TSLP induces platelet mitophagy and promotes thrombosis in Kawasaki disease 

TSLP induces platelet mitophagy and promotes thrombosis in Kawasaki disease | 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 Research Article TSLP induces platelet mitophagy and promotes thrombosis in Kawasaki disease Lanyan Fu, Daniel Thomas MacKeigan, Qing Gong, Di Che, Yufen Xu, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1628519/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Kawasaki disease (KD) is an acute systemic vasculitis primarily affecting infants and children with an unclear etiology. Coronary artery aneurysm (CAA) is a common manifestation in severe KD patents, which may lead to thrombotic cardiovascular events such as heart attack and stroke even years after onset. We and others have previously reported systemic platelet activation in KD patients. Thymic stromal lymphopoietin (TSLP) is a recently identified interleukin-7 (IL-7) like cytokine associated with promoting pathological inflammation and most notably, platelet activation. The present study is to investigate the role of TSLP in KD-associated thrombosis. Methods To discover potential proteins underlying platelet activation in KD, we conducted a protein chip assay of 34 cytokines and discovered a significant upregulation in thymus stromal lymphopoietin (TSLP). ELISA corroborated the upregulation of TSLP in another group of KD patients. Clinical samples (plasma and platelets) from KD patients and healthy controls were analyzed via flow cytometry, immunofluorescent confocal microscopy, western blot, immunoprecipitation and thrombosis assays to reveal the underlying mechanisms. Results Among the 34 cytokines, we discovered several were aberrantly expressed, nine of which were continuously elevated after IVIG treatment and maintained during the convalescence in KD patients compared to healthy controls, including TSLP. The upregulation of TSLP in KD patients was confirmed by ELISA, which showed a further increase of TSLP in convalescent patients complicated with thrombosis. The expression of TSLP receptors (TSLPRs) on platelets were also significantly upregulated in KD patients complicated with thrombosis. Platelet activation, apoptosis, and mitochondrial autophagy (mitophagy) were increased in KD patients, which increase was exacerbated in convalescent patients complicated with thrombosis. In vitro, TSLP induced platelet activation and platelet mitophagy in healthy blood donors as we observed in KD patients. TSLP, similar to mitophagy agonist CCCP, promoted thrombosis, which was attenuated by the mitophagy inhibitor Mdivi-1. Co-immunoprecipitation in TSLP-treated platelets revealed TSLPR bound to mitophagy regulators, Parkin and VDAC1, suggesting a potential novel TSLP-mediated platelet mitophagy pathway. Conclusions Our results demonstrate that TSLP induces platelet mitophagy via a novel TSLPR/Parkin/VDAC1 mitophagy pathway that promotes thrombosis in KD. These results suggest TSLP as a novel therapeutic target against KD-associated thrombosis. TSLP Platelet Mitophagy Thrombosis Kawasaki disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Kawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is a common pediatric vasculitis of medium and small muscular arteries that affects mainly children under 5 years old 1 , 2 . The main cause of death in KD is heart attack and stroke, which is often caused by occlusive thrombosis precipitated by coronary artery aneurysm (CAA) 3 , 4 . The risk of thrombosis persists up to years following initial onset and treatment. Indeed, our group and several others have reported elevated platelet activation in KD patients 5 – 7 , which may serve as a risk factor for thrombosis in KD patients with pre-existing CAA 8 , 9 . The pathogenesis of platelet activation and thrombosis in KD remains elusive, although increased thrombocytosis 10 , disordered platelet function 11 , and increased platelet-derived microparticles 12 have been reported. It is imperative to uncover its pathophysiology, particularly the mechanism that causes platelet activation, to identify novel therapeutic targets against KD-associated thrombosis. Three traditional antiplatelet pathways 13 are currently used for the clinical treatment of KD: thromboxane A2 pathway (such as aspirin), ADP pathway (such as clopidogrel), and platelet receptor pathway (such as abciximab) 14 , 15 . For KD patients with coronary artery aneurysm and thrombosis, combined treatment of antiplatelet and thrombolysis or anticoagulation therapy is usually used 1 . However, these processes cannot completely inhibit platelet activation and thrombosis, indicating that there may be other underlying platelet activation mechanisms regulated by unknown factors. Studies have reported that cytokines contribute to inflammation and thrombotic responses 16 , 17 . However, whether plasma cytokines contribute to platelet activation and thrombosis in KD is unknown. Thymic stromal lymphopoietin (TSLP) is an IL-7 like cytokine that is mainly derived from epithelial cells, fibroblasts and mast cells 18 . Accumulating evidence implicates the dysregulated expression of TSLP in multiple diseases such as asthma, allergic rhinitis, leukemia and atherosclerosis 19 – 22 . Recently, the roles of TSLP and TSLPR on platelets have been linked to platelet activation and thrombus formation through PI3K/Akt signaling 23 , 24 . However, the role of TSLP in aberrant platelet activation and thrombosis in KD has not been previously reported. Platelet autophagy is involved in the regulation of platelet activity 25 , 26 . Not surprisingly, mitophagy, a selective autophagy that regulates mitochondrial quality also occurs in platelets and is emerging as an important regulatory mechanism in platelets. Since the mitochondrion is essential for energy production, it is no surprise that enhanced mitophagy is linked with platelet autophagy and activation. Recent reports 27 demonstrate platelet mitophagy promotes thrombosis, however, the role of platelet mitophagy in KD platelet activation and thrombosis has never been explored. In the present study, we discovered that TSLP is significantly upregulated in KD and promotes platelet mitophagy and thrombosis via a novel TSLPR/Parkin/VDAC1-dependent signaling pathway. Our findings further elucidate the mechanism of thrombosis in KD and identify TSLP as a potential novel anti-thrombotic therapeutic target. Materials And Methods Study objects Samples of KD patients and age-matched healthy controls (HC) were collected from the Guangzhou Women and Children Medical Center in China, between July 2016 and March 2021(Supplemental Materials Table I). All specimens were stored in the clinical biological resource bank (Clinical Bio-bank) of this hospital. KD patients were diagnosed by our hospital cardiology physician, according to the latest version of the American Heart Association's 2017 revised diagnostic criteria and treatment guidelines 1 . Coronary artery abnormalities of KD patients were categorized according to their Z score: no coronary artery damage(Z 2 ~ < 2.5mm), large CAA (≥ 8.0 mm or Z ≥ 10 mm), medium CAA (< 8.0 mm and Z ≥ 5 ~ < 10 mm), and small CAA (Z ≥ 2.5 ~ < 5 mm). This study was approved by the Ethics Committee of Guangzhou Women and Children's Medical Center (Number: 2014073009 and 2018052105). All participants’ parents/guardians gave written informed consent in accordance with the Declaration of Helsinki. We categorized blood samples from KD patient samples into either of three stages, the acute phase (patients before Intravenous IgG (IVIG) and after IVIG treatment within 10 days), subacute phase (after treatment 11 days-1 mouth) and convalescence phase (Aspirin or other antiplatelet drug treatment more than 1–6 mouths). Preparation of human platelets Venous blood was drawn from healthy controls and KD patients then collect with sodium citrate or ACD (2-4mL) anticoagulant tube, and pretreated with 75nM prostaglandin E1 (PGE1; Catalog No. : HY-B0131, MEC) to prevent platelet activation. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 900 rpm at 22˚C for 10mins, platelet-poor plasma (PPP) was obtained by centrifugation of PRP at 3500 rpm at 22˚C for 10mins, and platelet pellets at the bottom of the tube 28 , 29 . Platelet counts in PRP were performed with an automatic blood cell analyzer (sysmex XS-500i). To prepare washed platelets, platelet pellets were gently washed twice with CGS buffer (0.123 M NaCl, 0.033 M D-glucose, 0.013 M trisodium citrate, pH 6.5) containing 75nM PGE1, then the washed platelets were suspended in modified Tyrode’s buffer (MTB; 2.5 mM Hepes, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO 3 , 5.5 mM D-glucose, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4) containing 75nM PGE1 at 3–5 × 10 8 platelets/mL, and incubated at room temperature for 1 h before use. Regents and antibodies Bafilomycin A1 (Baf-A1), mitochondrial division inhibitor-1 (Mdivi-1) and mitophagy agonist Carbonyl cyanide m-chlorophenyl hydrazone, Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were purchased from Selleckchem. Prostaglandin E1 (PGE1) was purchased from MCE. Recombinant human TSLP was purchased from Beyotime and peprotech. CaCl 2 (0.025M) and thrombin were purchased from Diagnostica Stago. Antibodies of β-actin, TSLP, LC3A/B, Bcl-xL, Bax, IgG, and Caspase-3 were purchased from Cell Signaling Technology. TSLPR, Parkin, and PINK1 antibodies were obtained from Abcam. Antibodies of TSLP receptors (TSLPR and IL-7R), VDAC1, Tom20 and protein A/G PLUS-Agarose immunoprecipitation reagents were purchased from Santa Cruz Biotechnology. The anti-CD41a and anti-CD62p were purchased from eBioscience. Human TSLP ELISA kit was purchased from R&D Systems, soluble CD40L (sCD40L) and soluble P-selectin (sP-selectin) ELISA kits were purchased from MultiSciences. MitoTracker antibody and MitoProbe™ TMRM Assay Kit were purchased from Thermofisher. Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Keygen Biotech. Multiplex Analysis and Quantification of Cytokines The Bio-Plex Pro Human Inflammation Group 34-Plex 171AL001M (Bio-Rad, Hercules, CA) kit was used to detect the cytokine content in plasma samples selecting by “Clinical Bio-bank” from KD patients and healthy control. The protocol was followed according to the manufacturer’s instructions. The Bio-Plex Verification Kit Version 3.0 (Bio-plex200, Bio-Rad, Hercules, CA) was used to verify the hydrodynamic performance, consistent optical alignment, double identification and identification of single bead signatures. The measurement was performed using the Bio-Plex protein array system integrated with Bio-Plex Manager software version 3.0 (Bio-Rad, Hercules, CA). ELISA ELISA was used to detect plasma concentrations of TSLP (R&D Systems), sCD40L and sP-selectin (MultiSciences). ELISA experiments were carried out according to the manufacturer's protocol as we previously described 5 . Briefly, PPP samples (40–100µL) were added into each well of a microtiter plate followed by 10–100µL of the detection antibody. Following 1-2h incubation at 37˚C, 50–100µL of streptavidin-HRP was added to each well and allowed to incubate for 30-60mins. The wells were subsequently washed and 50–100µL color reagent A and 50–100µL color Reagent B were added, gently vortexed, and incubated at 37˚C for 10 minutes in the dark. The reaction was quenched by adding 50µL Reagent C and plates were read at 450 nm with a Variskan Flash (Thermo Scientific). Measurement of platelet activation, mitochondrial membrane potential (ΔΨm), PS exposure by flow cytometry To assess platelet activation, PRP was prepared from human whole blood as described above. PRP (3–5µL) was labelled with-anti-CD41a and anti-CD62p, incubated at room temperature for 15-30mins. The reaction was stopped by adding PBS, and then analyzed by flow cytometry analysis (BD FACScanto II). Platelets were identified and gated by their characteristic forward and side scatter properties. A total of 10,000 platelets were measured from each sample as we previously described 30 , 31 . Platelets were pelleted from PRP and subsequently resuspended in MTB adjusted to a concentration of 3–5×10 8 platelets/mL. Platelets were incubated with 1µM tetramethylrhodamine methyl ester (TMRM) at 37˚C for 30 mins. Platelets were subsequently incubated with 5µL of Annexin V at room temperature for 15 mins. Flow cytometry was then used to analyze ΔΨm and PS externalization 32 – 34 . Platelets were identified and gated by their characteristic forward and side scatter properties. A total of 10,000 platelets were analyzed from each sample. Observation of autophagosomes by transmission electron microscopy The platelet pellet was prepared as above described, then washed twice and fixed with 2.5% glutaraldehyde solution and 1% osmium tetroxide solution. Finally, the platelets were dehydrated with an ethanol solution then embedded and cut into ultrathin 100 nm sections. After staining with a saturated uranyl acetate solution and a lead acetate solution, the platelets were observed and photographed under a transmission electron microscope. Immunofluorescence and confocal microscopy PRP was prepared from human whole blood as described above. MitoTracker® was incubated with platelets (100µL of PRP diluted in 1mL MTB) at room temperature for 15mins. Platelets were then pelleted by centrifugation at 3500 rpm for 10mins at 25˚C, resuspended in MTB (400µL) by centrifuged again at 7200 rpm for 3mins to adhere to the glass slide. Platelets were then fixed with 4% paraformaldehyde (100–200µL) for 15mins, washed three times with PBS, permeabilized for 10mins in 0.25% Triton X-100, washed 3 times with PBS and then blocked (5% BSA, 0.1% Triton X-100 in PBS) for 30mins at room temperature. After the sealing solution was removed, the platelets were incubated with a primary antibody against LC3, TSLPR (1% BSA in PBS 1:200) at 4˚C overnight. Next, a fluorescent secondary antibody (1% BSA in PBS 1:1000) was added to platelets and incubated for 2h at room temperature in the dark, then washed 3 times with PBS. After platelets were sealed with anti-fluorescence quenching liquid, the stained platelets were observed using a Zeiss LSM 800 confocal microscope with 63x oil immersion lens and photographed as we previously described 35 . Western blotting analysis Platelets from patients and healthy volunteers were obtained by gradient centrifugation. Healthy volunteers PRP were treated with recombinant human TSLP (200 ng/mL or 500 ng/mL) for 3h, and then lysed with an equal volume of lysis buffer on ice for 30mins. The protein concentration was determined using a BCA protein assay kit (Applygen Beijing) according to the manufacturer’s protocol. Each sample was loaded in equal proportion to a 10%-12% SDS gel and separated via polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane. Either TSLP, TSLPR, Caspase-3, Bax, Bcl-xL, PINK1, Parkin, LC3A/B, Tom20, β-actin, VDAC1 or IL-7R (1:2000) antibody was incubated with the membrane at 4˚C overnight, then washed and incubated with the corresponding secondary antibody for 2h at room temperature. Antibody binding was detected with the ECL detection reagent by Amersham image 600 Software and the bands were quantified with Quantity Image J as we previously described 36 . Immunoprecipitation analysis PRP was pretreated with or without Baf-A1 (400nM for 1h) and then treated with recombinant human TSLP (500 ng/mL for 3h). Platelets were then lysed and mixed with the specific target antibody for Parkin IP (1:50 dilution); VDAC1 IP (1:30 dilution) or TSLPR IP (1:30 dilution), and the IgG control group as a negative control, and incubated for 1h at 4˚C. Then, 20µL of resuspended A/G PLUS-Agarose was added to each sample and incubated at 4˚C on a rocker platform overnight. Following this incubation, samples were pelleted and washed 4 times before being loaded on SDS-PAGE. The subsequent steps were according to the aforementioned western blot protocol 37 . In vitro thrombosis and clot retraction assays 1) Whole blood thrombosis assay Glass tubes were coated with Sigmacote® and dried overnight. In one experiment, 200µL aliquots of PRP were incubated with a gradient concentration of recombinant human TSLP (0, 100, 200 or 500ng/mL) in 37˚C for 3h using 1.5 NIH unit/mL thrombin as a positive control. In another experiment, 200µL aliquots of PRP were incubated with CCCP (10µM) or Mdivi-1 (10µM) treatment for 1h followed by incubation with recombinant human TSLP for 3h. PRP samples were then diluted with 200µL PBS, 50µL whole blood and 100µL CaCl 2 (0.025M) incubated at 37˚C with images photographed at 30min/1h/1h30m/2h 38 . 2) Platelet-rich plasma clot retraction assay As described for whole blood thrombosis without the addition of whole blood. Each condition received 200µL PRP, 200µL PBS and 100µL CaCl 2 (0.025M) and was left to clot in 37˚C, with clot retraction photographed over time 39 . Statistical analyses All experiments were repeated at least 3 times, the data are presented as the mean ± SD. Differences between the experimental groups were assessed by a two-tailed unpaired t-test, and in datasets with 3 or more groups was performed by one-way analysis of variance followed by Tukey's test Differences were analyzed by GraphPad Prism 7 with p value less than 0.05 considered as statistically significant. Results KD patients with thrombosis showed increased platelet activation and apoptosis We previously reported that plasma levels of soluble CD40L (sCD40L) and soluble P-selectin (sP-selectin) were elevated in KD patients, and positively correlates with the degree of coronary artery damage in Kawasaki disease patients 5 . In the present study, we examined plasma from another group of KD convalescent patients and found a significant increase in the platelet activation marker P-selectin (CD62p) (Fig. 1 A ) . Similarly, sP-selectin and sCD40L were significantly elevated in the plasma of acute (patients before IVIG and after IVIG ) and convalescent patients relative to healthy controls (Fig. 1 B &C ) and significantly elevated in convalescent patients complicated with thrombosis relative to those without (Fig. 1 D &E ). These findings confirmed that baseline platelet activation is significantly greater in acute and convalescent KD patients, which is exacerbated in patients complicated with thrombosis. Previous reports have shown that platelet activation is associated with platelet mitochondrial dysfunction and control of the platelet life span 40 . When dysfunction of platelet mitochondria occurs, the mitochondrial membrane potential will decrease, the apoptosis protein will change, PS valgus, and calcium ion changes, which will eventually lead to platelet apoptosis 32 , 33 . Thus, we analyzed apoptotic signaling and mitochondrial dysfunction in platelets from KD patients. Using flow cytometry, we found a significant reduction in mitochondrial membrane potential (Δψ m) in platelets from KD patients relative to healthy controls (Fig. 2 A). Western blot analysis confirmed an upregulation in hallmark platelet apoptotic proteins including Bax, Caspase-3 and VDAC1, and the downregulation of the anti-apoptotic protein Bcl-xL. Notably, apoptotic markers were most enhanced in convalescent patients complicated with thrombosis ( Fig. 2 B ) . Consistent with these findings, flow cytometry analysis revealed a significant increase in the percentage of Annexin V-positive platelets in the KD convalescent patients compared to healthy controls (Fig. 2 C). Taken together, the above data suggests an elevation in both platelet activation and apoptosis in KD patients, especially in those complicated with thrombosis. Platelet autophagy is upregulated in KD Since we found increased apoptosis in KD patients, we next decided to investigate whether basal platelet autophagy was affected. Fluorescent confocal microscopy revealed that the platelet autophagy marker LC-3 was significantly increased in platelets from KD patients relative to healthy controls (Fig. 3 A). Electron microscopy also found an increase in the number of platelet autolysosomes in KD patient platelets (Fig. 3 B). Consistent with these data, western blot analysis showed an increase in the autophagy proteins PINK1, Parkin, Tom20 and LC3A/B in KD patients, especially in patients with thrombosis (Fig. 3 C). Collectively, these data confirm platelet autophagy is upregulated in patients with KD and is closely related to KD-related thrombosis. Plasma TSLP and platelet TSLP receptors were elevated in KD patients complicated with thrombosis Given the inflammatory nature of KD, we suspected dysregulated cytokines may contribute to platelet activation. To investigate this, we utilized a protein chip assay to quantify the expression of 34 cytokines in plasma from KD patients in acute, subacute and recovery phase comparison with healthy controls and febrile controls. Several were aberrantly expressed, nine of which were continuously elevated after treatment and maintained during the recovery phase in KD compared to healthy controls, including TSLP (Fig. 4 A &B ). Since TSLP/TSLPR has previously been linked to platelet activation and thrombosis 23 , we decided to narrow our focus on this protein. We divided the patients into two groups according to echocardiography, KD with thrombosis and those without thrombosis (Fig. 4 C). ELISA analysis corroborated the upregulation of plasma TSLP in acute (before IVIG and after IVIG) and convalescent phase KD patients in different group of samples (Fig. 4 D), which also showed a significant increase in convalescent KD patients complicated with thrombosis relative to those without (Fig. 4 D). Similarly, the expression of the TSLP receptors (TSLPR and IL-7R) on platelets was upregulated in KD patients, especially in patients with thrombosis (Fig. 4 E). TSLP promotes mitophagy-mediated platelet activation and apoptosis causing thrombosis in vitro We next investigated the effect of TSLP on platelet activation and mitophagy in vitro. TSLP treatment on healthy human platelets caused a significant increase in CD62p (Fig. 5 A) and dose-dependently accelerated human PRP and whole blood thrombosis ( Fig. 5 B ) . To investigate TSLP in mitophagy, fluorescent confocal microscopy was used to image the mitophagy marker LC-3 and found a significant increase in platelets following TSLP treatment ( Fig. 5 C ) . Western blot analysis also revealed that platelet mitophagy and apoptotic proteins were increased by TSLP treatment in a dose-dependent manner ( Fig. 5 D ) . Based on this apparent link with mitophagy, we used TSLP in combination or in comparison to the mitophagy agonist (CCCP) and mitophagy inhibitor (Mdivi-1) in a thrombosis assay. Interestingly, CCCP showed similar thrombosis to TSLP, whereas Mdivi-1 alleviated thrombosis induced by TSLP (Fig. 5 E). Taken together, these data suggest that TSLP induces platelet mitophagy causing downstream platelet activation and thrombosis. TSLPR binds Parkin and VDAC1 to trigger TSLP induced platelet mitophagy Multiple studies have demonstrated that the PINK1/Parkin signalling pathway is involved in the regulation of mitophagy 41 – 43 . Thus, we explored whether TSLP regulates KD platelet mitophagy through the PINK1/Parkin signaling pathway. In vitro, TSLP dose-dependently increased the expression of TSLPR and IL-7R in platelets ( Fig. 6 A ) . TSLP also significantly increased the expression of PINK1, Parkin and VDAC1 in platelets (Fig. 6 A &B ). Furthermore, pretreatment with the late-stage autophagy inhibitor Baf-A1 did not prevent TSLP-induced elevation of PINK1, Parkin, VDAC1 and TSLPR (Fig. 6 B). Together, these results suggest that TSLP may promote platelet mitophagy through the PINK1/Parkin pathway. Based on a study that showed TSLP may cause TSLPR internalization 44 , we hypothesized that TSLP caused TSLPR internalization and translocation to the mitochondria where it directly promotes mitophagy. Indeed, immunoprecipitation (IP) results show that Parkin and VDAC1 both independently bound TSLPR in platelets and their binding to each other and TSLPR was significantly enhanced following TSLP treatment (Fig. 6 C &D ). This result is consistent with previous reports 45 , in which Parkin complexes with VDAC1 to induce mitophagy. Further evidence for the TSLPR-Parkin-VDAC1 interaction comes from an additional IP using TSLPR to pull down Parkin and VDAC1 ( Fig. 6 E ) , effectively demonstrating a triple co-IP (Fig. 6 C-E). Immunofluorescent confocal imaging confirmed TSLPR upregulation and colocalization to the mitochondria in platelets following TSLP-treatment ( Fig. 6 F ) . Taken together, our results show that TSLP induces platelet mitophagy through the TSLPR binding with Parkin and VDAC1. Discussion In the current study, we describe a novel mechanism of platelet activation and thrombosis induced by TSLP in KD. Using clinical samples, we found significantly upregulated expression of plasma TSLP in KD patients relative to healthy controls, which was exacerbated in patients complicated with thrombosis. Furthermore, TSLP receptor (TSLPR and IL-7R) expression was significantly enhanced on platelets of KD patients complicated with thrombosis. Interestingly, we found increased platelet mitophagy and apoptosis in KD patients complicated with thrombosis, which TSLP induced in vitro. Lastly, TSLPR bound to mitophagy regulators Parkin and VDAC1 respectively following TSLP treatment, suggesting a novel TSLP-mediated mitophagy pathway in platelets. Taken together, our findings uncover a novel mechanism of platelet activation and thrombosis in KD and suggest TSLP as a novel anti-thrombotic target. Our work identifies that upregulated TSLP expression at least partially underlies platelet activation and thrombosis in KD. TSLP-induced platelet mitophagy and activation likely promotes thrombosis by promoting platelets to directly bind its ligands mediating platelet aggregation 46 – 48 , and to harbor phosphatidylserine (PS) on its surface, which promotes a hypercoagulable state through cell-based thrombin generation 34 , 49 . Current anti-thrombotic therapies may not effectively inhibit TSLP-induced thrombosis in KD given its unique mechanism, which may explain the persistent platelet activation and thrombosis in treated patients. A previous report identified that TSLP activates platelets through the PI3K/AKT pathway. However, since we found that the mitophagy inhibitor Mdivi-1 significantly attenuated TSLP-mediated in vitro thrombosis, we propose that its effect on PI3K/AKT signaling may be downstream of its induction of mitophagy 23 , 50 . Mitophagy is mainly regulated by the PINK1/Parkin signaling pathway 51 . Recombinant Voltage Dependent Anion Channel Protein 1(VDAC1) is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis 52 . Interestingly, we found that TSLPR bound to Parkin and VDAC1 and TSLP treatment enhanced the independent binding of TSLPR/Parkin/VDAC1. TSLP binding TSLPR is reported to cause receptor internalization 44 , which is consistent with our findings of TSLPR co-localization with the mitochondria in platelets following TSLP treatment. This TSLPR/Parkin/VDAC1 protein complex may be an important driver of TSLP-mediated platelet mitophagy in KD. Several reports suggest platelet activation may be a major driver of inflammation in KD 8 , 53 . Platelets contain and release several proinflammatory cytokines upon activation such as TNF-α, IFN-β, and IL-6 that may propagate an inflammatory state 40 , 54 . Activated platelets also express P-selectin, which promotes the formation of platelet-leukocyte aggregates, an important contributor in the progression of KD 55 , 56 . Inflammatory responses, platelet activation and thrombosis are inextricably linked. We propose that in addition to its role in thrombosis, TSLP may also contribute to systemic inflammation through its activation of platelets and release of pro-inflammatory factors. Recent studies have shown that TSLP upregulates inflammatory responses by inducing autophagy in T cells, which partially validating our hypothesis 57 . To our knowledge, we are the first to report evidence of increased platelet apoptosis in KD. It is interesting to note that the therapeutic mechanism of IVIG in KD is unclear, yet has previously been reported to inhibit platelet apoptosis in immune thrombocytopenia (ITP) 58 . Thus, it is conceivable that IVIG partially exerts its therapeutic effect in KD through its inhibition of platelet apoptosis. The cause of platelet apoptosis in KD remains unclear, however, considering the intimate link of platelet apoptosis and mitophagy, TSLP likely has a faciliatory role. The origin of TSLP in KD remains unclear. A variety of stimuli and cytokines (IL-4, IL-13, IL-5, NF-κB, and TNF-α etc.) can activate TSLP production 59 . Interestingly, a recent report found that TSLP production in human dermal microvascular endothelial cells is also triggered by activated platelets in an IL-1β dependent manner 60 . Based on this report, TSLP may activate platelets, which triggers endothelial cells to produce TSLP creating a positive feedback cycle that drives TSLP production and platelet activation. Thus, TSLP neutralization may effectively normalize its plasma concentration and reduce platelets activation. Many experts suggest targeting TSLP-mediated signaling as a novel therapeutic strategy against allergic diseases to neutralize its inflammatory function 61 . Interestingly, KD disease is marked by a persistent inflammatory state over many months that share several features to allergic disease inflammation including abnormal type 2 inflammation, Th17/Treg imbalance, and other immunopathogenesis 62 . In addition, anti-TSLP monoclonal antibodies are already used for the treatment of severe asthma, such as Tezepelumab and CSJ117 63 . Thus, TSLP may serve as a novel therapeutic target that effectively treats the inflammatory and thrombotic risks associated with KD. A limitation of our research is that we did not use the TSLPR knockout KD mouse model to verify our results. However, our study utilized a large number of rare human clinical specimens, including convalesce stage KD patients with thrombosis, to identify the pivotal role of TSLP in KD. Our results demonstrate a close relationship between TSLP and thrombosis in vivo and in vitro. TSLP induced platelet mitophagy and activation via the TSLPR/Parkin/VDAC1 signaling pathway to promote thrombosis in KD. Our findings highlight TSLP as an important contributor and novel therauptic target for KD-associated thrombosis. Abbreviations KD Kawasaki disease TSLP Thymus stromal lymphopoietin P TSLPR Thymus stromal lymphopoietin receptor INK1/Parkin PTEN induced putative kinase 1/Parkin VDAC1 Recombinant Voltage Dependent Anion Channel Protein 1 PS Phosphatidylserine IVIG Intravenous IgG CAA Coronary artery aneurysm Baf-A1 Bafilomycin A1 Mdivi-1 Mitochondrial division inhibitor-1 CCCP Mitophagy agonist Carbonyl cyanide m-chlorophenyl hydrazone, Carbonyl cyanide 3-chlorophenylhydrazone PGE1 Prostaglandin E1 PRP Platelet-rich plasma PPP platelet-poor plasma. Declarations Funding This study was funded by the Guangdong Basic and Applied Basic Research Foundation (grant numbers 2021B1515230003), the Guangdong Natural Science Fund, China (grant numbers 2019A1515012061, 202102020829, 2022A1515012558), the Guangzhou Science and Technology Program Project, China (grant numbers 201904010486, 202102010197, 202102020829), the Subject Construction Project of Guangzhou Medical University (grant numbers 02-410-2206062), Postdoctoral Research Initiation Fund from Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center (grant numbers 3001162, 3001178-04) and Canadian Institutes of Health Research Foundation grant (grant numbers 389035). Acknowledgments The authors would like to thank the Clinical Biological Resource Bank of Guangzhou Women and Children’s Medical Center for providing all the clinical samples. We are grateful to all patients and volunteers for donating specimens. Conflicts of Interest Statement The authors report no conflicts of interest. Data Availability The data used to support the findings of this study are available from the corresponding author upon request. E-mail: [email protected] . Author contributions All authors contributed significantly to this work. L.Y.F, D.T.M, and Q.G designed the study, performed most of the experiments, analyzed data, interpreted results, and wrote the manuscript. H.Y.Y performed Flow cytometer experiments, analyzed data, and interpreted results. D.C and C.N.C performed immunofluorescence and confocal microscopic analyses. L.P, Y.F.X, H.Z.Z, L.Z, Z.Y.J and W.Z.P provided project resources. L.Y.F, K.N.C performed immunoprecipitation experiments, analyzed data, and interpreted results and Picture editing. E.C and H.Y.Y performed article revision and language polish. G.X.Q and H.Y.N designed and supervised the study, interpreted results, and wrote the manuscript. All authors reviewed the manuscript. In addition, all authors have read and approved the manuscript. Ethics statement This study was approved by the Medical Ethics Committee of Guangzhou Women and Children’s Medical Center (2014073009 and 2018052105) and was conducted according to the International Ethical Guidelines for Research Involving Human Subjects stated in the Declaration of Helsinki. Informed written consent was obtained from the guardians of the patients and controls. References McCrindle BW, Rowley AH, Newburger JW, et al. Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association. Circulation. 2017;135(17):e927-e999. Newburger JW, Takahashi M, Burns JC. Kawasaki Disease. J Am Coll Cardiol. 2016;67(14):1738–1749. Miura M, Kobayashi T, Kaneko T, et al. Association of Severity of Coronary Artery Aneurysms in Patients With Kawasaki Disease and Risk of Later Coronary Events. JAMA Pediatr. 2018;172(5):e180030. Fukazawa R, Kobayashi T, Mikami M, et al. Nationwide Survey of Patients With Giant Coronary Aneurysm Secondary to Kawasaki Disease 1999–2010 in Japan. Circ J. 2017;82(1):239–246. Pi L, Che D, Long H, et al. Immature platelets and antiplatelet therapy response to aspirin in Kawasaki disease. Drug Des Devel Ther. 2018;12:1353–1362. Zhang Y, Wang Y, Zhang L, et al. Reduced Platelet miR-223 Induction in Kawasaki Disease Leads to Severe Coronary Artery Pathology Through a miR-223/PDGFRbeta Vascular Smooth Muscle Cell Axis. Circ Res. 2020;127(7):855–873. Ishikawa T, Iwashima S. Endothelial dysfunction in children within 5 years after onset of Kawasaki disease. J Pediatr. 2013;163(4):1117–1121. Arora K, Guleria S, Jindal AK, Rawat A, Singh S. Platelets in Kawasaki disease: Is this only a numbers game or something beyond? Genes Dis. 2020;7(1):62–66. Rinder HM, Schuster JE, Rinder CS, Wang C, Schweidler HJ, Smith BR. Correlation of thrombosis with increased platelet turnover in thrombocytosis. Blood. 1998;91(4):1288–1294. Straface E, Gambardella L, Metere A, et al. Oxidative stress and defective platelet apoptosis in naive patients with Kawasaki disease. Biochem Biophys Res Commun. 2010;392(3):426–430. Pietraforte D, Gambardella L, Marchesi A, et al. Platelets in Kawasaki patients: two different populations with different mitochondrial functions. Int J Cardiol. 2014;172(2):526–528. Jin J, Wang J, Lu Y, et al. Platelet-Derived Microparticles: A New Index of Monitoring Platelet Activation and Inflammation in Kawasaki Disease. Indian J Pediatr. 2019;86(3):250–255. Xu XR, Carrim N, Neves MA, et al. Platelets and platelet adhesion molecules: novel mechanisms of thrombosis and anti-thrombotic therapies. Thromb J. 2016;14(Suppl 1):29. Rowley AH, Shulman ST. Pathogenesis and management of Kawasaki disease. Expert Rev Anti Infect Ther. 2010;8(2):197–203. Shulman ST, Rowley AH. Kawasaki disease: insights into pathogenesis and approaches to treatment. Nat Rev Rheumatol. 2015;11(8):475–482. Mussbacher M, Salzmann M, Brostjan C, et al. Cell Type-Specific Roles of NF-kappaB Linking Inflammation and Thrombosis. Front Immunol. 2019;10:85. Rawish E, Nording H, Munte T, Langer HF. Platelets as Mediators of Neuroinflammation and Thrombosis. Front Immunol. 2020;11:548631. Allakhverdi Z, Comeau MR, Jessup HK, et al. Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells. J Exp Med. 2007;204(2):253–258. Lin SC, Huang JJ, Wang JY, Chuang HC, Chiang BL, Ye YL. Upregulated thymic stromal lymphopoietin receptor expression in children with asthma. Eur J Clin Invest. 2016;46(6):511–519. Wang WW, Pan YL, Yu HW, Zhang B, Shao SW. Histamine H4 receptor regulates Th2-cytokine profile through thymic stromal lymphopoietin in allergic rhinitis. Eur Arch Otorhinolaryngol. 2019. Savino AM, Izraeli S. On mice and humans: the role of thymic stromal lymphopoietin in human B-cell development and leukemia. Haematologica. 2016;101(4):391–393. Yu K, Dong Q, Mao X, et al. Disruption of the TSLP-TSLPR-LAP signaling between epithelial and dendritic cells through hyperlipidemia contributes to regulatory T-Cell defects in atherosclerotic mice. Atherosclerosis. 2015;238(2):278–288. Dong J, Lin J, Wang B, et al. Inflammatory cytokine TSLP stimulates platelet secretion and potentiates platelet aggregation via a TSLPR-dependent PI3K/Akt signaling pathway. Cell Physiol Biochem. 2015;35(1):160–174. Mao Y, Peng Y, Zeng Q, et al. A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor. PLoS One. 2015;10(10):e0141464. Feng W, Chang C, Luo D, et al. Dissection of autophagy in human platelets. Autophagy. 2014;10(4):642–651. Ouseph MM, Huang Y, Banerjee M, et al. Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis. Blood. 2015;126(10):1224–1233. Zhou H, Li D, Zhu P, et al. Melatonin suppresses platelet activation and function against cardiac ischemia/reperfusion injury via PPARgamma/FUNDC1/mitophagy pathways. J Pineal Res. 2017;63(4). Li BX, Dai X, Xu XR, et al. In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbalpha. Sci Rep. 2021;11(1):11663. Zeng Z, Xia L, Fan X, et al. Platelet-derived miR-223 promotes a phenotypic switch in arterial injury repair. J Clin Invest. 2019;129(3):1372–1386. Xu XR, Wang Y, Adili R, et al. Apolipoprotein A-IV binds alphaIIbbeta3 integrin and inhibits thrombosis. Nat Commun. 2018;9(1):3608. Yang H, Lang S, Zhai Z, et al. Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression. Blood. 2009;114(2):425–436. Gyulkhandanyan AV, Allen DJ, Mykhaylov S, et al. Mitochondrial Inner Membrane Depolarization as a Marker of Platelet Apoptosis: Disclosure of Nonapoptotic Membrane Depolarization. Clin Appl Thromb Hemost. 2017;23(2):139–147. Leytin V, Mykhaylov S, Starkey AF, et al. Intravenous immunoglobulin inhibits anti-glycoprotein IIb-induced platelet apoptosis in a murine model of immune thrombocytopenia. Br J Haematol. 2006;133(1):78–82. Li C, Piran S, Chen P, et al. The maternal immune response to fetal platelet GPIbalpha causes frequent miscarriage in mice that can be prevented by intravenous IgG and anti-FcRn therapies. J Clin Invest. 2011;121(11):4537–4547. Wang K, Gong Q, Zhan Y, et al. Blockage of Autophagic Flux and Induction of Mitochondria Fragmentation by Paroxetine Hydrochloride in Lung Cancer Cells Promotes Apoptosis via the ROS-MAPK Pathway. Front Cell Dev Biol. 2019;7:397. Li S, Fu L, Tian T, et al. Disrupting SOD1 activity inhibits cell growth and enhances lipid accumulation in nasopharyngeal carcinoma. Cell Commun Signal. 2018;16(1):28. Li L, Yao YC, Gu XQ, et al. Plasminogen kringle 5 induces endothelial cell apoptosis by triggering a voltage-dependent anion channel 1 (VDAC1) positive feedback loop. J Biol Chem. 2014;289(47):32628–32638. Aleman MM, Byrnes JR, Wang JG, et al. Factor XIII activity mediates red blood cell retention in venous thrombi. J Clin Invest. 2014;124(8):3590–3600. Zhang Z, Shen C, Fang M, et al. Novel contact-kinin inhibitor sylvestin targets thromboinflammation and ameliorates ischemic stroke. Cell Mol Life Sci. 2022;79(5):240. Davizon-Castillo P, McMahon B, Aguila S, et al. TNF-alpha-driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging. Blood. 2019;134(9):727–740. Sliter DA, Martinez J, Hao L, et al. Parkin and PINK1 mitigate STING-induced inflammation. Nature. 2018;561(7722):258–262. Lin Q, Li S, Jiang N, et al. PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation. Redox Biol. 2019;26:101254. Quinn PMJ, Moreira PI, Ambrosio AF, Alves CH. PINK1/PARKIN signalling in neurodegeneration and neuroinflammation. Acta Neuropathol Commun. 2020;8(1):189. Kitajima M, Lee HC, Nakayama T, Ziegler SF. TSLP enhances the function of helper type 2 cells. Eur J Immunol. 2011;41(7):1862–1871. Geisler S, Holmstrom KM, Skujat D, et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol. 2010;12(2):119–131. Ni H, Denis CV, Subbarao S, et al. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest. 2000;106(3):385–392. MacKeigan DT, Ni T, Shen C, et al. Updated Understanding of Platelets in Thrombosis and Hemostasis: The Roles of Integrin PSI Domains and their Potential as Therapeutic Targets. Cardiovasc Hematol Disord Drug Targets. 2020;20(4):260–273. Wang Y, Gallant RC, Ni H. Extracellular matrix proteins in the regulation of thrombus formation. Curr Opin Hematol. 2016;23(3):280–287. Roberts HR, Hoffman M, Monroe DM. A cell-based model of thrombin generation. Semin Thromb Hemost. 2006;32 Suppl 1:32–38. Yu X, Peng Y, Liang H, et al. TSLP/TSLPR promote angiogenesis following ischemic stroke via activation of the PI3K/AKT pathway. Mol Med Rep. 2018;17(2):3411–3417. Barazzuol L, Giamogante F, Brini M, Cali T. PINK1/Parkin Mediated Mitophagy, Ca(2+) Signalling, and ER-Mitochondria Contacts in Parkinson's Disease. Int J Mol Sci. 2020;21(5). Ham SJ, Lee D, Yoo H, Jun K, Shin H, Chung J. Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination. Proc Natl Acad Sci U S A. 2020;117(8):4281–4291. Corrigan JJ, Jr. Kawasaki disease and the plight of the platelet. Am J Dis Child. 1986;140(12):1223–1224. Semple JW, Italiano JE, Jr., Freedman J. Platelets and the immune continuum. Nat Rev Immunol. 2011;11(4):264–274. Ueno K, Nomura Y, Morita Y, Eguchi T, Masuda K, Kawano Y. Circulating platelet-neutrophil aggregates play a significant role in Kawasaki disease. Circ J. 2015;79(6):1349–1356. Vignesh P, Rawat A, Shandilya JK, Singh Sachdeva MU, Ahluwalia J, Singh S. Monocyte platelet aggregates in children with Kawasaki disease- a preliminary study from a tertiary care centre in North-West India. Pediatr Rheumatol Online J. 2021;19(1):25. Han NR, Moon PD, Nam SY, et al. TSLP up-regulates inflammatory responses through induction of autophagy in T cells. FASEB J. 2022;36(2):e22148. Leytin V. Apoptosis in the anucleate platelet. Blood Rev. 2012;26(2):51–63. Takai T. TSLP expression: cellular sources, triggers, and regulatory mechanisms. Allergol Int. 2012;61(1):3–17. Truchetet ME, Demoures B, Eduardo Guimaraes J, et al. Platelets Induce Thymic Stromal Lymphopoietin Production by Endothelial Cells: Contribution to Fibrosis in Human Systemic Sclerosis. Arthritis Rheumatol. 2016;68(11):2784–2794. Cianferoni A, Spergel J. The importance of TSLP in allergic disease and its role as a potential therapeutic target. Expert Rev Clin Immunol. 2014;10(11):1463–1474. Sugitani Y, Furuno K, Sueishi K, Hara T. Macrophages and cytotoxic T cells infiltrate the destructed mitral tissue in Kawasaki disease. BMJ Case Rep . 2018;2018. Nakajima S, Kabata H, Kabashima K, Asano K. Anti-TSLP antibodies: Targeting a master regulator of type 2 immune responses. Allergol Int. 2020;69(2):197–203. Additional Declarations No competing interests reported. Supplementary Files SupplementalMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted 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-1628519","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":104437553,"identity":"6db452d6-cac8-4d5f-bb70-7ecf59e88e2f","order_by":0,"name":"Lanyan Fu","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lanyan","middleName":"","lastName":"Fu","suffix":""},{"id":104437554,"identity":"563fc634-5e61-48f7-bb84-2b261502e61d","order_by":1,"name":"Daniel Thomas MacKeigan","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"Thomas","lastName":"MacKeigan","suffix":""},{"id":104437555,"identity":"1b7c3043-1e9d-482b-b132-d4dbbf599da1","order_by":2,"name":"Qing Gong","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Gong","suffix":""},{"id":104437556,"identity":"3f814870-e417-4915-b159-f041c8429b9e","order_by":3,"name":"Di Che","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Che","suffix":""},{"id":104437557,"identity":"80579305-4df0-4b30-8c29-da06b178121f","order_by":4,"name":"Yufen Xu","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yufen","middleName":"","lastName":"Xu","suffix":""},{"id":104437558,"identity":"3c3a8552-8cef-4a4f-8b58-c94e4c5af3c1","order_by":5,"name":"Lei Pi","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Pi","suffix":""},{"id":104437559,"identity":"3a39529a-897b-4bb8-8340-28bdf8b4ade1","order_by":6,"name":"Chaonan Sun","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chaonan","middleName":"","lastName":"Sun","suffix":""},{"id":104437560,"identity":"c12e7d0d-9835-4d92-aa56-0dfb04f1282b","order_by":7,"name":"Hongyan Yu","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Yu","suffix":""},{"id":104437561,"identity":"8a4d4d5f-0487-4724-b54f-177149ed1cad","order_by":8,"name":"Kaining Chen","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kaining","middleName":"","lastName":"Chen","suffix":""},{"id":104437562,"identity":"85515029-be7e-41d4-aaf7-28dbf78140d2","order_by":9,"name":"Huazhong Zhou","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huazhong","middleName":"","lastName":"Zhou","suffix":""},{"id":104437563,"identity":"4134efe4-64eb-46e1-bf6f-1ac7589ed2fe","order_by":10,"name":"Zhiyong Jiang","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhiyong","middleName":"","lastName":"Jiang","suffix":""},{"id":104437564,"identity":"0d036303-27be-4055-a3c4-966f3ec59439","order_by":11,"name":"Zhouping Wang","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhouping","middleName":"","lastName":"Wang","suffix":""},{"id":104437565,"identity":"f10f785a-9a7e-40a5-b8ef-10c1a7c99c2b","order_by":12,"name":"Li Zhang","email":"","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhang","suffix":""},{"id":104437566,"identity":"5a3f803d-dcf8-491e-9f22-a7454f5e82f7","order_by":13,"name":"Eric G. Cerenzia","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eric","middleName":"G.","lastName":"Cerenzia","suffix":""},{"id":104437567,"identity":"0598d1b3-32e3-4308-be06-8693b5aec282","order_by":14,"name":"Heyu Ni","email":"","orcid":"","institution":"University of Toronto, Canadian Blood Services Centre for Innovation, St. Michael's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Heyu","middleName":"","lastName":"Ni","suffix":""},{"id":104437568,"identity":"db841d44-f69e-48bc-8ab6-8c2cda760bf7","order_by":15,"name":"Xiaoqiong Gu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYJCCA0DMA8SMD0jWwmxAsm1sEkQpk4/IMTzwo8Jahl+6/Vrl1za7aP4G5mMfv+DRYngjLeFgz5l0Hsk5Z8puy7Yl5844wJY8WwaflhnJBw7wth3mMbiRk3ZbctuB3A0MPMbM+JxoOCOx4eBfqJZiorTISyQfOAyxJf0Y40eoFsYPeLQY8DxLOCwD8suMHGZpxn9AvxxmS2bGo4NBvj3H+OObCmt7fon0hx9/nLHL7W9vPsz4A58tB8AUyFgeA2YeKBvCwGVLA1wL+wO44XhtGQWjYBSMghEHAATmUIUsg2qfAAAAAElFTkSuQmCC","orcid":"","institution":"Guangzhou Women and Children’s Medical Center, Guangzhou Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaoqiong","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2022-05-06 06:59:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1628519/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1628519/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":21396226,"identity":"d6898621-0ecc-4d3d-a15f-ec093ae6ee8c","added_by":"auto","created_at":"2022-05-12 15:25:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":105848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlatelet activation makers were elevated in KD patients with thrombosis. A:\u003c/strong\u003e Flow cytometry was used to detect platelet activation marker CD62p and to analyze the percentage of CD62p positive platelets in HC (n=8) vs KD (n=22) (HC vs KD\u003cem\u003e \u003c/em\u003e***\u003cem\u003eP \u003c/em\u003e=0.0004). \u003cstrong\u003eB-C:\u003c/strong\u003e ELISA detection of soluble P-selectin (sP-selectin) and soluble CD40L (sCD40L), respectively in the plasma of acute(Before IVIG n=8 and After IVIG n=23) and convalescent phase (n=75) patients compared with HCs (n=36) (HC vs KD/Convalescence\u003cem\u003e \u003c/em\u003e**\u003cem\u003eP\u003c/em\u003e=0.004 and****\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001; After IVIG vs KD/Convalescence **\u003cem\u003eP\u003c/em\u003e=0.007 and****\u003cem\u003eP\u003c/em\u003e =0.0001 ) , \u003cstrong\u003eD-E: \u003c/strong\u003eELISA detection of sP-selectin and sCD40L in the plasma of convalescent patients with or without thrombosis compared to healthy controls (HC vs KD/No thrombosis (n=60),\u003cem\u003e \u003c/em\u003e*\u003cem\u003eP \u003c/em\u003e=0.048 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001; KD/No thrombosis vs KD/Thrombosis (n=15) *\u003cem\u003eP \u003c/em\u003e=0.018 and **\u003cem\u003eP\u003c/em\u003e =0.001). Notes: HC: healthy controls; KD: Kawasaki disease; KD/Thrombosis: Convalescent KD patients with thrombosis; KD/No thrombosis: Convalescent KD patients without thrombosis. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001 using t tests (nonparametric tests) and one-way ANOVA (multiple comparisons) test to analyze in datasets with three groups. The graph represents data from at least three replicates. All data are presented as mean ± SD.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1628519/v1/fabdbd4b9d023da20ea6f318.png"},{"id":21396232,"identity":"c972e178-e5ef-4faf-af83-ea9b754cb4fb","added_by":"auto","created_at":"2022-05-12 15:25:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired mitochondrial function in KD patients. A: \u003c/strong\u003eTMRM positive platelets (Ψm) were measured by flow cytometry in HC (n =5) or KD (n = 10) with CCCP (5µL) as a positive control for mitophagy (HC vs KD **\u003cem\u003eP \u003c/em\u003e=0.006). \u003cstrong\u003eB: \u003c/strong\u003eWestern blot analysis of the expression of apoptosis-related proteins in platelets of each group. Quantification of HC (n=4) and KD with thrombosis (n=6) and without thrombosis (n=6) in platelets.\u003cstrong\u003e C:\u003c/strong\u003e Flow cytometry analysis and quantification showing the percentage of Annexin-V positive platelets in HC (n = 9) and KD (n = 14) patient (HC vs KD ***\u003cem\u003eP\u003c/em\u003e=0.001). *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001 using t tests (nonparametric tests). Notes: ns: not significant. The graph represents data from at least three replicates. All data are presented as mean ± SD. \u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1628519/v1/b8112ddbf93a37c1ecc3ec29.png"},{"id":21396227,"identity":"c844e856-845c-4537-8ee2-bf122c5598aa","added_by":"auto","created_at":"2022-05-12 15:25:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":227869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlatelet autophagy in KD was greater in those with thrombosis compared to those without thrombosis. A:\u003c/strong\u003e Confocal microscopy was used to observe the fluorescence intensity of the autophagy marker LC3 in KD patients (n=4) compared with HCs (n=4) (HC vs KD *\u003cem\u003eP \u003c/em\u003e=0.023). \u003cstrong\u003eB:\u003c/strong\u003e Morphological alterations in platelets (KD and HC group n=3) were detected by transmission electron microscopy (the red arrow indicates autophagosomes) (HC vs KD **\u003cem\u003eP \u003c/em\u003e=0.001). \u003cstrong\u003eC:\u003c/strong\u003e Western blot analysis of the expression of autophagy-related proteins in platelets of each group, and quantification of the expression of HC (n=4) and KD with thrombosis (n=6) and without thrombosis (n=6) in platelets. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001 using t tests (nonparametric tests) and one-way ANOVA (multiple comparisons) to analyze datasets with three groups. Actin served as a loading control. The graph represents data from at least three replicates. All data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1628519/v1/11e05207d8ad1b45e9d2b3a7.png"},{"id":21396233,"identity":"055d194c-e75a-4efb-8659-8abd2a616392","added_by":"auto","created_at":"2022-05-12 15:25:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of Plasma TSLP and platelet TSLP receptors increased in KD patients complicated with thrombosis.\u003c/strong\u003e \u003cstrong\u003eA: \u003c/strong\u003eHeatmap of 34 differentially expressed inflammatory factors in plasma from healthy control (HC) (n=20), Febrile illness (n=30), KD patients in acute (n=74), subacute (n=47) and convalescence (n=32).\u003cstrong\u003eB: \u003c/strong\u003eProtein chip assay was used to detect plasma TSLP inflammatory factors of KD patients in the acute (Before IVIG n=35 and After IVIG n=39 ), subacute and convalescent (n=32) patients relative to HCs (n=20) (all****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). \u003cstrong\u003eC:\u003c/strong\u003e KD patients were divided into four groups, small CAA (SCAA), medium CAA (MCAA)and giant CAA (GCAA) combined thrombosis and normal coronary artery (NCAL) without thrombosis according to coronary artery Z-worst. The dashed red circle is a coronary aneurysm, and the position indicated by the arrow is the coronary artery. Representative echocardiogram images of corresponding patients with KD/Thrombosis and KD/No thrombosis were shown. \u003cstrong\u003eD:\u003c/strong\u003e Verified the expression of TSLP in the plasma of another group of KD patients by ELISA in the acute (Before IVIG n=22 and After IVIG n=39) and convalescent (n=95) patients relative to HCs (n=63) (all****P \u0026lt; 0.0001), Convalescent KD patients with thrombosis (n=38) compared with without thrombosis (n=57) (KD/No thrombosis vs KD/Thrombosis **\u003cem\u003eP\u003c/em\u003e =0.005).\u003cstrong\u003eE:\u003c/strong\u003e Western blot analysis and quantification of TSLP receptor (TSLPR and IL-7R) in platelets of HCs (n=4), KD with thrombosis (n=6), and KD without thrombosis (n=6).\u003cem\u003e \u003c/em\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 using t tests (and nonparametric tests). Notes: HC: healthy control, KD/Thrombosis: Convalescent KD patients with thrombosis; KD/No thrombosis: Convalescent KD patients without thrombosis. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001 using one-way ANOVA (multiple comparisons) to analyze in datasets with three groups. The graph represents data from at least three replicates. All data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1628519/v1/ee199cfbd90f733b3f03cf2a.png"},{"id":21396905,"identity":"3150a143-cd9b-4886-b41e-850a343ff346","added_by":"auto","created_at":"2022-05-12 15:30:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":318446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP promotes thrombus formation in vitro by promoting mitochondrial autophagy and activates platelets. A: \u003c/strong\u003eHuman platelets were stimulated with TSLP (500ng/mL) and the platelet activation marker CD62p was detected by flow cytometry (control vs TSLP *\u003cem\u003eP\u003c/em\u003e=0.014).\u003cstrong\u003eB: \u003c/strong\u003eHuman platelet-rich plasma (PRP) was stimulated with different concentrations of TSLP (100, 200 and 500ng/mL) for 3h while clot retraction was periodically photographed. Thrombin (1.5 NIH unit/mL)-treated PRP served as positive control\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eC: \u003c/strong\u003eHuman platelets were treated with TSLP, and the fluorescence intensity of autophagy marker LC3 protein was observed under confocal microscopy (control vs TSLP **\u003cem\u003eP\u003c/em\u003e=0.006). \u003cstrong\u003eD:\u003c/strong\u003e Western blot analysis and quantification of the expression of mitophagy and apoptosis-related proteins in platelets after TSLP (200ng/ mL and 500ng/ mL) treatment for 3h. \u003cstrong\u003eE: \u003c/strong\u003eThrombosis assay was compared after pretreatment with an autophagy agonist (CCCP 10μm) or inhibitor (Mdivi-1 10μm) in combination with TSLP or with TSLP (500ng/mL).*\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001 using t tests to analyze two independent experiments (nonparametric tests) and one-way ANOVA to analyze in datasets with three or more groups. The graph represents data from at least three replicates. All data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1628519/v1/7191326cdc22d8fb663731aa.png"},{"id":21396231,"identity":"1846d0e7-b41d-4fc9-8c9f-c59a755eb5fa","added_by":"auto","created_at":"2022-05-12 15:25:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":222208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLPR binds with Parkin and VDAC1 in platelet mitochondria to regulate mitophagy induced by TSLP. A:\u003c/strong\u003e Western blot detection and quantification of the expression of TSLP receptors (TSLPR and IL-7R) in platelets treated with TSLP (200ng/mL and 500ng/mL) for 3h. \u003cstrong\u003eB: \u003c/strong\u003eHuman platelets were treated with Baf-A1 (400nM) for 1h, and then TSLP (500ng/mL) for 3h. The expression of mitophagy-associated proteins in platelets was detected and quantified by Western blot. \u003cstrong\u003eC-D:\u003c/strong\u003e Human platelets were treated with TSLP (500ng/mL) for 3h, and prepared for immunoprecipitation of Parkin and VDAC1, respectively. \u003cstrong\u003eE: \u003c/strong\u003eHuman platelets were treated with Baf-A1 (400nM) for 1h, then treated with TSLP (500ng/mL) for 3h, and prepared for immunoprecipitation of TSLPR. \u003cstrong\u003eF: \u003c/strong\u003eHuman platelets were treated with Baf-A1 (400nM) for 1h, then treated with TSLP (500ng/mL) for 3h, stained with Mitotracker and TSLPR antibody, then imaged by confocal microscopy. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 using one-way ANOVA to analyze in datasets with three or more groups. The graph represents data form at least three replicates. All data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-1628519/v1/82eb3d9e93580015b97c266d.png"},{"id":21396907,"identity":"82e20619-71ca-4a61-a0dc-a5c8c78fa2c6","added_by":"auto","created_at":"2022-05-12 15:30:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1793946,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1628519/v1/19ac9498-b981-4ea9-8fd2-79ae5cfc8b4e.pdf"},{"id":21396906,"identity":"bbf8a295-5cd9-40c4-8acb-9e3e3523dab8","added_by":"auto","created_at":"2022-05-12 15:30:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":178621,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-1628519/v1/9b181b4ed65db9b4ef98d9dd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"TSLP induces platelet mitophagy and promotes thrombosis in Kawasaki disease ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is a common pediatric vasculitis of medium and small muscular arteries that affects mainly children under 5 years old\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The main cause of death in KD is heart attack and stroke, which is often caused by occlusive thrombosis precipitated by coronary artery aneurysm (CAA)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The risk of thrombosis persists up to years following initial onset and treatment. Indeed, our group and several others have reported elevated platelet activation in KD patients\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, which may serve as a risk factor for thrombosis in KD patients with pre-existing CAA\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The pathogenesis of platelet activation and thrombosis in KD remains elusive, although increased thrombocytosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, disordered platelet function\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and increased platelet-derived microparticles\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e have been reported. It is imperative to uncover its pathophysiology, particularly the mechanism that causes platelet activation, to identify novel therapeutic targets against KD-associated thrombosis.\u003c/p\u003e \u003cp\u003eThree traditional antiplatelet pathways\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e are currently used for the clinical treatment of KD: thromboxane A2 pathway (such as aspirin), ADP pathway (such as clopidogrel), and platelet receptor pathway (such as abciximab)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For KD patients with coronary artery aneurysm and thrombosis, combined treatment of antiplatelet and thrombolysis or anticoagulation therapy is usually used\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, these processes cannot completely inhibit platelet activation and thrombosis, indicating that there may be other underlying platelet activation mechanisms regulated by unknown factors. Studies have reported that cytokines contribute to inflammation and thrombotic responses\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, whether plasma cytokines contribute to platelet activation and thrombosis in KD is unknown.\u003c/p\u003e \u003cp\u003eThymic stromal lymphopoietin (TSLP) is an IL-7 like cytokine that is mainly derived from epithelial cells, fibroblasts and mast cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence implicates the dysregulated expression of TSLP in multiple diseases such as asthma, allergic rhinitis, leukemia and atherosclerosis\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Recently, the roles of TSLP and TSLPR on platelets have been linked to platelet activation and thrombus formation through PI3K/Akt signaling\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, the role of TSLP in aberrant platelet activation and thrombosis in KD has not been previously reported.\u003c/p\u003e \u003cp\u003ePlatelet autophagy is involved in the regulation of platelet activity\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Not surprisingly, mitophagy, a selective autophagy that regulates mitochondrial quality also occurs in platelets and is emerging as an important regulatory mechanism in platelets. Since the mitochondrion is essential for energy production, it is no surprise that enhanced mitophagy is linked with platelet autophagy and activation. Recent reports\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e demonstrate platelet mitophagy promotes thrombosis, however, the role of platelet mitophagy in KD platelet activation and thrombosis has never been explored.\u003c/p\u003e \u003cp\u003eIn the present study, we discovered that TSLP is significantly upregulated in KD and promotes platelet mitophagy and thrombosis via a novel TSLPR/Parkin/VDAC1-dependent signaling pathway. Our findings further elucidate the mechanism of thrombosis in KD and identify TSLP as a potential novel anti-thrombotic therapeutic target.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy objects\u003c/h2\u003e \u003cp\u003eSamples of KD patients and age-matched healthy controls (HC) were collected from the Guangzhou Women and Children Medical Center in China, between July 2016 and March 2021(Supplemental Materials Table I). All specimens were stored in the clinical biological resource bank (Clinical Bio-bank) of this hospital. KD patients were diagnosed by our hospital cardiology physician, according to the latest version of the American Heart Association's 2017 revised diagnostic criteria and treatment guidelines\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Coronary artery abnormalities of KD patients were categorized according to their Z score: no coronary artery damage(Z\u0026thinsp;\u0026lt;\u0026thinsp;2 mm), coronary artery dilation(Z\u0026thinsp;\u0026gt;\u0026thinsp;2\u0026thinsp;~\u0026thinsp;\u0026lt;\u0026thinsp;2.5mm), large CAA (\u0026ge;\u0026thinsp;8.0 mm or Z\u0026thinsp;\u0026ge;\u0026thinsp;10 mm), medium CAA (\u0026lt;\u0026thinsp;8.0 mm and Z\u0026thinsp;\u0026ge;\u0026thinsp;5\u0026thinsp;~\u0026thinsp;\u0026lt;\u0026thinsp;10 mm), and small CAA (Z\u0026thinsp;\u0026ge;\u0026thinsp;2.5\u0026thinsp;~\u0026thinsp;\u0026lt;\u0026thinsp;5 mm). This study was approved by the Ethics Committee of Guangzhou Women and Children's Medical Center (Number: 2014073009 and 2018052105). All participants\u0026rsquo; parents/guardians gave written informed consent in accordance with the Declaration of Helsinki. We categorized blood samples from KD patient samples into either of three stages, the acute phase (patients before Intravenous IgG (IVIG) and after IVIG treatment within 10 days), subacute phase (after treatment 11 days-1 mouth) and convalescence phase (Aspirin or other antiplatelet drug treatment more than 1\u0026ndash;6 mouths).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of human platelets\u003c/h2\u003e \u003cp\u003eVenous blood was drawn from healthy controls and KD patients then collect with sodium citrate or ACD (2-4mL) anticoagulant tube, and pretreated with 75nM prostaglandin E1 (PGE1; Catalog No. : HY-B0131, MEC) to prevent platelet activation. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 900 rpm at 22˚C for 10mins, platelet-poor plasma (PPP) was obtained by centrifugation of PRP at 3500 rpm at 22˚C for 10mins, and platelet pellets at the bottom of the tube\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Platelet counts in PRP were performed with an automatic blood cell analyzer (sysmex XS-500i). To prepare washed platelets, platelet pellets were gently washed twice with CGS buffer (0.123 M NaCl, 0.033 M D-glucose, 0.013 M trisodium citrate, pH 6.5) containing 75nM PGE1, then the washed platelets were suspended in modified Tyrode\u0026rsquo;s buffer (MTB; 2.5 mM Hepes, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 5.5 mM D-glucose, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 7.4) containing 75nM PGE1 at 3\u0026ndash;5 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e platelets/mL, and incubated at room temperature for 1 h before use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRegents and antibodies\u003c/h2\u003e \u003cp\u003eBafilomycin A1 (Baf-A1), mitochondrial division inhibitor-1 (Mdivi-1) and mitophagy agonist Carbonyl cyanide m-chlorophenyl hydrazone, Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were purchased from Selleckchem. Prostaglandin E1 (PGE1) was purchased from MCE. Recombinant human TSLP was purchased from Beyotime and peprotech. CaCl\u003csub\u003e2\u003c/sub\u003e (0.025M) and thrombin were purchased from Diagnostica Stago. Antibodies of β-actin, TSLP, LC3A/B, Bcl-xL, Bax, IgG, and Caspase-3 were purchased from Cell Signaling Technology. TSLPR, Parkin, and PINK1 antibodies were obtained from Abcam. Antibodies of TSLP receptors (TSLPR and IL-7R), VDAC1, Tom20 and protein A/G PLUS-Agarose immunoprecipitation reagents were purchased from Santa Cruz Biotechnology. The anti-CD41a and anti-CD62p were purchased from eBioscience. Human TSLP ELISA kit was purchased from R\u0026amp;D Systems, soluble CD40L (sCD40L) and soluble P-selectin (sP-selectin) ELISA kits were purchased from MultiSciences. MitoTracker antibody and MitoProbe\u0026trade; TMRM Assay Kit were purchased from Thermofisher. Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Keygen Biotech.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMultiplex Analysis and Quantification of Cytokines\u003c/h2\u003e \u003cp\u003eThe Bio-Plex Pro Human Inflammation Group 34-Plex 171AL001M (Bio-Rad, Hercules, CA) kit was used to detect the cytokine content in plasma samples selecting by \u0026ldquo;Clinical Bio-bank\u0026rdquo; from KD patients and healthy control. The protocol was followed according to the manufacturer\u0026rsquo;s instructions. The Bio-Plex Verification Kit Version 3.0 (Bio-plex200, Bio-Rad, Hercules, CA) was used to verify the hydrodynamic performance, consistent optical alignment, double identification and identification of single bead signatures. The measurement was performed using the Bio-Plex protein array system integrated with Bio-Plex Manager software version 3.0 (Bio-Rad, Hercules, CA).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eELISA was used to detect plasma concentrations of TSLP (R\u0026amp;D Systems), sCD40L and sP-selectin (MultiSciences). ELISA experiments were carried out according to the manufacturer's protocol as we previously described\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Briefly, PPP samples (40\u0026ndash;100\u0026micro;L) were added into each well of a microtiter plate followed by 10\u0026ndash;100\u0026micro;L of the detection antibody. Following 1-2h incubation at 37˚C, 50\u0026ndash;100\u0026micro;L of streptavidin-HRP was added to each well and allowed to incubate for 30-60mins. The wells were subsequently washed and 50\u0026ndash;100\u0026micro;L color reagent A and 50\u0026ndash;100\u0026micro;L color Reagent B were added, gently vortexed, and incubated at 37˚C for 10 minutes in the dark. The reaction was quenched by adding 50\u0026micro;L Reagent C and plates were read at 450 nm with a Variskan Flash (Thermo Scientific).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of platelet activation, mitochondrial membrane potential (ΔΨm), PS exposure by flow cytometry\u003c/h2\u003e \u003cp\u003eTo assess platelet activation, PRP was prepared from human whole blood as described above. PRP (3\u0026ndash;5\u0026micro;L) was labelled with-anti-CD41a and anti-CD62p, incubated at room temperature for 15-30mins. The reaction was stopped by adding PBS, and then analyzed by flow cytometry analysis (BD FACScanto II). Platelets were identified and gated by their characteristic forward and side scatter properties. A total of 10,000 platelets were measured from each sample as we previously described\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePlatelets were pelleted from PRP and subsequently resuspended in MTB adjusted to a concentration of 3\u0026ndash;5\u0026times;10\u003csup\u003e8\u003c/sup\u003e platelets/mL. Platelets were incubated with 1\u0026micro;M tetramethylrhodamine methyl ester (TMRM) at 37˚C for 30 mins. Platelets were subsequently incubated with 5\u0026micro;L of Annexin V at room temperature for 15 mins. Flow cytometry was then used to analyze ΔΨm and PS externalization\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Platelets were identified and gated by their characteristic forward and side scatter properties. A total of 10,000 platelets were analyzed from each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eObservation of autophagosomes by transmission electron microscopy\u003c/h2\u003e \u003cp\u003eThe platelet pellet was prepared as above described, then washed twice and fixed with 2.5% glutaraldehyde solution and 1% osmium tetroxide solution. Finally, the platelets were dehydrated with an ethanol solution then embedded and cut into ultrathin 100 nm sections. After staining with a saturated uranyl acetate solution and a lead acetate solution, the platelets were observed and photographed under a transmission electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence and confocal microscopy\u003c/h2\u003e \u003cp\u003ePRP was prepared from human whole blood as described above. MitoTracker\u0026reg; was incubated with platelets (100\u0026micro;L of PRP diluted in 1mL MTB) at room temperature for 15mins. Platelets were then pelleted by centrifugation at 3500 rpm for 10mins at 25˚C, resuspended in MTB (400\u0026micro;L) by centrifuged again at 7200 rpm for 3mins to adhere to the glass slide. Platelets were then fixed with 4% paraformaldehyde (100\u0026ndash;200\u0026micro;L) for 15mins, washed three times with PBS, permeabilized for 10mins in 0.25% Triton X-100, washed 3 times with PBS and then blocked (5% BSA, 0.1% Triton X-100 in PBS) for 30mins at room temperature. After the sealing solution was removed, the platelets were incubated with a primary antibody against LC3, TSLPR (1% BSA in PBS 1:200) at 4˚C overnight. Next, a fluorescent secondary antibody (1% BSA in PBS 1:1000) was added to platelets and incubated for 2h at room temperature in the dark, then washed 3 times with PBS. After platelets were sealed with anti-fluorescence quenching liquid, the stained platelets were observed using a Zeiss LSM 800 confocal microscope with 63x oil immersion lens and photographed as we previously described\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003e Platelets from patients and healthy volunteers were obtained by gradient centrifugation. Healthy volunteers PRP were treated with recombinant human TSLP (200 ng/mL or 500 ng/mL) for 3h, and then lysed with an equal volume of lysis buffer on ice for 30mins. The protein concentration was determined using a BCA protein assay kit (Applygen Beijing) according to the manufacturer\u0026rsquo;s protocol. Each sample was loaded in equal proportion to a 10%-12% SDS gel and separated via polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane. Either TSLP, TSLPR, Caspase-3, Bax, Bcl-xL, PINK1, Parkin, LC3A/B, Tom20, β-actin, VDAC1 or IL-7R (1:2000) antibody was incubated with the membrane at 4˚C overnight, then washed and incubated with the corresponding secondary antibody for 2h at room temperature. Antibody binding was detected with the ECL detection reagent by Amersham image 600 Software and the bands were quantified with Quantity Image J as we previously described\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eImmunoprecipitation analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003ePRP was pretreated with or without Baf-A1 (400nM for 1h) and then treated with recombinant human TSLP (500 ng/mL for 3h). Platelets were then lysed and mixed with the specific target antibody for Parkin IP (1:50 dilution); VDAC1 IP (1:30 dilution) or TSLPR IP (1:30 dilution), and the IgG control group as a negative control, and incubated for 1h at 4˚C. Then, 20\u0026micro;L of resuspended A/G PLUS-Agarose was added to each sample and incubated at 4˚C on a rocker platform overnight. Following this incubation, samples were pelleted and washed 4 times before being loaded on SDS-PAGE. The subsequent steps were according to the aforementioned western blot protocol\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro thrombosis and clot retraction assays\u003c/span\u003e \u003c/p\u003e \u003cp\u003e1) Whole blood thrombosis assay\u003c/p\u003e \u003cp\u003eGlass tubes were coated with Sigmacote\u0026reg; and dried overnight. In one experiment, 200\u0026micro;L aliquots of PRP were incubated with a gradient concentration of recombinant human TSLP (0, 100, 200 or 500ng/mL) in 37˚C for 3h using 1.5 NIH unit/mL thrombin as a positive control. In another experiment, 200\u0026micro;L aliquots of PRP were incubated with CCCP (10\u0026micro;M) or Mdivi-1 (10\u0026micro;M) treatment for 1h followed by incubation with recombinant human TSLP for 3h. PRP samples were then diluted with 200\u0026micro;L PBS, 50\u0026micro;L whole blood and 100\u0026micro;L CaCl\u003csub\u003e2\u003c/sub\u003e (0.025M) incubated at 37˚C with images photographed at 30min/1h/1h30m/2h\u003csup\u003e38\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e2) Platelet-rich plasma clot retraction assay\u003c/p\u003e \u003cp\u003eAs described for whole blood thrombosis without the addition of whole blood. Each condition received 200\u0026micro;L PRP, 200\u0026micro;L PBS and 100\u0026micro;L CaCl\u003csub\u003e2\u003c/sub\u003e (0.025M) and was left to clot in 37˚C, with clot retraction photographed over time\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll experiments were repeated at least 3 times, the data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Differences between the experimental groups were assessed by a two-tailed unpaired t-test, and in datasets with 3 or more groups was performed by one-way analysis of variance followed by Tukey's test Differences were analyzed by GraphPad Prism 7 with \u003cem\u003ep\u003c/em\u003e value less than 0.05 considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eKD patients with thrombosis showed increased platelet activation and apoptosis\u003c/h2\u003e \u003cp\u003eWe previously reported that plasma levels of soluble CD40L (sCD40L) and soluble P-selectin (sP-selectin) were elevated in KD patients, and positively correlates with the degree of coronary artery damage in Kawasaki disease patients\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In the present study, we examined plasma from another group of KD convalescent patients and found a significant increase in the platelet activation marker P-selectin (CD62p) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Similarly, sP-selectin and sCD40L were significantly elevated in the plasma of acute (patients before IVIG and after IVIG ) and convalescent patients relative to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e\u0026amp;C\u003c/b\u003e) and significantly elevated in convalescent patients complicated with thrombosis relative to those without (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e\u0026amp;E\u003c/b\u003e). These findings confirmed that baseline platelet activation is significantly greater in acute and convalescent KD patients, which is exacerbated in patients complicated with thrombosis.\u003c/p\u003e \u003cp\u003ePrevious reports have shown that platelet activation is associated with platelet mitochondrial dysfunction and control of the platelet life span\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. When dysfunction of platelet mitochondria occurs, the mitochondrial membrane potential will decrease, the apoptosis protein will change, PS valgus, and calcium ion changes, which will eventually lead to platelet apoptosis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Thus, we analyzed apoptotic signaling and mitochondrial dysfunction in platelets from KD patients. Using flow cytometry, we found a significant reduction in mitochondrial membrane potential (Δψ m) in platelets from KD patients relative to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blot analysis confirmed an upregulation in hallmark platelet apoptotic proteins including Bax, Caspase-3 and VDAC1, and the downregulation of the anti-apoptotic protein Bcl-xL. Notably, apoptotic markers were most enhanced in convalescent patients complicated with thrombosis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Consistent with these findings, flow cytometry analysis revealed a significant increase in the percentage of Annexin V-positive platelets in the KD convalescent patients compared to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Taken together, the above data suggests an elevation in both platelet activation and apoptosis in KD patients, especially in those complicated with thrombosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePlatelet autophagy is upregulated in KD\u003c/h2\u003e \u003cp\u003eSince we found increased apoptosis in KD patients, we next decided to investigate whether basal platelet autophagy was affected. Fluorescent confocal microscopy revealed that the platelet autophagy marker LC-3 was significantly increased in platelets from KD patients relative to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Electron microscopy also found an increase in the number of platelet autolysosomes in KD patient platelets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Consistent with these data, western blot analysis showed an increase in the autophagy proteins PINK1, Parkin, Tom20 and LC3A/B in KD patients, especially in patients with thrombosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Collectively, these data confirm platelet autophagy is upregulated in patients with KD and is closely related to KD-related thrombosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePlasma TSLP and platelet TSLP receptors were elevated in KD patients complicated with thrombosis\u003c/h2\u003e \u003cp\u003eGiven the inflammatory nature of KD, we suspected dysregulated cytokines may contribute to platelet activation. To investigate this, we utilized a protein chip assay to quantify the expression of 34 cytokines in plasma from KD patients in acute, subacute and recovery phase comparison with healthy controls and febrile controls. Several were aberrantly expressed, nine of which were continuously elevated after treatment and maintained during the recovery phase in KD compared to healthy controls, including TSLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e\u0026amp;B\u003c/b\u003e). Since TSLP/TSLPR has previously been linked to platelet activation and thrombosis\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we decided to narrow our focus on this protein. We divided the patients into two groups according to echocardiography, KD with thrombosis and those without thrombosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). ELISA analysis corroborated the upregulation of plasma TSLP in acute (before IVIG and after IVIG) and convalescent phase KD patients in different group of samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), which also showed a significant increase in convalescent KD patients complicated with thrombosis relative to those without (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Similarly, the expression of the TSLP receptors (TSLPR and IL-7R) on platelets was upregulated in KD patients, especially in patients with thrombosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTSLP promotes mitophagy-mediated platelet activation and apoptosis causing thrombosis in vitro\u003c/h2\u003e \u003cp\u003eWe next investigated the effect of TSLP on platelet activation and mitophagy in vitro. TSLP treatment on healthy human platelets caused a significant increase in CD62p (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and dose-dependently accelerated human PRP and whole blood thrombosis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. To investigate TSLP in mitophagy, fluorescent confocal microscopy was used to image the mitophagy marker LC-3 and found a significant increase in platelets following TSLP treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Western blot analysis also revealed that platelet mitophagy and apoptotic proteins were increased by TSLP treatment in a dose-dependent manner \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Based on this apparent link with mitophagy, we used TSLP in combination or in comparison to the mitophagy agonist (CCCP) and mitophagy inhibitor (Mdivi-1) in a thrombosis assay. Interestingly, CCCP showed similar thrombosis to TSLP, whereas Mdivi-1 alleviated thrombosis induced by TSLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Taken together, these data suggest that TSLP induces platelet mitophagy causing downstream platelet activation and thrombosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTSLPR binds Parkin and VDAC1 to trigger TSLP induced platelet mitophagy\u003c/h2\u003e \u003cp\u003eMultiple studies have demonstrated that the PINK1/Parkin signalling pathway is involved in the regulation of mitophagy\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. Thus, we explored whether TSLP regulates KD platelet mitophagy through the PINK1/Parkin signaling pathway. In vitro, TSLP dose-dependently increased the expression of TSLPR and IL-7R in platelets \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. TSLP also significantly increased the expression of PINK1, Parkin and VDAC1 in platelets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e\u0026amp;B\u003c/b\u003e). Furthermore, pretreatment with the late-stage autophagy inhibitor Baf-A1 did not prevent TSLP-induced elevation of PINK1, Parkin, VDAC1 and TSLPR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Together, these results suggest that TSLP may promote platelet mitophagy through the PINK1/Parkin pathway.\u003c/p\u003e \u003cp\u003eBased on a study that showed TSLP may cause TSLPR internalization\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, we hypothesized that TSLP caused TSLPR internalization and translocation to the mitochondria where it directly promotes mitophagy. Indeed, immunoprecipitation (IP) results show that Parkin and VDAC1 both independently bound TSLPR in platelets and their binding to each other and TSLPR was significantly enhanced following TSLP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e\u0026amp;D\u003c/b\u003e). This result is consistent with previous reports\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, in which Parkin complexes with VDAC1 to induce mitophagy. Further evidence for the TSLPR-Parkin-VDAC1 interaction comes from an additional IP using TSLPR to pull down Parkin and VDAC1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e, effectively demonstrating a triple co-IP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-E). Immunofluorescent confocal imaging confirmed TSLPR upregulation and colocalization to the mitochondria in platelets following TSLP-treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Taken together, our results show that TSLP induces platelet mitophagy through the TSLPR binding with Parkin and VDAC1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the current study, we describe a novel mechanism of platelet activation and thrombosis induced by TSLP in KD. Using clinical samples, we found significantly upregulated expression of plasma TSLP in KD patients relative to healthy controls, which was exacerbated in patients complicated with thrombosis. Furthermore, TSLP receptor (TSLPR and IL-7R) expression was significantly enhanced on platelets of KD patients complicated with thrombosis. Interestingly, we found increased platelet mitophagy and apoptosis in KD patients complicated with thrombosis, which TSLP induced in vitro. Lastly, TSLPR bound to mitophagy regulators Parkin and VDAC1 respectively following TSLP treatment, suggesting a novel TSLP-mediated mitophagy pathway in platelets. Taken together, our findings uncover a novel mechanism of platelet activation and thrombosis in KD and suggest TSLP as a novel anti-thrombotic target.\u003c/p\u003e \u003cp\u003eOur work identifies that upregulated TSLP expression at least partially underlies platelet activation and thrombosis in KD. TSLP-induced platelet mitophagy and activation likely promotes thrombosis by promoting platelets to directly bind its ligands mediating platelet aggregation\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and to harbor phosphatidylserine (PS) on its surface, which promotes a hypercoagulable state through cell-based thrombin generation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Current anti-thrombotic therapies may not effectively inhibit TSLP-induced thrombosis in KD given its unique mechanism, which may explain the persistent platelet activation and thrombosis in treated patients. A previous report identified that TSLP activates platelets through the PI3K/AKT pathway. However, since we found that the mitophagy inhibitor Mdivi-1 significantly attenuated TSLP-mediated in vitro thrombosis, we propose that its effect on PI3K/AKT signaling may be downstream of its induction of mitophagy\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMitophagy is mainly regulated by the PINK1/Parkin signaling pathway\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Recombinant Voltage Dependent Anion Channel Protein 1(VDAC1) is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Interestingly, we found that TSLPR bound to Parkin and VDAC1 and TSLP treatment enhanced the independent binding of TSLPR/Parkin/VDAC1. TSLP binding TSLPR is reported to cause receptor internalization\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, which is consistent with our findings of TSLPR co-localization with the mitochondria in platelets following TSLP treatment. This TSLPR/Parkin/VDAC1 protein complex may be an important driver of TSLP-mediated platelet mitophagy in KD.\u003c/p\u003e \u003cp\u003eSeveral reports suggest platelet activation may be a major driver of inflammation in KD\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Platelets contain and release several proinflammatory cytokines upon activation such as TNF-α, IFN-β, and IL-6 that may propagate an inflammatory state\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Activated platelets also express P-selectin, which promotes the formation of platelet-leukocyte aggregates, an important contributor in the progression of KD\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Inflammatory responses, platelet activation and thrombosis are inextricably linked. We propose that in addition to its role in thrombosis, TSLP may also contribute to systemic inflammation through its activation of platelets and release of pro-inflammatory factors. Recent studies have shown that TSLP upregulates inflammatory responses by inducing autophagy in T cells, which partially validating our hypothesis\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo our knowledge, we are the first to report evidence of increased platelet apoptosis in KD. It is interesting to note that the therapeutic mechanism of IVIG in KD is unclear, yet has previously been reported to inhibit platelet apoptosis in immune thrombocytopenia (ITP)\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Thus, it is conceivable that IVIG partially exerts its therapeutic effect in KD through its inhibition of platelet apoptosis. The cause of platelet apoptosis in KD remains unclear, however, considering the intimate link of platelet apoptosis and mitophagy, TSLP likely has a faciliatory role.\u003c/p\u003e \u003cp\u003eThe origin of TSLP in KD remains unclear. A variety of stimuli and cytokines (IL-4, IL-13, IL-5, NF-κB, and TNF-α etc.) can activate TSLP production\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Interestingly, a recent report found that TSLP production in human dermal microvascular endothelial cells is also triggered by activated platelets in an IL-1β dependent manner\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Based on this report, TSLP may activate platelets, which triggers endothelial cells to produce TSLP creating a positive feedback cycle that drives TSLP production and platelet activation. Thus, TSLP neutralization may effectively normalize its plasma concentration and reduce platelets activation.\u003c/p\u003e \u003cp\u003eMany experts suggest targeting TSLP-mediated signaling as a novel therapeutic strategy against allergic diseases to neutralize its inflammatory function\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Interestingly, KD disease is marked by a persistent inflammatory state over many months that share several features to allergic disease inflammation including abnormal type 2 inflammation, Th17/Treg imbalance, and other immunopathogenesis\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In addition, anti-TSLP monoclonal antibodies are already used for the treatment of severe asthma, such as Tezepelumab and CSJ117\u003csup\u003e63\u003c/sup\u003e. Thus, TSLP may serve as a novel therapeutic target that effectively treats the inflammatory and thrombotic risks associated with KD.\u003c/p\u003e \u003cp\u003eA limitation of our research is that we did not use the TSLPR knockout KD mouse model to verify our results. However, our study utilized a large number of rare human clinical specimens, including convalesce stage KD patients with thrombosis, to identify the pivotal role of TSLP in KD.\u003c/p\u003e \u003cp\u003eOur results demonstrate a close relationship between TSLP and thrombosis in vivo and in vitro. TSLP induced platelet mitophagy and activation via the TSLPR/Parkin/VDAC1 signaling pathway to promote thrombosis in KD. Our findings highlight TSLP as an important contributor and novel therauptic target for KD-associated thrombosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKawasaki disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTSLP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThymus stromal lymphopoietin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP TSLPR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThymus stromal lymphopoietin receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eINK1/Parkin\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePTEN induced putative kinase 1/Parkin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVDAC1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRecombinant Voltage Dependent Anion Channel Protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphatidylserine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIVIG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntravenous IgG\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCoronary artery aneurysm\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBaf-A1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBafilomycin A1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMdivi-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial division inhibitor-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCCP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitophagy agonist Carbonyl cyanide m-chlorophenyl hydrazone, Carbonyl cyanide 3-chlorophenylhydrazone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGE1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProstaglandin E1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlatelet-rich plasma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplatelet-poor plasma.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Guangdong Basic and Applied Basic Research Foundation (grant numbers 2021B1515230003), the Guangdong Natural Science Fund, China (grant numbers 2019A1515012061, 202102020829, 2022A1515012558), the Guangzhou Science and Technology Program Project, China (grant numbers 201904010486, 202102010197, 202102020829), the Subject Construction Project of Guangzhou Medical University (grant numbers 02-410-2206062), Postdoctoral Research Initiation Fund from Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center (grant numbers 3001162, 3001178-04) and Canadian Institutes of Health Research Foundation grant (grant numbers 389035).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Clinical Biological Resource Bank of Guangzhou Women and Children’s Medical Center for providing all the clinical samples. We are grateful to all patients and volunteers for donating specimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u0026nbsp;E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed significantly to this work.\u0026nbsp;L.Y.F,\u0026nbsp;D.T.M, and Q.G designed the study, performed most of the experiments, analyzed data, interpreted results, and wrote the manuscript. H.Y.Y performed\u0026nbsp;Flow cytometer\u0026nbsp;experiments, analyzed data, and interpreted results. D.C\u0026nbsp;and\u0026nbsp;C.N.C performed immunofluorescence and confocal microscopic analyses. L.P, Y.F.X, H.Z.Z,\u0026nbsp;L.Z, Z.Y.J\u0026nbsp;and\u0026nbsp;W.Z.P\u0026nbsp;provided project resources. L.Y.F,\u0026nbsp;K.N.C\u0026nbsp;performed immunoprecipitation\u0026nbsp;experiments, analyzed data, and interpreted results\u0026nbsp;and Picture editing. E.C and\u0026nbsp;H.Y.Y performed article revision and language polish.\u0026nbsp;G.X.Q and H.Y.N designed and supervised the study, interpreted results, and wrote the manuscript. All authors reviewed the manuscript. In addition, all authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Committee of Guangzhou Women and Children’s Medical Center (2014073009 and 2018052105) and was conducted according to the International Ethical Guidelines for Research Involving Human Subjects stated in the Declaration of Helsinki. Informed written consent was obtained from the guardians of the patients and controls.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcCrindle BW, Rowley AH, Newburger JW, et al. Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association. Circulation. 2017;135(17):e927-e999.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewburger JW, Takahashi M, Burns JC. Kawasaki Disease. J Am Coll Cardiol. 2016;67(14):1738\u0026ndash;1749.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiura M, Kobayashi T, Kaneko T, et al. Association of Severity of Coronary Artery Aneurysms in Patients With Kawasaki Disease and Risk of Later Coronary Events. JAMA Pediatr. 2018;172(5):e180030.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukazawa R, Kobayashi T, Mikami M, et al. Nationwide Survey of Patients With Giant Coronary Aneurysm Secondary to Kawasaki Disease 1999\u0026ndash;2010 in Japan. Circ J. 2017;82(1):239\u0026ndash;246.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePi L, Che D, Long H, et al. Immature platelets and antiplatelet therapy response to aspirin in Kawasaki disease. Drug Des Devel Ther. 2018;12:1353\u0026ndash;1362.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang Y, Zhang L, et al. Reduced Platelet miR-223 Induction in Kawasaki Disease Leads to Severe Coronary Artery Pathology Through a miR-223/PDGFRbeta Vascular Smooth Muscle Cell Axis. Circ Res. 2020;127(7):855\u0026ndash;873.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshikawa T, Iwashima S. Endothelial dysfunction in children within 5 years after onset of Kawasaki disease. J Pediatr. 2013;163(4):1117\u0026ndash;1121.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArora K, Guleria S, Jindal AK, Rawat A, Singh S. Platelets in Kawasaki disease: Is this only a numbers game or something beyond? Genes Dis. 2020;7(1):62\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRinder HM, Schuster JE, Rinder CS, Wang C, Schweidler HJ, Smith BR. Correlation of thrombosis with increased platelet turnover in thrombocytosis. Blood. 1998;91(4):1288\u0026ndash;1294.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStraface E, Gambardella L, Metere A, et al. Oxidative stress and defective platelet apoptosis in naive patients with Kawasaki disease. Biochem Biophys Res Commun. 2010;392(3):426\u0026ndash;430.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePietraforte D, Gambardella L, Marchesi A, et al. Platelets in Kawasaki patients: two different populations with different mitochondrial functions. Int J Cardiol. 2014;172(2):526\u0026ndash;528.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin J, Wang J, Lu Y, et al. Platelet-Derived Microparticles: A New Index of Monitoring Platelet Activation and Inflammation in Kawasaki Disease. Indian J Pediatr. 2019;86(3):250\u0026ndash;255.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu XR, Carrim N, Neves MA, et al. Platelets and platelet adhesion molecules: novel mechanisms of thrombosis and anti-thrombotic therapies. Thromb J. 2016;14(Suppl 1):29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRowley AH, Shulman ST. Pathogenesis and management of Kawasaki disease. Expert Rev Anti Infect Ther. 2010;8(2):197\u0026ndash;203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShulman ST, Rowley AH. Kawasaki disease: insights into pathogenesis and approaches to treatment. Nat Rev Rheumatol. 2015;11(8):475\u0026ndash;482.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMussbacher M, Salzmann M, Brostjan C, et al. Cell Type-Specific Roles of NF-kappaB Linking Inflammation and Thrombosis. Front Immunol. 2019;10:85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRawish E, Nording H, Munte T, Langer HF. Platelets as Mediators of Neuroinflammation and Thrombosis. Front Immunol. 2020;11:548631.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllakhverdi Z, Comeau MR, Jessup HK, et al. Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells. J Exp Med. 2007;204(2):253\u0026ndash;258.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin SC, Huang JJ, Wang JY, Chuang HC, Chiang BL, Ye YL. Upregulated thymic stromal lymphopoietin receptor expression in children with asthma. Eur J Clin Invest. 2016;46(6):511\u0026ndash;519.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang WW, Pan YL, Yu HW, Zhang B, Shao SW. Histamine H4 receptor regulates Th2-cytokine profile through thymic stromal lymphopoietin in allergic rhinitis. Eur Arch Otorhinolaryngol. 2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavino AM, Izraeli S. On mice and humans: the role of thymic stromal lymphopoietin in human B-cell development and leukemia. Haematologica. 2016;101(4):391\u0026ndash;393.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu K, Dong Q, Mao X, et al. Disruption of the TSLP-TSLPR-LAP signaling between epithelial and dendritic cells through hyperlipidemia contributes to regulatory T-Cell defects in atherosclerotic mice. Atherosclerosis. 2015;238(2):278\u0026ndash;288.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong J, Lin J, Wang B, et al. Inflammatory cytokine TSLP stimulates platelet secretion and potentiates platelet aggregation via a TSLPR-dependent PI3K/Akt signaling pathway. Cell Physiol Biochem. 2015;35(1):160\u0026ndash;174.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMao Y, Peng Y, Zeng Q, et al. A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor. PLoS One. 2015;10(10):e0141464.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng W, Chang C, Luo D, et al. Dissection of autophagy in human platelets. Autophagy. 2014;10(4):642\u0026ndash;651.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOuseph MM, Huang Y, Banerjee M, et al. Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis. Blood. 2015;126(10):1224\u0026ndash;1233.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou H, Li D, Zhu P, et al. Melatonin suppresses platelet activation and function against cardiac ischemia/reperfusion injury via PPARgamma/FUNDC1/mitophagy pathways. J Pineal Res. 2017;63(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi BX, Dai X, Xu XR, et al. In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbalpha. Sci Rep. 2021;11(1):11663.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng Z, Xia L, Fan X, et al. Platelet-derived miR-223 promotes a phenotypic switch in arterial injury repair. J Clin Invest. 2019;129(3):1372\u0026ndash;1386.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu XR, Wang Y, Adili R, et al. Apolipoprotein A-IV binds alphaIIbbeta3 integrin and inhibits thrombosis. Nat Commun. 2018;9(1):3608.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Lang S, Zhai Z, et al. Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression. Blood. 2009;114(2):425\u0026ndash;436.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGyulkhandanyan AV, Allen DJ, Mykhaylov S, et al. Mitochondrial Inner Membrane Depolarization as a Marker of Platelet Apoptosis: Disclosure of Nonapoptotic Membrane Depolarization. Clin Appl Thromb Hemost. 2017;23(2):139\u0026ndash;147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeytin V, Mykhaylov S, Starkey AF, et al. Intravenous immunoglobulin inhibits anti-glycoprotein IIb-induced platelet apoptosis in a murine model of immune thrombocytopenia. Br J Haematol. 2006;133(1):78\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Piran S, Chen P, et al. The maternal immune response to fetal platelet GPIbalpha causes frequent miscarriage in mice that can be prevented by intravenous IgG and anti-FcRn therapies. J Clin Invest. 2011;121(11):4537\u0026ndash;4547.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Gong Q, Zhan Y, et al. Blockage of Autophagic Flux and Induction of Mitochondria Fragmentation by Paroxetine Hydrochloride in Lung Cancer Cells Promotes Apoptosis via the ROS-MAPK Pathway. Front Cell Dev Biol. 2019;7:397.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Fu L, Tian T, et al. Disrupting SOD1 activity inhibits cell growth and enhances lipid accumulation in nasopharyngeal carcinoma. Cell Commun Signal. 2018;16(1):28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi L, Yao YC, Gu XQ, et al. Plasminogen kringle 5 induces endothelial cell apoptosis by triggering a voltage-dependent anion channel 1 (VDAC1) positive feedback loop. J Biol Chem. 2014;289(47):32628\u0026ndash;32638.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAleman MM, Byrnes JR, Wang JG, et al. Factor XIII activity mediates red blood cell retention in venous thrombi. J Clin Invest. 2014;124(8):3590\u0026ndash;3600.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Shen C, Fang M, et al. Novel contact-kinin inhibitor sylvestin targets thromboinflammation and ameliorates ischemic stroke. Cell Mol Life Sci. 2022;79(5):240.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavizon-Castillo P, McMahon B, Aguila S, et al. TNF-alpha-driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging. Blood. 2019;134(9):727\u0026ndash;740.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSliter DA, Martinez J, Hao L, et al. Parkin and PINK1 mitigate STING-induced inflammation. Nature. 2018;561(7722):258\u0026ndash;262.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin Q, Li S, Jiang N, et al. PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation. Redox Biol. 2019;26:101254.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuinn PMJ, Moreira PI, Ambrosio AF, Alves CH. PINK1/PARKIN signalling in neurodegeneration and neuroinflammation. Acta Neuropathol Commun. 2020;8(1):189.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKitajima M, Lee HC, Nakayama T, Ziegler SF. TSLP enhances the function of helper type 2 cells. Eur J Immunol. 2011;41(7):1862\u0026ndash;1871.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeisler S, Holmstrom KM, Skujat D, et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol. 2010;12(2):119\u0026ndash;131.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi H, Denis CV, Subbarao S, et al. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest. 2000;106(3):385\u0026ndash;392.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMacKeigan DT, Ni T, Shen C, et al. Updated Understanding of Platelets in Thrombosis and Hemostasis: The Roles of Integrin PSI Domains and their Potential as Therapeutic Targets. Cardiovasc Hematol Disord Drug Targets. 2020;20(4):260\u0026ndash;273.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Gallant RC, Ni H. Extracellular matrix proteins in the regulation of thrombus formation. Curr Opin Hematol. 2016;23(3):280\u0026ndash;287.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts HR, Hoffman M, Monroe DM. A cell-based model of thrombin generation. Semin Thromb Hemost. 2006;32 Suppl 1:32\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu X, Peng Y, Liang H, et al. TSLP/TSLPR promote angiogenesis following ischemic stroke via activation of the PI3K/AKT pathway. Mol Med Rep. 2018;17(2):3411\u0026ndash;3417.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarazzuol L, Giamogante F, Brini M, Cali T. PINK1/Parkin Mediated Mitophagy, Ca(2+) Signalling, and ER-Mitochondria Contacts in Parkinson's Disease. Int J Mol Sci. 2020;21(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHam SJ, Lee D, Yoo H, Jun K, Shin H, Chung J. Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination. Proc Natl Acad Sci U S A. 2020;117(8):4281\u0026ndash;4291.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorrigan JJ, Jr. Kawasaki disease and the plight of the platelet. Am J Dis Child. 1986;140(12):1223\u0026ndash;1224.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemple JW, Italiano JE, Jr., Freedman J. Platelets and the immune continuum. Nat Rev Immunol. 2011;11(4):264\u0026ndash;274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUeno K, Nomura Y, Morita Y, Eguchi T, Masuda K, Kawano Y. Circulating platelet-neutrophil aggregates play a significant role in Kawasaki disease. Circ J. 2015;79(6):1349\u0026ndash;1356.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVignesh P, Rawat A, Shandilya JK, Singh Sachdeva MU, Ahluwalia J, Singh S. Monocyte platelet aggregates in children with Kawasaki disease- a preliminary study from a tertiary care centre in North-West India. Pediatr Rheumatol Online J. 2021;19(1):25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan NR, Moon PD, Nam SY, et al. TSLP up-regulates inflammatory responses through induction of autophagy in T cells. FASEB J. 2022;36(2):e22148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeytin V. Apoptosis in the anucleate platelet. Blood Rev. 2012;26(2):51\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakai T. TSLP expression: cellular sources, triggers, and regulatory mechanisms. Allergol Int. 2012;61(1):3\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTruchetet ME, Demoures B, Eduardo Guimaraes J, et al. Platelets Induce Thymic Stromal Lymphopoietin Production by Endothelial Cells: Contribution to Fibrosis in Human Systemic Sclerosis. Arthritis Rheumatol. 2016;68(11):2784\u0026ndash;2794.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCianferoni A, Spergel J. The importance of TSLP in allergic disease and its role as a potential therapeutic target. Expert Rev Clin Immunol. 2014;10(11):1463\u0026ndash;1474.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugitani Y, Furuno K, Sueishi K, Hara T. Macrophages and cytotoxic T cells infiltrate the destructed mitral tissue in Kawasaki disease. \u003cem\u003eBMJ Case Rep\u003c/em\u003e. 2018;2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakajima S, Kabata H, Kabashima K, Asano K. Anti-TSLP antibodies: Targeting a master regulator of type 2 immune responses. Allergol Int. 2020;69(2):197\u0026ndash;203.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TSLP, Platelet, Mitophagy, Thrombosis, Kawasaki disease","lastPublishedDoi":"10.21203/rs.3.rs-1628519/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1628519/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eKawasaki disease (KD) is an acute systemic vasculitis primarily affecting infants and children with an unclear etiology. Coronary artery aneurysm (CAA) is a common manifestation in severe KD patents, which may lead to thrombotic cardiovascular events such as heart attack and stroke even years after onset. We and others have previously reported systemic platelet activation in KD patients. Thymic stromal lymphopoietin (TSLP) is a recently identified interleukin-7 (IL-7) like cytokine associated with promoting pathological inflammation and most notably, platelet activation. The present study is to investigate the role of TSLP in KD-associated thrombosis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo discover potential proteins underlying platelet activation in KD, we conducted a protein chip assay of 34 cytokines and discovered a significant upregulation in thymus stromal lymphopoietin (TSLP). ELISA corroborated the upregulation of TSLP in another group of KD patients. Clinical samples (plasma and platelets) from KD patients and healthy controls were analyzed via flow cytometry, immunofluorescent confocal microscopy, western blot, immunoprecipitation and thrombosis assays to reveal the underlying mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong the 34 cytokines, we discovered several were aberrantly expressed, nine of which were continuously elevated after IVIG treatment and maintained during the convalescence in KD patients compared to healthy controls, including TSLP. The upregulation of TSLP in KD patients was confirmed by ELISA, which showed a further increase of TSLP in convalescent patients complicated with thrombosis. The expression of TSLP receptors (TSLPRs) on platelets were also significantly upregulated in KD patients complicated with thrombosis. Platelet activation, apoptosis, and mitochondrial autophagy (mitophagy) were increased in KD patients, which increase was exacerbated in convalescent patients complicated with thrombosis. In vitro, TSLP induced platelet activation and platelet mitophagy in healthy blood donors as we observed in KD patients. TSLP, similar to mitophagy agonist CCCP, promoted thrombosis, which was attenuated by the mitophagy inhibitor Mdivi-1. Co-immunoprecipitation in TSLP-treated platelets revealed TSLPR bound to mitophagy regulators, Parkin and VDAC1, suggesting a potential novel TSLP-mediated platelet mitophagy pathway.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results demonstrate that TSLP induces platelet mitophagy via a novel TSLPR/Parkin/VDAC1 mitophagy pathway that promotes thrombosis in KD. These results suggest TSLP as a novel therapeutic target against KD-associated thrombosis.\u003c/p\u003e","manuscriptTitle":"TSLP induces platelet mitophagy and promotes thrombosis in Kawasaki disease ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-12 15:25:44","doi":"10.21203/rs.3.rs-1628519/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd170f99-3c60-41d4-b4db-200213891fff","owner":[],"postedDate":"May 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-12T15:25:46+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-12 15:25:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1628519","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1628519","identity":"rs-1628519","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}