet formation continues with ongoing thrombin generation and fibrin polymerization to stabilize the clot structure. Despite this, no current drugs have been found to target cellular processes specific to procoagulant platelet formation without causing systemic adverse effects.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e This knowledge gap in current treatment strategies highlights the potential of developing novel therapies to target procoagulant platelets, thereby reducing the risk of ischemic stroke and other thromboembolic events without impairing normal hemostasis.\u003c/p\u003e\u003cp\u003eMultiple \u003cem\u003ein vitro\u003c/em\u003e studies have investigated potential therapeutic targets to modulate procoagulant platelet formation. Rapamycin, also known as sirolimus, is a macrolide compound that inhibits the mechanistic/mammalian target of rapamycin (mTOR) pathway, a key regulator of cell growth, metabolism, and immune responses.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e By inhibiting mTOR complex 1 (mTORC1), rapamycin increases autophagy, suppresses protein synthesis, reduces inflammation, and modulates cellular metabolism, making it a promising candidate for a range of clinical conditions.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Rapamycin is used to treat various conditions such as preventing organ transplant rejection, and cancer, and has recently been approved by the US Food and Drug Agency for the treatment of feline HCM. Low dose delayed release rapamycin (LDDRR) has been shown to have cardioprotective effects in HCM-affected cats by reducing or halting the progression of left ventricular hypertrophy.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Findings in humans suggest that similar mTOR inhibition may also help reduce the progression of hypertrophy.\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Additionally, a multi-omic study found that rapamycin-treated cats with HCM-had downregulations in proteins associated with the complements, inflammation, coagulation cascade and von Willebrand Factors, suggesting that rapamycin may have anti-thrombotic effects.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe effects of rapamycin on platelet function remain poorly understood with most studies limited to \u003cem\u003ein vitro\u003c/em\u003e models, which yielded conflicting results. \u003cem\u003eIn vitro\u003c/em\u003e treatment of human platelets with rapamycin increases platelet aggregation and secretion in response to ADP and thrombin in one study while another study showed that collagen response is modulated by reducing platelet aggregation and spreading.\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e An \u003cem\u003ein vitro\u003c/em\u003e study in human platelets demonstrated that rapamycin reduces procoagulant platelet formation by protecting mitochondrial membrane potential (ΔΨm) and limiting phosphatidylserine (PS) externalization to prevent excessive thrombin generation via an mTORC1-independent mechanism.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Moreover, the effects of rapamycin on platelet function and procoagulant platelet formation have yet to be demonstrated in an ex vivo large animal model. Given the potential antithrombotic and cardioprotective effects of rapamycin in HCM, further research is crucial to explore its potential as a therapeutic strategy for preventing thrombosis in both cats and humans with HCM. Herein, we conducted the first \u003cem\u003eex vivo\u003c/em\u003e study assessing the pharmacodynamic effects of LDDRR on platelet aggregation, activation and procoagulant platelet tendency in domestic cats. We hypothesized that following a month-long treatment of once weekly rapamycin, platelet activation and formation of procoagulant phenotypes would be safely reduced without compromising platelet aggregation. Furthermore, by utilizing a previously validated scoring system to evaluate procoagulant tendency in cats, we hypothesized that rapamycin would modulate the overall platelet procoagulant tendency at all time points after rapamycin treatment.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003ePatient outcome\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNone of the 8 cats studied exhibited any notable adverse effects secondary to low dose delayed-release rapamycin (LDDRR) given once every 7 days for 4 weeks\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ethroughout the study period. Table 1 summarizes the selected hematology variables before and after rapamycin treatment. Figure 1 summarizes a modified experimental approach to characterize procoagulant platelet phenotypes in cats.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. \u003cstrong\u003eSelected hematological findings in 8 cats before and 3, 24, and 48 hours after treatment with delay-release rapamycin.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"780\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e24h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e48h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 320px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline vs 3h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline vs 24h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline vs 48h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWhite Blood Cells x 10\u003csup\u003e9\u003c/sup\u003e/L\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Ref: 3.50-20.70)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e5.20 (4.53-6.69)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4.93 (4.03-5.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e5.28 (4.89-5.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e5.22 (4.30-6.52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLymphocytes x 10\u003csup\u003e9\u003c/sup\u003e/L\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Ref: 0.83-9.10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e2.81 (1.84-4.17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.54 (1.22-2.69)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e2.08 (1.44-2.99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.11 (1.59-2.84)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonocytes x 10\u003csup\u003e9\u003c/sup\u003e/L\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Ref: 0.09-1.21)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.11 (0.06-0.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.23 (0.18-0.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.24 (0.17-0.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.22 (0.08-0.30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeutrophils x 10\u003csup\u003e9\u003c/sup\u003e/L\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Ref: 1.63-13.37)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e2.51 (\u0026plusmn;1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e2.84 (\u0026plusmn;0.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e2.88 (\u0026plusmn;0.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e2.75 (\u0026plusmn;0.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHematocrit, % (Ref: 33.70-55.40)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e35.09 (\u0026plusmn;4.96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e35.21 (\u0026plusmn;8.21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e36.06 (\u0026plusmn;3.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e35.18 (\u0026plusmn;3.95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlatelets x 10\u003csup\u003e9\u003c/sup\u003e/L\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Ref: 125-618)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e248.6 (\u0026plusmn;115.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e309.5 (\u0026plusmn;124.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e325.5 (\u0026plusmn;63.56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e291.1 (\u0026plusmn;61.60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Platelet Volume, fl\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Ref: 8.6-14.9)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e9.54 (\u0026plusmn;0.59)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e9.35 (\u0026plusmn;0.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e9.47 (\u0026plusmn;0.62)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e9.52 (\u0026plusmn;0.82)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeutrophil-to-lymphocyte ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.91 (0.53-1.17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.55 (1.01-2.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e1.36 (0.82-2.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e1.39 (0.83-1.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlatelet-to-lymphocyte ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e110.7 (\u0026plusmn;86.66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e188.9 (\u0026plusmn;100.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e165.6 (\u0026plusmn;80.89)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e136.9 (\u0026plusmn;50.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRapamycin did not affect adenosine diphosphate induced platelet aggregation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePlatelet aggregation in response to adenosine diphosphate (ADP) at 24 and 48 hrs, measured by whole blood impedance platelet aggregometry in heparinized blood, was variable among cats (co-efficient of variance =16.3%, 18.1%, respectively). No differences in any aggregometry variables were noted following treatment with LDDRR at 24 and 48 hrs compared to baseline (area under the curve (AUC) p=0.10, aggregation unit (AU) p =0.19, velocity (AU/min) p =0.08) (Figure 2). Of the 8 cats, 4 (50%) had increased aggregation (AUC), 2 (25%) had decreased aggregation (AUC) and 2 (25%) had no changes in aggregation (AUC) at 24 hrs. No significant difference in aggregation at baseline and 24 hrs after dosing was found (AUC \u003csub\u003ebaseline\u003c/sub\u003e= 111.8 \u0026plusmn; 21.02 vs AUC\u003csub\u003e24 hrs\u003c/sub\u003e= 125.8 \u0026plusmn; 18.24, p=0.11). At 48 hrs, 3 of the 8 cats (37.5%) had increased aggregation but, overall, this difference did not differ from baseline measurements (AUC\u003csub\u003e48 hrs.\u003c/sub\u003e: 131.8 \u0026plusmn; 25.94, p=0.09) (Figure 2A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLow dose delayed-release rapamycin prevented the loss of platelet mitochondrial membrane potential in the presence of potent agonists\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eProtective effects on platelet mitochondrial membrane potential (\u0026Delta;\u0026Psi;m) were detected at 3, 24 and 48 hrs after the last dose of rapamycin when \u0026Delta;\u0026Psi;m was measured as percent change (%) in tetramethylrhodamine methyl ester (TMRM)-positive platelets (Figure 3C, D). Rapamycin prevented the loss of \u0026Delta;\u0026Psi;m induced by thrombin at 3 hrs (baseline= -15.6%, IQR -23.8 to -0.4 vs 3 hrs = -3.6%, IQR -7.2 to 1.1, p=0.04), 24 hrs (-2.5%, IQR -6.9 to 2.1, p=0.02) and 48 hrs (-1.2%, IQR -5.2 to 0.5, p=0.04) post LDDRR treatment. With the addition of collagen to thrombin-activated cells, \u0026Delta;\u0026Psi;m was also preserved at 3 hrs (-8.5%, IQR -12.5 to -1.3, p=0.02), 24 hrs (-5.1%, IQR -12.6 to 0.2, p=0.02) and 48 hrs (-6.2%, IQR -16.9 to -2.5, p=0.02) when compared to baseline (-25.3%, IQR -45.7 to -16.5). Similarly, protective effects were found upon stimulation with thrombin and CVX (baseline= -75.6%, IQR -82.4 to -53.4 vs 3 hrs= -28.5%, IQR -61.3 to -17.3, p=0.02) (24 hrs= -34.8%, IQR -53.3 to -15.4, p=0.02) (48 hrs = -44.9%, IQR -60.4 to -20.8, p=0.02) (Figure 3D). When the loss of \u0026Delta;\u0026Psi;m was measured as fold change (log 10) in TMRM median fluorescence intensity (MFI FC \u003csub\u003elog 10\u003c/sub\u003e), we did not detect any significant differences in \u0026Delta;\u0026Psi;m in response to thrombin in the presence or absence of collagen or CVX at all time points (Figure 3E). Platelets treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), which disrupts the proton gradient across the mitochondrial inner membrane, leading to a loss of \u0026Delta;\u0026Psi;m, served as negative controls. Surprisingly, treatment of platelets with CCCP at 3 (MFI FC Log 10 = -0.02 \u0026plusmn; 0.1;) and 24 hrs (-0.009 \u0026plusmn; 0.1) after the last dose of LDDRR showed significant reductions in the loss of \u0026Delta;\u0026Psi;m when compared to baseline (-0.1 \u0026plusmn; 0.1) (p=0.03, p=0.03, respectively) (Figure 3E).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhosphatidylserine externalization was significantly modulated at 24 hours after rapamycin dosing\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the measurements of\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026Delta;\u0026Psi;m, exogenous calcium was added to the remaining aliquots of PRP, and phosphatidylserine (PS) externalization was assessed by detecting the percent change (%) in annexin V-positive platelets over time (Figure 4C, D). At 24 hrs after the last dose of LDDRR, externalization of PS in response to thrombin (Change PS \u003csub\u003ebaseline\u003c/sub\u003e = 39.7% \u0026plusmn; 53.0 vs Change PS \u003csub\u003e24 hrs\u003c/sub\u003e = -5.8% \u0026plusmn; 31.4, p=0.006) and a combination of thrombin and COL (Change PS \u003csub\u003ebaseline\u003c/sub\u003e = 45.4%, IQR 12.8 to 152.8 vs Change PS \u003csub\u003e24 hrs\u003c/sub\u003e = 11.4%, IQR -4.4 to 22.1, p=0.02) were significantly decreased (Figure 4D). However, changes in PS on platelets were widely variable among cats at 3 and 48 hrs after LDDRR dosing. Overall, rapamycin had no significant effect on PS externalization when platelets were stimulated with thrombin and CVX (p=0.14) (Figure 4D).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRapamycin modulated alpha granule secretion in response to adenosine diphosphate and thrombin in the presence of collagen\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFinal aliquots of platelets were assessed for P-selectin (CD62P) in response to ADP, and thrombin in the presence or absence of COL or CVX. Response was evaluated based on fold change (log 10) in median fluorescence intensity between baseline and at 3, 24 and 48 hrs after LDDRR treatments. We found that alpha-granule secretion in response to thrombin and COL at 3 hours was modulated compared to baseline (MFI FC \u003csub\u003ebaseline\u0026nbsp;\u003c/sub\u003e= 0.09 \u0026plusmn; 0.08 vs MFI FC \u003csub\u003e3 hrs\u0026nbsp;\u003c/sub\u003e= -0.02 \u0026plusmn; 0.1, p=0.006). Response to ADP was also significantly decreased at 24 (MFI FC\u003csub\u003e\u0026nbsp;24h\u003c/sub\u003e= 0.006 \u0026plusmn; 0.05) and 48 hrs (MFI FC = 0.004\u0026plusmn;0.06) compared to baseline (MFI FC \u003csub\u003ebaseline\u003c/sub\u003e = 0.07 \u0026plusmn; 0.06, p=0.004, p=0.007, respectively) (Figure 5 C, D). No significant changes in P-selectin expression were noted in platelets stimulated with thrombin in the presence or absence of CVX (p=0.17, p=0.21, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEx vivo effects of rapamycin on the tendency of procoagulant platelet formation in individual cats\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFigure 6 summarizes the distribution of platelet procoagulant tendency scores in the 8 cats using the previously established scoring system.\u003csup\u003e35\u003c/sup\u003e Significant reductions in procoagulant tendency scores among individual cats were recorded over time when platelets were stimulated with thrombin or thrombin + COL (p=0.04 and p=0.004, respectively) in comparison to baseline. Most reductions in procoagulant platelet tendency scores were noted at 24 hrs post dose compared to baseline especially when thrombin with or without COL were used as agonists. None of the cats had scores greater than 2 in the thrombin group at all time points. \u0026nbsp;Only 1 out of 8 cats (12.5%) had a tendency score \u0026ge; 2 at 24 hrs in the thrombin and COL group compared to 5/8 (62.5%) at baseline. Cats in the thrombin + CVX group had slight decreases in their tendency scores at 3 and 24 hrs after LDDDR treatment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we conducted the first \u003cem\u003eex vivo\u003c/em\u003e study evaluating the effects of LDDRR on platelet aggregation, activation and procoagulant phenotypes in domestic cats. We found that LDDRR treatment in cats was well tolerated and the aggregation response to ADP remained unchanged after treatment. However, ADP-mediated platelet activation, measured by P-selectin upregulation, was modulated. One of the procoagulant platelet markers, ΔΨm, was protected at all time points after LDDRR, following \u003cem\u003eex vivo\u003c/em\u003e treatments of platelets with strong agonists. Additionally, the other procoagulant phenotype, PS, was reduced at 24h after LDDRR. Lastly, P-selectin as a marker of procoagulant platelets also was significantly decreased in the presence of thrombin and COL shortly after rapamycin treatment. Using a novel scoring system by factoring in all 3 procoagulant markers, we found that LDDRR was most efficacious at reducing the platelet procoagulant tendency scores at 24 hrs and to a lesser extent. At 48 hrs post dose.\u003c/p\u003e\u003cp\u003eOur complete blood count (CBC) showed an increasing trend in platelet count was noticed at 3 and 24 hrs compared to the baseline. Since feline platelets are highly subjected to \u003cem\u003ein vitro\u003c/em\u003e activation and aggregation, which can lead to falsely low platelet counts on automated analyzers, the modulation in platelet activation by rapamycin might have decreased \u003cem\u003ein vitro\u003c/em\u003e aggregate, thereby increasing the platelet count on CBC. Another possibility is that rapamycin might have induced apoptosis, triggering platelet generation or turnover. However, research regarding rapamycin\u0026rsquo;s effect on apoptosis seems to be cell dependent. For instance, while rapamycin induces apoptosis in dendritic cells, it inhibits apoptosis in monocytes and macrophages.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Its apoptotic effect on platelets remains unexplored.\u003c/p\u003e\u003cp\u003eA significant drop in lymphocyte count was observed at 3 hrs, followed by recovery at 24 and 48 hrs. The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are commonly used as non-specific indicators of systemic inflammation and stress. Trends of increased NLR and PLR were noted following rapamycin treatment. Since overall neutrophil count did not increase after LDDRR, the observed increase in NLR and PLR was likely due to decreased lymphocyte counts rather than systemic inflammation or stress. In healthy cats, NLR typically ranges from 1.5 to 2.8, suggesting that the observed increase in NLR is unlikely to be clinically significance. PLR, on the other hand, exceeded the reported reference range (100 to 164) in cats, likely due to both increased platelet counts and decreased lymphocytes.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e However, the transient decrease in lymphocyte count following rapamycin treatments remains unclear. One possibility is that rapamycin enhances autophagy, and in conjunction with mTORC inhibition, affects specific lymphocytes sub-sets, leading to a transient decrease in lymphocyte count. Because mTOR activity is crucial for T cell proliferation, rapamycin disrupts the cell cycle and reduces the proliferation of T cells, ultimately reducing peripheral lymphocyte numbers.\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Further studies in clinical cats with underlying inflammation are needed to delineate the clinical significance of these hematological changes.\u003c/p\u003e\u003cp\u003eIn agreement with multiple human studies, our study did not observe any changes in ADP-mediated platelet aggregation.\u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e In a study by Pelekanou et al., human platelets pre-treated with rapamycin had similar aggregation response upon stimulation with ADP and collagen when compared to baseline.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e​ Additionally, in kidney transplant patients, short-term rapamycin treatment did not affect \u003cem\u003ein vitro\u003c/em\u003e aggregation response to SFLLRN, a thrombin-receptor activating peptide (PAR-1) agonist.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e These findings are clinically significant as they indicate that LDDRR does not negatively impact platelet aggregation. One possible explanation for this observation is that autophagy is not essential for platelet aggregation.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e A study using Atg7 knockout mice, which lack the key platelet-specific gene critical for autophagy, has shown that their platelets maintain normal aggregation in response to ADP, collagen and thrombin.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Another plausible explanation is that low dose, short-term rapamycin treatment used in our study partially inhibits mTORC1 while sparing mTORC2, which regulates pathways involved in the platelet aggregation and cytoskeletal rearrangement. However, evidence regarding rapamycin's effect on platelet aggregation remains conflicting. Several studies have reported that rapamycin enhances platelet aggregation.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e These discrepancies may be due to differences in aggregation assays, as light transmission aggregometry may be more sensitive in detecting subtle changes in platelet function. Also, the use of different agonists such as collagen and thrombin could provide a more comprehensive assessment of aggregation.\u003c/p\u003e\u003cp\u003eTo study procoagulant phenotypes, we utilized a modified approach to evaluate a panel of 3 markers by flow cytometry.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e These markers include ΔΨm loss, PS externalization and P-selectin expression. This novel technique of evaluating procoagulant markers sequentially instead of simultaneous detection was developed specifically to study procoagulant platelets in cats due to several reasons.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e First, feline platelets are more susceptible to \u003cem\u003ein vitro\u003c/em\u003e activation due to their sensitivity to subtle changes in ambient temperature, causing cold-induced activation, irreversible shape change and microaggregates.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Unlike human and murine platelets, when feline platelets are exposed to low concentrations of exogenous calcium, they can undergo spontaneous aggregation in the absence of strong agonists. The sensitive nature of feline platelets, therefore, prevents the simultaneous detections of PS, P-selectin and ΔΨm.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe first procoagulant marker evaluated was ΔΨm after stimulation with strong platelet agonists. We showed that LDDRR prevented the loss of platelet ΔΨm with thrombin, COL or CVX stimulation at all time points. This preservation of ΔΨm is significant since ΔΨm loss is a marker of mitochondrial permeability transition pore (mPTP) opening. Potent agonists like thrombin and COL bind to their respective receptors, initiating a signaling cascade that transiently increases intracellular calcium, which facilitates calcium entry into the mitochondria via an electrochemical gradient across the inner mitochondrial membrane. Located on the inner mitochondrial membrane, the mPTP, which is regulated by cyclophilin D (CypD), opens to allow calcium efflux to the cytosol. This results in sustained cytosolic hypercalcemia, which is crucial to mediate intracellular signaling to promote procoagulant platelet formation.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Our data align with findings by Śledź et al., who assessed ΔΨm using the potentiometric dye, JC-1, in healthy human platelets, and found that \u003cem\u003ein vitro\u003c/em\u003e rapamycin prevented the loss of ΔΨm in an mTORC1-independent manner.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e The exact mechanisms underlying these observations in platelets are unclear, but rapamycin has been shown to reduce mitochondrial oxidative stress by reducing reactive oxygen species, promoting mPTP opening and calcium efflux. Rapamycin has also been shown to induce mitophagy, a specific type of autophagy, in other cell types, to facilitate removal of damaged or dysfunctional mitochondria improving mitochondrial function.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e Clinically, individuals, especially elderly with age-related decline in mitochondrial efficiency, and those with cardiovascular diseases, are prone to mitochondrial dysfunction and loss of platelet ΔΨm.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e In these groups, impaired mitochondrial function may exaggerate generation of procoagulant platelets, thereby increasing the risk of thrombotic complications. Thus, therapeutic strategies such as rapamycin, which preserves mitochondrial integrity, may hold promise when used in conjunction with traditional antithrombotic therapies.\u003c/p\u003e\u003cp\u003eFollowing mPTP opening, supramaximal calcium stimulates the activation of TMEM16F, a scramblase enzyme, which promotes the translocation of the negatively charged PS from the inner to the outer side of the plasma membrane. As mentioned previously, the exposed PS facilitates thrombin generation by creating a surface for coagulation enzymes binding.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e While LDDRR prevented ΔΨm loss induced by potent agonists at all timepoints, PS externalization was reduced only at 24 hrs but reverted to baseline at 48 hrs. Consistent with our findings, Śledź et al., which measured PS externalization in a similar fashion after thrombin and CVX stimulation, found that \u003cem\u003ein vitro\u003c/em\u003e rapamycin treatment partially modulated PS externalization in a dose-dependent manner.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Since plasma concentration of rapamycin declines after peak concentration at 3 hrs post administration, this decrease in plasma concentration could explain the incomplete inhibition of PS externalization. However, the protective effect of rapamycin on mitochondrial membrane integrity lasted beyond 24 hrs, indicating that, in addition to mitochondria, there were other sources of calcium that could facilitate PS exposure and procoagulant platelet generation. One plausible explanation which could have caused this discrepancy in our results is the methodology used to assess PS flip. Following the measurement of ΔΨm, more exogenous calcium was added to PRP prior to quantifying PS expression. This exogenous calcium could enter the platelet cytosol via a process called store-operated calcium entry (SOCE), which serves to elevate intracellular calcium and, in part, plays a vital role in sustained supramaximal hypercalcemia.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e Previous activation of platelets with PAR (thrombin) and glycoprotein VI agonists (COL, CVX) could have depleted calcium stores in the dense tubular system via inositol triphosphate-mediated release, activating Orai1 channels on the plasma membrane to promote calcium entry into the cytosol.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e While mitochondria play a crucial role in providing calcium necessary for the sustained elevation of intracellular calcium required for procoagulant platelet formation, it is important to note that other mechanisms, such as SOCE, also synergistically elevate calcium to achieve supramaximal calcium levels.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e This influx of extracellular calcium might explain the externalization of PS despite ΔΨm hyperpolarization under rapamycin treatment. Further experiments are needed to test this hypothesis. Nevertheless, rapamycin\u0026rsquo;s significant effect on reducing the PS exposure is crucial as it contributes to pathogenic or abnormal thrombin generation.\u003c/p\u003e\u003cp\u003eP-selectin, a transmembrane protein found on the platelet alpha granules, was utilized, not only as a marker of alpha granule secretion, but also to distinguish between apoptotic and procoagulant platelets, since PS and ΔΨm loss are also features of apoptotic platelets.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e However, while procoagulant platelets have surface expression of P-selectin, apoptotic platelets do not undergo alpha-granule release, and is, therefore, recommended as a surface marker by the International Society of Thrombosis and Haemostasis.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e P-selectin, as a marker of procoagulant platelet, was significantly decreased in thrombin and COL-treated platelets 3 hrs after LDDRR treatment. Several studies have investigated the effects of rapamycin on P-selectin expression in platelets with varying results. One study in human platelets demonstrated that rapamycin did not have any immediate effects on thrombin-induced P-selectin expression.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e Conversely, another study found that rapamycin decreases platelet P-selectin expression in older mice in a venous thrombosis model.\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e These findings suggest that rapamycin's effects on P-selectin expression may be age and species-dependent. The lack of significant modulation in P-selectin expression did not parallel with the decreases in PS externalization and ΔΨm upon stimulation with the respective agonists throughout the study period. Despite not evaluating all procoagulant markers simultaneously, this suggests that LDDRR might shift platelets from a procoagulant phenotype to a classical activation maintaining their response to agonists. Since we observed P-selectin upregulation in response to all procoagulant agonist groups before and after LDDRR treatment, it was unlikely that platelets underwent apoptosis. However, further studies utilizing more specific apoptotic markers will be needed to further rule out apoptosis due to strong agonist stimulations. These findings demonstrate the importance of evaluating multiple markers to accurately define procoagulant phenotypes.\u003c/p\u003e\u003cp\u003eWhile P-selectin upregulation in response to strong agonists was preserved after LDDRR, platelet activation induced by ADP was significantly decreased at 24 and 48 hrs. The variation in rapamycin\u0026rsquo;s effects on P-selectin expression across different agonists may stem from the downstream signaling pathways initiated by agonists. Intracellular signaling pathways activated by ADP may be more prone to rapamycin modulation than those mediated by thrombin and GPVI agonist. One plausible explanation is that ADP-mediated platelet activation occurs primarily through the receptor, P2Y\u003csub\u003e12\u003c/sub\u003e, which activates the Akt signaling cascade, rather than PKC that thrombin utilizes.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e No studies to date have examined the specific effects of mTORC1 inhibition on ADP-mediated activation. However, our results suggest that there are agonist-specific differences in the regulation of mTOR signaling, requiring further investigations. If rapamycin reduces ADP-mediated platelet priming or activation through an mTORC1-dependent mechanism, then rapamycin may elicit synergistic platelet inhibitory effects of P2Y\u003csub\u003e12\u003c/sub\u003e inhibitors like clopidogrel and prasugrel. Like human beings, cats also have reduced responses to P2Y\u003csub\u003e12\u003c/sub\u003e inhibitors secondary to pharmacogenomic variations and other co-morbidities resulting in reduced efficacy and increased on-treatment thrombotic events. As mentioned previously, LDDRR is conditionally approved for the treatment of feline HCM. Given that clopidogrel is the recommended first-line antithrombotic prevention in HCM cats, future studies should focus on evaluating the clinical benefits of dual therapy consisting of rapamycin and P2Y\u003csub\u003e12\u003c/sub\u003e inhibitors. It could enhance the therapeutic efficacy of clopidogrel by further attenuation of the ADP signaling pathway.\u003c/p\u003e\u003cp\u003eWe further validated our previously established scoring system to assess platelet procoagulant tendency in cats by demonstrating reductions in procoagulant scores at 24 hrs and, to a lower extent, 48 hrs, after LDDRR treatment. These findings suggest that rapamycin may provide short-term therapeutic benefits in preventing thrombosis associated with HCM. To better understand the physiological relevance of these findings, additional assessments such as thrombin generation, thrombus formation under shear stress and viscoelastic evaluations should be performed before and after LDDRR treatments. These assays would provide deeper insights into rapamycin\u0026rsquo;s effects on clot formation and fibrinolysis. Our data also suggest that the anti-procoagulant effects of rapamycin maybe transient, emphasizing the need to investigate the pharmacodynamics beyond 48 hrs. This would help determine the optimal dosing regimen to maintain sustained suppression of procoagulant platelet formation. Furthermore, additional studies should explore the potential interactions between LDDRR and antithrombotics, which may prolong the suppression of procoagulant platelet formation and enhance overall therapeutic efficacy.\u003c/p\u003e\u003cp\u003eThis study has several limitations. Rather than characterizing and quantifying procoagulant platelets by assessing all three markers simultaneously, we developed a scoring system that integrates data from each marker individually to evaluate the platelet procoagulant tendency in individual cats. Quantifying procoagulant platelets in cats presents a challenge due to the inherent sensitivity of feline platelets. While a stepwise evaluation of each procoagulant phenotype may be more physiological, it carries notable limitations. For instance, analyzing PS and P-selectin separately may impair our ability to distinguish between apoptotic and procoagulant platelets, as PS exposure occurs in both states. During protocol development, simultaneous detection of PS and P-selectin was attempted, but this led to suboptimal P-selectin antibody binding and in vitro clot formation. The co-detection of P-selectin or other activation markers such as CD40L with PS may better differentiate apoptotic from procoagulant platelets in feline samples in addressing this limitation. Additionally, the small sample size may underpower the detection of notable trends due to Type II error. Platelet function testing was performed in clinically healthy cats, which might not fully reflect the platelet hyperresponsiveness seen in HCM affected cats. Although mitochondrial membrane potential and PS externalization were assessed, rapamycin\u0026rsquo;s mechanism of action was not explored. Future studies are needed to investigate the mechanistic approach.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis was the first \u003cem\u003eex vivo\u003c/em\u003e study in a large animal model investigating the anti-platelet effects of rapamycin. Not only was rapamycin safe and well-tolerated, it also was effective in suppressing procoagulant platelet tendency. Considering that rapamycin did not alter platelet aggregation and no cats in our study had any adverse effects of hemorrhage, the modulation in procoagulant tendency by rapamycin suggests that rapamycin may prevent pathologic thrombosis without affecting hemostasis. Given the benefit of LDDRR in reversing HCM, clinical trials evaluating the additional antithrombotic effect of rapamycin in HCM-affected cats and individuals at risk of arterial thrombosis and stroke are warranted.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/p\u003e\u003cp\u003eEight cats (4 intact males and 4 intact females) from a colony of Maine Coon/outbred mixed domestic cats from the Feline HCM Research Colony at University of California, Davis and North Carolina State University were enrolled for this study. Physical examinations, complete blood count using an automated hematology analyzer (HM5, Zoetis, Parsippany, NJ) and transthoracic echocardiogram were performed by the corresponding author and board-certified cardiologist (JAS) to ensure that all cats were clinically healthy prior to the study. No cats showed any echocardiographic evidence of HCM at the time of the study. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of California, Davis (protocol number: 21895) and carried out in accordance with relevant guidelines and regulations. All methods were reported in compliance with the ARRIVE guidelines.\u003c/p\u003e\u003cp\u003e\u003cem\u003eDrug administration\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCats were treated with low-dose delayed release rapamycin (LDDRR) 0.3 mg/kg rapamycin orally every 7 days for 4 consecutive weeks according to previously published study and pharmacokinetic data from the manufacturer.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e At the time of the study, formulation of rapamycin provided by the manufacturer was not commercially available for veterinary use while conditional approval is sought from the FDA Center for Veterinary Medicine. During the study period, all cats were fed the same diet and housed in groups with environmental enrichment. Cats were monitored twice daily for any potential adverse effects such as gastrointestinal signs (vomiting, diarrhea, inappetence) and bleeding diathesis (hematomas, hematuria, hematemesis, petechia and ecchymosis) throughout the study period and up to 1 week after drug administration.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBlood collection\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFour to 6 ml of blood was then collected from either the medial saphenous or jugular vein using a 21- or 23-gauge butterfly needle. Blood was transferred immediately to tubes containing 3.2% trisodium citrate or sodium heparin. Each tube was gently inverted and inspected for any possible clotting. Blood sampling occurred 24 hrs before the first dose of delay-released rapamycin, and at 3, 24 and 48 hrs after the last dose in week 4.\u003c/p\u003e\u003cp\u003e\u003cem\u003eWhole blood impedance platelet aggregometry\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePlatelet aggregation was measured by whole blood impedance platelet aggregometry (Multiplate, Roche, Mannheim, Germany) at 24 and 48 hrs after the last dose of rapamycin as previously described. Briefly, heparinized whole blood is diluted with equal volumes of pre-warmed 0.9% NaCl and incubated at 37\u0026deg;C for 3 minutes under physiologic shear generated by Teflon-coated magnetic stir bars at 800-rpm. ADP (6.25 \u0026micro;M, MilliporeSigma, Burlingon, MA) was then added and platelet aggregation, measured as electrical impedance over a 6-minute period, was reported as maximum aggregation, velocity (AU/min) and area under the curve (AUC; AU x minutes).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePlatelet rich plasma isolation\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWithin 30 minutes of blood collection, citrated whole blood was placed in a 37\u0026deg;C bead bath for 30 minutes to facilitate red blood cell sedimentation. Platelet rich plasma (PRP) was then generated by centrifugation at 300 \u0026times; g (21\u0026deg;C, no brakes) for 5 minutes. PRP was assessed for \u0026ldquo;swirling\u0026rdquo; to ensure that platelets were still in their resting discoid shape. An automated hematology analyzer (HM5, Zoetis, Parsippany, NJ) was used to measure platelet counts in PRP and further confirmed by manual blood smear analysis. Prior to activation, PRP was rested for an additional 30 minutes at 37\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cem\u003eProcoagulant platelet characterization\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) externalization and P-selectin expression were analyzed consecutively by flow cytometry to identify and characterize procoagulant platelets as previously described.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e In brief, platelets were first standardized to 1x10\u003csup\u003e7\u003c/sup\u003e cells/ml using Tyrode HEPES (pH 7.2, 5.5 mM dextrose, 1mM CaCl\u003csub\u003e2\u003c/sub\u003e) to a final volume of 200 \u0026micro;l. Tetramethylrhodamine methyl ester (TMRM) (320nM, Invitrogen, Eugene, OR) was added to PRP and placed in 21% oxygen at 37\u0026deg;C under gentle rocking for 15 minutes. TMRM-loaded platelets were subsequently stimulated with bovine alpha thrombin (0.001U/ml, 5 minutes at 37\u0026deg;C, Haematologic Technologies, Inc., Essex Junction, VT) prior to further activation with equine type I collagen (COL) (4 \u0026micro;g/ml, CHRONO-LOG Corp., Havertown, PA), or convulxin (CVX) (100 ng/ml, Cayman chemicals, Ann Arbor, MI) for an additional 5 minutes. Unstimulated platelets served as positive control while platelets treated with either 20 \u0026micro;M carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Invitrogen, Eugene, OR) or 10 \u0026micro;M A23187 (Millipore Sigma, Burlington, MA) served as negative controls (Figs.\u0026nbsp;2A - C). Platelets were then analyzed live by flow cytometry (Beckman-Coulter FC500, Beckman-Coulter Inc., Miami, FL). Shortly after, the remaining aliquots of platelets were loaded with additional 1mM CaCl\u003csub\u003e2\u003c/sub\u003e (final concentration 2mM). To quantify PS externalization, platelets were labelled with annexin V conjugated to fluorescein isothiocyanate (FITC) (1:200, Cat: 556419, BD Pharmingen, San Jose, CA) for 45 minutes at 37\u0026deg;C followed by fixation with 0.1% methanol free paraformaldehyde (Thermo Scientific, Waltham, MA). Unstimulated platelets and platelets treated with A23187 served as the negative and positive controls, respectively.\u003c/p\u003e\u003cp\u003eThe final marker, P-selectin, was analyzed in a separate aliquot of PRP, standardized to 1x10\u003csup\u003e7\u003c/sup\u003e cells/ml with Tyrode HEPES (pH 7.2, 5mM dextrose, no divalent cations). Resting platelets and adenosine diphosphate (ADP) (20 \u0026micro;M, Sigma-Aldrich, St. Louis, MO) treated platelets served as negative and positive controls, respectively. After platelet activation with the aforementioned agonists, rat anti-mouse monoclonal antibody conjugated to FITC specific to P-selectin (CD62P) (1:200, Clone: RB40.34, BD Biosciences, San Diego, CA) along with mouse anti-human monoclonal antibody conjugated to allophycocyanin against β3-integrin (CD61) (1:1000, Clone:VI-PL2, eBioscience, San Deigo, CA) were added to the platelets. Platelets then were incubated for 45 minutes at 37\u0026deg;C followed by fixation similar to annexin V.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFlow Cytometry Analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePlatelets were identified by forward- and side-scatter properties previously established using 0.9 \u0026micro;m and 3 \u0026micro;m calibration beads in the log scale as described and 10,000 events were analyzed for each experimental condition (Figs.\u0026nbsp;3A, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Gating of TMRM, PS and P-selectin positive platelets (Figs.\u0026nbsp;3B, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The magnitude in the loss of ΔΨm in response to agonists was calculated using Equations 1 and 2 using values of TMRM median fluorescence intensity (MFI) and the percentage of TMRM-positive cells (Fig.\u0026nbsp;3E, F). Externalization of PS in response to agonists was evaluated as change in percentage increase using the following Eq.\u0026nbsp;3. Lastly, agonist-induced upregulation of P-selectin was quantified using Eq.\u0026nbsp;4. All flow cytometry data were assessed using commercially available software (FlowJo, Tree Str Inc., Ashland, OR).\u003c/p\u003e\u003cp\u003eEquation 1:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:TMRM\\:MFI\\:Fold\\:Change\\:\\left(\\text{log}10\\right)=\\left(\\text{log}10\\:MFI\\:activated\\right)-\\left(\\text{log}10\\:MFI\\:resting\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eEquation 2:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:TMRM\\:\\left(\\%\\:change\\right)=\\frac{\\left(\\%TMRM\\:activated\\right)-\\left(\\%TMRM\\:resting\\right)}{\\left(\\%TMRM\\:resting\\right)}\\:\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eEquation 3:\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:Annexin\\:V\\:\\left(\\%\\:change\\right)=\\frac{\\left(\\%AV\\:activated\\right)-\\left(\\%AV\\:resting\\right)}{\\left(\\%AV\\:resting\\right)}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eEquation 4:\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:P-selectin\\:MFI\\:Fold\\:Change\\:\\left(\\text{log}10\\right)=\\left(\\text{log}10\\:MFI\\:activated\\right)-\\left(\\text{log}10\\:MFI\\:resting\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eScoring system for identifying the tendency of procoagulant platelet formation in cats\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe procoagulant tendency of platelets in each cat was assessed using a previously established scoring system.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e In brief, the distributions of the 3 procoagulant markers were assessed after \u003cem\u003ein vitro\u003c/em\u003e treatments with the 3 agonist groups (thrombin, thrombin\u0026thinsp;+\u0026thinsp;COL and thrombin\u0026thinsp;+\u0026thinsp;CVX. Arbitrary cut-offs of each procoagulant marker were assigned based on the medians and interquartile ranges of platelet responses generated in a population of 8 healthy cats. A score of 1 was assigned to the specific phenotype if the response to the agonist group was above or below the 40th percentile of the data generated in the population. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e outlines the proposed scoring system for assessing procoagulant tendency in individual cats. A score of 0 was assigned if P-selectin upregulation did not exceed the anticipated increase as it was categorized as an apoptotic response. A total score of 3 in an individual was considered to have the highest procoagulant tendency while a score of 0 was considered to have apoptotic or the lowest procoagulant tendency.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eCriteria for determining the tendency of procoagulant scores in individual cats.\u003c/b\u003e Median fluorescence intensity (MFI), tetramethyl rhodamine methyl ester (TMRM), interquartile range (IQR).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProcoagulant marker\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (IQR)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAssigned score\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1. P-selectin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1 (0.005 to 0.13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1: If MFI FC (log10)\u0026thinsp;\u0026ge;\u0026thinsp;0.05 from resting\u003c/p\u003e\u003cp\u003e0: If MFI FC (log10)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 from resting\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2. Mitochondrial membrane potential-TMRM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-32.4% (-54.2% to -17.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1: If change (%) in ΔΨm \u0026ge; -22.5% from resting\u003c/p\u003e\u003cp\u003e0: If Change (%) in ΔΨm \u0026lt; -22.5% from resting\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3. Phosphatidylserine-Annexin V\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45.4% (12.7\u0026ndash;152.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1: If change (%) in PS\u0026thinsp;\u0026ge;\u0026thinsp;30% from resting\u003c/p\u003e\u003cp\u003e0: If Change (%) in PS\u0026thinsp;\u0026lt;\u0026thinsp;30% from resting\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eBased on preliminary data, sample size calculations showed that a minimum of 6 cats were needed to detect the lowest detectable effect of 30% in platelet activation with 80% power and an alpha-priori of 0.05. Due to the high plausibility of \u003cem\u003ein vitro\u003c/em\u003e activation and spontaneous aggregation when processing feline platelets, a total of 8 cats were included. The normality of data was determined by D\u0026rsquo;Agostino and Pearson test. Data that were normally distributed were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation while non-parametric data were presented as median and interquartile range (IQR). Paired parametric and non-parametric paired data were compared using \u003cem\u003et\u003c/em\u003e-tests and Wilcoxon signed-rank test, respectively, as appropriate based on normality. Inter-individual variability was assessed by co-efficient of variation, which is calculated by the ratio of standard deviation to mean. Repeated measures ANOVA and Kruskal-Wallis one-way analysis of variance were utilized to analyze the treatment effect over time with post-hoc analyses by Tukey test. Fisher\u0026rsquo;s exact test was used in categorical data analysis to compare procoagulant tendency scores among cats. Data were considered statistically significant if p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analyses were conducted using commercially available software (Prism 10.0, GraphPad Software, La Jolla, CA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding and disclosures: This study was funded by TriviumVet (Waterford, Ireland); industry sponsor did not have any influence over data analysis, final results or conclusion of this study. Funding for M.S. was provided in part by both University of California, Davis and TriviumVet. Authors S.F. and L.G. are employees of TriviumVet and J.A.S serves as a scientific advisory board member for TriviumVet. The authors, N.N. and R. H. L.L. declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eFunding and disclosures: This study was funded by TriviumVet (Waterford, Ireland); industry sponsor did not have any influence over data analysis, final results or conclusion of this study. Funding for M.S. was provided in part by both University of California, Davis and TriviumVet. Authors S.F. and L.G. are employees of TriviumVet and J.A.S serves as a scientific advisory board member for TriviumVet. The remaining authors, N.N. and R.H.L.L. declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eAuthor Contribution\u003c/p\u003e\n\u003cp\u003eM.S, J.A.S, S.F, L.G and R.H.L.L conceived the research. J.A.S, N.N, M.S. and R.H.L.L selected the study subjects. M.S., N.N, R.H.L.L collected the data. M.S., N.N, R.H.L.L., J.A.S. analyzed the data. M.S and R.H.L.L drafted the manuscript and all authors contributed to the revision of the manuscript and approved of the final version of the manuscript for submission. The remaining authors, N.N. and R.H.L.L. declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Carina Gonzalez for her assistance with the data collection for this study.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe authors declare that all data supporting the findings of this study are available within the article or from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePayne, J. R. \u0026amp; Brodbelt, D. C. Luis Fuentes, V. Cardiomyopathy prevalence in 780 apparently healthy cats in rehoming centres (the CatScan study). \u003cem\u003eJ. Vet. Cardiol.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (Suppl 1), 244\u0026ndash;257. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jvc.2015.03.008\u003c/span\u003e\u003cspan address=\"10.1016/j.jvc.2015.03.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaige, C. F., Abbott, J. A., Elvinger, F. \u0026amp; Pyle, R. L. Prevalence of cardiomyopathy in apparently healthy cats. \u003cem\u003eJ. Am. Vet. Med. Assoc.\u003c/em\u003e \u003cb\u003e234\u003c/b\u003e, 1398\u0026ndash;1403. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2460/javma.234.11.1398\u003c/span\u003e\u003cspan address=\"10.2460/javma.234.11.1398\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFreeman, L. M., Rush, J. E., Stern, J. A., Huggins, G. S. \u0026amp; Maron, M. S. Feline Hypertrophic Cardiomyopathy: A Spontaneous Large Animal Model of Human HCM. \u003cem\u003eCardiol. Res.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 139\u0026ndash;142. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14740/cr578w\u003c/span\u003e\u003cspan address=\"10.14740/cr578w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharpe, A. N. et al. Pharmacokinetics of a single dose of Aficamten (CK-274) on cardiac contractility in a A31P MYBPC3 hypertrophic cardiomyopathy cat model. \u003cem\u003eJ. Vet. Pharmacol. Ther.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 52\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jvp.13103\u003c/span\u003e\u003cspan address=\"10.1111/jvp.13103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuis Fuentes, V. et al. ACVIM consensus statement guidelines for the classification, diagnosis, and management of cardiomyopathies in cats. \u003cem\u003eJ. Vet. Intern. Med.\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e, 1062\u0026ndash;1077. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jvim.15745\u003c/span\u003e\u003cspan address=\"10.1111/jvim.15745\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaron, B. J. et al. Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e92\u003c/b\u003e, 785\u0026ndash;789. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/01.cir.92.4.785\u003c/span\u003e\u003cspan address=\"10.1161/01.cir.92.4.785\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1995).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOlivotto, I. et al. Impact of atrial fibrillation on the clinical course of hypertrophic cardiomyopathy. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e104\u003c/b\u003e, 2517\u0026ndash;2524. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/hc4601.097997\u003c/span\u003e\u003cspan address=\"10.1161/hc4601.097997\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2001).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiontis, K. C. et al. Atrial fibrillation in hypertrophic cardiomyopathy: prevalence, clinical correlations, and mortality in a large high-risk population. \u003cem\u003eJ. Am. Heart Assoc.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, e001002. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/JAHA.114.001002\u003c/span\u003e\u003cspan address=\"10.1161/JAHA.114.001002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eC\u0026ocirc;t\u0026eacute;, E. et al. Atrial fibrillation in cats: 50 cases (1979\u0026ndash;2002). \u003cem\u003eJ. Am. Vet. Med. Assoc.\u003c/em\u003e \u003cb\u003e225\u003c/b\u003e, 256\u0026ndash;260. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2460/javma.2004.225.256\u003c/span\u003e\u003cspan address=\"10.2460/javma.2004.225.256\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHogan, D. F. Feline Cardiogenic Arterial Thromboembolism: Prevention and Therapy. \u003cem\u003eVet. Clin. North. Am. Small Anim. Pract.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 1065\u0026ndash;1082. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cvsm.2017.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.cvsm.2017.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTablin, F. et al. Platelet activation in cats with hypertrophic cardiomyopathy. \u003cem\u003eJ. Vet. Intern. Med.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 411\u0026ndash;418. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jvim.12325\u003c/span\u003e\u003cspan address=\"10.1111/jvim.12325\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, R. H., Stern, J. A., Ho, V., Tablin, F. \u0026amp; Harris, S. P. Platelet Activation and Clopidogrel Effects on ADP-Induced Platelet Activation in Cats with or without the A31P Mutation in MYBPC3. \u003cem\u003eJ. Vet. Intern. Med.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 1619\u0026ndash;1629. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jvim.14568\u003c/span\u003e\u003cspan address=\"10.1111/jvim.14568\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTan, A. W. K. et al. Platelet Priming and Activation in Naturally Occurring Thermal Burn Injuries and Wildfire Smoke Exposure Is Associated With Intracardiac Thrombosis and Spontaneous Echocardiographic Contrast in Feline Survivors. \u003cem\u003eFront. Vet. Sci.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 892377. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2022.892377\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2022.892377\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDimitrow, P. P., Undas, A., Bober, M., Tracz, W. \u0026amp; Dubiel, J. S. Obstructive hypertrophic cardiomyopathy is associated with enhanced thrombin generation and platelet activation. \u003cem\u003eHeart\u003c/em\u003e \u003cb\u003e94\u003c/b\u003e, e21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/hrt.2007.126896\u003c/span\u003e\u003cspan address=\"10.1136/hrt.2007.126896\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeikert, U., K\u0026uuml;hl, U., Schultheiss, H. P. \u0026amp; Rauch, U. Platelet activation is increased in patients with cardiomyopathy: myocardial inflammation and platelet reactivity. \u003cem\u003ePlatelets\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 487\u0026ndash;491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/0953710021000057857\u003c/span\u003e\u003cspan address=\"10.1080/0953710021000057857\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIcli, A. et al. Increased mean platelet volume in hypertrophic cardiomyopathy. \u003cem\u003eAngiology\u003c/em\u003e \u003cb\u003e65\u003c/b\u003e, 420\u0026ndash;424. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/0003319713483919\u003c/span\u003e\u003cspan address=\"10.1177/0003319713483919\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, R. H. L. et al. Circulating neutrophil extracellular traps in cats with hypertrophic cardiomyopathy and cardiogenic arterial thromboembolism. \u003cem\u003eJ. Vet. Intern. Med.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 490\u0026ndash;502. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jvim.16676\u003c/span\u003e\u003cspan address=\"10.1111/jvim.16676\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAgbani, E. O. \u0026amp; Poole, A. W. Procoagulant platelets: generation, function, and therapeutic targeting in thrombosis. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e, 2171\u0026ndash;2179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood-2017-05-787259\u003c/span\u003e\u003cspan address=\"10.1182/blood-2017-05-787259\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eProdan, C. I., Vincent, A. S. \u0026amp; Dale, G. L. Coated-platelet levels are elevated in patients with transient ischemic attack. \u003cem\u003eTransl Res.\u003c/em\u003e \u003cb\u003e158\u003c/b\u003e, 71\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.trsl.2011.02.010\u003c/span\u003e\u003cspan address=\"10.1016/j.trsl.2011.02.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePasalic, L. et al. Novel assay demonstrates that coronary artery disease patients have heightened procoagulant platelet response. \u003cem\u003eJ. Thromb. Haemost\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e, 1198\u0026ndash;1210. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jth.14008\u003c/span\u003e\u003cspan address=\"10.1111/jth.14008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaiser, R. et al. Procoagulant platelet activation promotes venous thrombosis. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e144\u003c/b\u003e, 2546\u0026ndash;2553. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood.2024025476\u003c/span\u003e\u003cspan address=\"10.1182/blood.2024025476\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOmmen, S. R. et al. AHA/ACC Guideline for the Diagnosis and Treatment of Patients With Hypertrophic Cardiomyopathy: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. \u003cem\u003eCirculation\u003c/em\u003e 142, e558-e631 (2020). (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/CIR.0000000000000937\u003c/span\u003e\u003cspan address=\"10.1161/CIR.0000000000000937\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlais, M. C. et al. Consensus on the Rational Use of Antithrombotics in Veterinary Critical Care (CURATIVE): Domain 3-Defining antithrombotic protocols. \u003cem\u003eJ. Vet. Emerg. Crit. Care (San Antonio)\u003c/em\u003e. \u003cb\u003e29\u003c/b\u003e, 60\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/vec.12795\u003c/span\u003e\u003cspan address=\"10.1111/vec.12795\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHogan, D. F. et al. Secondary prevention of cardiogenic arterial thromboembolism in the cat: The double-blind, randomized, positive-controlled feline arterial thromboembolism; clopidogrel vs. aspirin trial (FAT CAT). \u003cem\u003eJ. Vet. Cardiol.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (Suppl 1), 306\u0026ndash;317. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jvc.2015.10.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jvc.2015.10.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSciarretta, S., Forte, M., Frati, G. \u0026amp; Sadoshima, J. New Insights Into the Role of mTOR Signaling in the Cardiovascular System. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cb\u003e122\u003c/b\u003e, 489\u0026ndash;505. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/CIRCRESAHA.117.311147\u003c/span\u003e\u003cspan address=\"10.1161/CIRCRESAHA.117.311147\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaplan, J. L. et al. Delayed-release rapamycin halts progression of left ventricular hypertrophy in subclinical feline hypertrophic cardiomyopathy: results of the RAPACAT trial. \u003cem\u003eJ. Am. Vet. Med. Assoc.\u003c/em\u003e \u003cb\u003e261\u003c/b\u003e, 1628\u0026ndash;1637. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2460/javma.23.04.0187\u003c/span\u003e\u003cspan address=\"10.2460/javma.23.04.0187\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShioi, T. et al. Rapamycin attenuates load-induced cardiac hypertrophy in mice. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e107\u003c/b\u003e, 1664\u0026ndash;1670. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/01.CIR.0000057979.36322.88\u003c/span\u003e\u003cspan address=\"10.1161/01.CIR.0000057979.36322.88\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaoletti, E. et al. Effect of sirolimus on left ventricular hypertrophy in kidney transplant recipients: a 1-year nonrandomized controlled trial. \u003cem\u003eAm. J. Kidney Dis.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e, 324\u0026ndash;330. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1053/j.ajkd.2008.04.018\u003c/span\u003e\u003cspan address=\"10.1053/j.ajkd.2008.04.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcMullen, J. R. et al. Inhibition of mTOR signaling with rapamycin regresses established cardiac hypertrophy induced by pressure overload. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e109\u003c/b\u003e, 3050\u0026ndash;3055. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/01.CIR.0000130641.08705.45\u003c/span\u003e\u003cspan address=\"10.1161/01.CIR.0000130641.08705.45\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRivas, V. N. et al. Multi-Omic, Histopathologic, and Clinicopathologic Effects of Once-Weekly Oral Rapamycin in a Naturally Occurring Feline Model of Hypertrophic Cardiomyopathy: A Pilot Study. \u003cem\u003eAnim. (Basel)\u003c/em\u003e. 13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani13203184\u003c/span\u003e\u003cspan address=\"10.3390/ani13203184\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, Q., Huang, K. S., Chen, M. \u0026amp; Huang, D. J. Rapamycin enhances platelet aggregation induced by adenosine diphosphate in vitro. \u003cem\u003ePlatelets\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 428\u0026ndash;431. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/09537100903114552\u003c/span\u003e\u003cspan address=\"10.1080/09537100903114552\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAslan, J. E., Tormoen, G. W., Loren, C. P., Pang, J. \u0026amp; McCarty, O. J. S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e118\u003c/b\u003e, 3129\u0026ndash;3136. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood-2011-02-331579\u003c/span\u003e\u003cspan address=\"10.1182/blood-2011-02-331579\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBabinska, A. et al. Enhancement of human platelet aggregation and secretion induced by rapamycin. \u003cem\u003eNephrol. Dial Transpl.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 3153\u0026ndash;3159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ndt/13.12.3153\u003c/span\u003e\u003cspan address=\"10.1093/ndt/13.12.3153\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eŚledź, K. M. et al. Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity. \u003cem\u003eBiochem. Pharmacol.\u003c/em\u003e \u003cb\u003e177\u003c/b\u003e, 113975. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcp.2020.113975\u003c/span\u003e\u003cspan address=\"10.1016/j.bcp.2020.113975\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShaverdian, M., Nguyen, N. \u0026amp; Li, R. H. L. A novel technique to characterize procoagulant platelet formation and evaluate platelet procoagulant tendency in cats by flow cytometry. \u003cem\u003eFront. Vet. Sci.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 1480756. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2024.1480756\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2024.1480756\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCastedo, M., Ferri, K. F. \u0026amp; Kroemer, G. Mammalian target of rapamycin (mTOR): pro- and anti-apoptotic. \u003cem\u003eCell. Death Differ.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 99\u0026ndash;100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sj.cdd.4400978\u003c/span\u003e\u003cspan address=\"10.1038/sj.cdd.4400978\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDonato, G., Pennisi, M. G., Persichetti, M. F., Archer, J. \u0026amp; Masucci, M. A. Retrospective Comparative Evaluation of Selected Blood Cell Ratios, Acute Phase Proteins, and Leukocyte Changes Suggestive of Inflammation in Cats. \u003cem\u003eAnim. (Basel)\u003c/em\u003e. 13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani13162579\u003c/span\u003e\u003cspan address=\"10.3390/ani13162579\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKay, J. E., Kromwel, L., Doe, S. E. \u0026amp; Denyer, M. Inhibition of T and B lymphocyte proliferation by rapamycin. \u003cem\u003eImmunology\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e, 544\u0026ndash;549 (1991).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBattaglia, M. et al. Rapamycin promotes expansion of functional CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;FOXP3\u0026thinsp;+\u0026thinsp;regulatory T cells of both healthy subjects and type 1 diabetic patients. \u003cem\u003eJ. Immunol.\u003c/em\u003e \u003cb\u003e177\u003c/b\u003e, 8338\u0026ndash;8347. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4049/jimmunol.177.12.8338\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.177.12.8338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThomson, A. W., Turnquist, H. R. \u0026amp; Raimondi, G. Immunoregulatory functions of mTOR inhibition. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 324\u0026ndash;337. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nri2546\u003c/span\u003e\u003cspan address=\"10.1038/nri2546\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePelekanou, V., Trezise, A. E., Moore, A. L. \u0026amp; Kay, J. E. FK 506 and rapamycin do not affect platelet aggregation or mitochondrial function. \u003cem\u003eTranspl. Proc.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 3200\u0026ndash;3201 (1991).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eL\u0026oacute;pez, E. et al. Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients. \u003cem\u003eJ. Cell. Mol. Med.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 636\u0026ndash;647. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jcmm.12044\u003c/span\u003e\u003cspan address=\"10.1111/jcmm.12044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHollerbach, A. et al. Platelet Activation by Antiphospholipid Antibodies Depends on Epitope Specificity and is Prevented by mTOR Inhibitors. \u003cem\u003eThromb. Haemost\u003c/em\u003e. \u003cb\u003e119\u003c/b\u003e, 1147\u0026ndash;1153. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1055/s-0039-1685453\u003c/span\u003e\u003cspan address=\"10.1055/s-0039-1685453\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOuseph, M. M. et al. Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e126\u003c/b\u003e, 1224\u0026ndash;1233. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood-2014-09-598722\u003c/span\u003e\u003cspan address=\"10.1182/blood-2014-09-598722\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJosefsson, E. C., Ramstr\u0026ouml;m, S., Thaler, J. \u0026amp; Lordkipanidz\u0026eacute;, M. \u0026amp; group, C. s. Consensus report on markers to distinguish procoagulant platelets from apoptotic platelets: communication from the Scientific and Standardization Committee of the ISTH. \u003cem\u003eJ Thromb Haemost\u003c/em\u003e 21, 2291\u0026ndash;2299 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jtha.2023.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jtha.2023.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTablin, F., Walker, N. J., Klein, S. D., Field, C. L. \u0026amp; Crowe, J. H. Animal models for studies on cold-induced platelet activation in human beings. \u003cem\u003eJ. Lab. Clin. Med.\u003c/em\u003e \u003cb\u003e135\u003c/b\u003e, 339\u0026ndash;346. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1067/mlc.2000.105619\u003c/span\u003e\u003cspan address=\"10.1067/mlc.2000.105619\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, Q. et al. Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke. \u003cem\u003eBiochem. Biophys. Res. Commun.\u003c/em\u003e \u003cb\u003e444\u003c/b\u003e, 182\u0026ndash;188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2014.01.032\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2014.01.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePanel, M., Ghaleh, B. \u0026amp; Morin, D. Mitochondria and aging: A role for the mitochondrial transition pore? \u003cem\u003eAging Cell.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, e12793. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/acel.12793\u003c/span\u003e\u003cspan address=\"10.1111/acel.12793\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKadenbach, B., Ramzan, R., Moosdorf, R. \u0026amp; Vogt, S. The role of mitochondrial membrane potential in ischemic heart failure. \u003cem\u003eMitochondrion\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 700\u0026ndash;706. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mito.2011.06.001\u003c/span\u003e\u003cspan address=\"10.1016/j.mito.2011.06.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMillington-Burgess, S. L. \u0026amp; Harper, M. T. Cytosolic and mitochondrial Ca. \u003cem\u003ePlatelets\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 855\u0026ndash;862. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/09537104.2021.1881951\u003c/span\u003e\u003cspan address=\"10.1080/09537104.2021.1881951\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang, H. et al. Bcl-2 family proteins are essential for platelet survival. \u003cem\u003eCell. Death Differ.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 943\u0026ndash;951. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sj.cdd.4402081\u003c/span\u003e\u003cspan address=\"10.1038/sj.cdd.4402081\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHua, V. M. et al. Necrotic platelets provide a procoagulant surface during thrombosis. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e126\u003c/b\u003e, 2852\u0026ndash;2862. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood-2015-08-663005\u003c/span\u003e\u003cspan address=\"10.1182/blood-2015-08-663005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoore, S. F., Hunter, R. W. \u0026amp; Hers, I. Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets. \u003cem\u003eJ. Thromb. Haemost\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, 748\u0026ndash;760. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jth.12552\u003c/span\u003e\u003cspan address=\"10.1111/jth.12552\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, J. et al. mTORC1 promotes aging-related venous thrombosis in mice via elevation of platelet volume and activation. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e128\u003c/b\u003e, 615\u0026ndash;624. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood-2015-10-672964\u003c/span\u003e\u003cspan address=\"10.1182/blood-2015-10-672964\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWoulfe, D., Jiang, H., Mortensen, R., Yang, J. \u0026amp; Brass, L. F. Activation of Rap1B by G(i) family members in platelets. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e277\u003c/b\u003e, 23382\u0026ndash;23390. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M202212200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M202212200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"rapamycin, procoagulant platelets, cardiogenic arterial thromboembolism, hypertrophic cardiomyopathy, mitochondrial membrane potential, phosphatidylserine","lastPublishedDoi":"10.21203/rs.3.rs-7068900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7068900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCardiogenic arterial thromboembolism (CATE) is a complication of hypertrophic cardiomyopathy (HCM) with a high mortality rate. Despite anti-platelet drugs use, on-treatment recurrence rate remains high indicating a critical need to discover novel therapies. Studies in both humans and cats show that low dose delayed release rapamycin (LDDRR) can reduce the progression of left ventricular hypertrophy. However, its effect on platelets is unclear. In this study we assessed the \u003cem\u003eex vivo\u003c/em\u003e effects of LDDRR on platelet aggregation, alpha-granule secretion indicated by an increase in P-selectin, and procoagulant platelet phenotypes including loss of mitochondrial membrane potential (ΔΨm) and phosphatidylserine (PS) exposure. Cats were treated with 0.3 mg/kg LDDRR orally every 7 days for 4 consecutive weeks. Blood was collected at 3, 24 and 48 hours after the last dose. While LDDRR had no effect on aggregation, it significantly decreased P-selectin expression in thrombin\u0026thinsp;+\u0026thinsp;COL (3 hrs), and ADP (24, 48 hrs) samples. LDDRR had protective effects on ΔΨm in all agonists at all times. PS exposure was reduced at 24 hrs in thrombin\u0026thinsp;\u0026plusmn;\u0026thinsp;COL samples. Our study indicated that LDDRR in cats can safely modulate platelet activation, procoagulant phenotypes and tendency in varying degrees making it an effective candidate to prevent CATE.\u003c/p\u003e","manuscriptTitle":"Ex vivo effects of low dose delayed release rapamycin on agonist-induced platelet aggregation, activation and procoagulant platelet phenotypes in domestic cats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 09:17:34","doi":"10.21203/rs.3.rs-7068900/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-06T11:50:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T16:20:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T13:18:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321972391548390211851584112293342635745","date":"2026-01-27T18:03:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42840903942314922775982478469306412735","date":"2026-01-05T05:52:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-14T23:10:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70374003885269677677967921672126347997","date":"2025-10-31T12:31:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-30T16:40:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-30T10:02:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-31T14:16:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-30T16:39:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-30T16:35:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6053158e-22a9-4962-b4f7-56bc9302871b","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57751783,"name":"Health sciences/Cardiology"},{"id":57751784,"name":"Health sciences/Diseases"},{"id":57751785,"name":"Biological sciences/Drug discovery"},{"id":57751786,"name":"Health sciences/Medical research"},{"id":57751787,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-13T16:08:46+00:00","versionOfRecord":{"articleIdentity":"rs-7068900","link":"https://doi.org/10.1038/s41598-026-46991-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-11 15:58:52","publishedOnDateReadable":"April 11th, 2026"},"versionCreatedAt":"2025-11-11 09:17:34","video":"","vorDoi":"10.1038/s41598-026-46991-z","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46991-z","workflowStages":[]},"version":"v1","identity":"rs-7068900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7068900","identity":"rs-7068900","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}