Biomedical application of TiO2NPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets

Biomedical application of TiO2NPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets | 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 Biomedical application of TiO 2 NPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets Yiying Bian, Qiushuo Jin, Jinrui He, Thien Ngo, OK-Nam Bae, Jingbo Pi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4187973/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Aug, 2024 Read the published version in Cell Biology and Toxicology → Version 1 posted 12 You are reading this latest preprint version Abstract Background Titanium dioxide nanoparticles (TiO 2 NPs) are widely used in medical application. However, the relevant health risk has not been completely assessed, the potential of inducing arterial thrombosis (AT) in particular. Methods Alterations in platelet function and susceptibility to arterial thrombosis induced by TiO 2 NPs were examined using peripheral blood samples from healthy adult males and an in vivo mouse model, respectively. Results Here, using human platelets (hPLTs) freshly isolated from health volunteers, we demonstrated TiO 2 NP treatment triggered the procoagulant activity of hPLTs through phosphatidylserine exposure and microvesicles generation. In addition, TiO 2 NP treatment increased the levels of glycoprotein IIb/IIIa and P-selectin leading to aggregation and activation of hPLTs, which were aggravated by providing physiology-mimicking conditions, including introduction of thrombin, collagen, and high shear stress. Interestingly, intracellular calcium levels in hPLTs were increased upon TiO 2 NP treatment, which were crucial in TiO 2 NP-induced hPLT procoagulant activity, activation and aggregation. Moreover, using mice in vivo models, we further confirmed that TiO 2 NP treatment a reduction in mouse platelet (mPLT) counts, disrupted blood flow, and exacerbated carotid arterial thrombosis with enhanced deposition of mPLT. Conclusions Together, our study provides evidence for an ignored health risk caused by TiO 2 NPs, specifically TiO 2 NP treatment augments procoagulant activity, activation and aggregation of PLTs via calcium-dependent mechanism and thus increases the risk of AT. Titanium dioxide nanoparticles (TiO2NPs) Platelet (PLT) Calcium Arterial thrombosis (AT) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The titanium dioxide nanoparticles (TiO 2 NPs) that are smaller than 100 nm are widely utilized in biomedical fields such as photoimaging, drug delivery and biological analysis due to their antimicrobial properties, photocatalytic activity, excellent biocompatibility, and corrosion resistance(Najahi-Missaoui et al. 2020 ; Zhao and Castranova 2011 ). Often, intravenous injection is the norm, allowing the distribution of these nanoparticles to each organ via the bloodstream for therapeutic purposes. However, this process frequently leads to unexpected organ toxicity, including liver, kidney, and brain injuries(Shi et al. 2013 ). Given the significance of blood cells as the primary and essential contact component in the blood circulation process, it is noteworthy that toxicological research on these cells remains relatively scarce. Hence, the potential health risk of TiO 2 NP treatment, particularly from a perspective focusing on blood cells, is becoming a large concern(Lee et al. 2019 ; Naserzadeh et al. 2018 ; Qi et al. 2022 ; Shakeel et al. 2016 ). The thrombosis, which is responsible for one in four deaths(2014), can be primarily categorized into venous, arterial, and microvascular thrombosis(Brandtner et al. 2021 ; Petri 2020 ). Our previous study have demonstrated that TiO 2 NPs have the ability to initiate procoagulant activity in red blood cells, ultimately culminating in the development of venous thrombosis(Bian et al. 2021 ). Although some reports have proposed that TiO 2 NPs can target platelets and cause microclots in microcirculation, their effects appear to be primarily restricted to platelet aggregation in vitro (Haberl et al. 2015 ). Nonetheless, in-depth thorough investigation of the underlying mechanism and the presence of other forms of platelet dysfunction still remain unexplored. Recently, the activation and aggregation of platelets, as indicated by an elevated expression of the integrin αIIbβ3 (GPIIb/IIIa complex, CD41/CD61) and the transmembrane protein P-selectin within the alpha granules, have been strongly linked to thrombotic disorders(Huang et al. 2019 ; Qiao et al. 2018 ; Yeini and Satchi-Fainaro 2022 ). In addition, recent studies have indicated that the pro-coagulant activity of platelets (PLTs) plays a crucial role in the development of thrombosis, which is primarily attributed to the level of phosphatidylserine (PS) exposed on the outer membrane surface and the release of microvesicles (MVs)(Pang et al. 2018 ; Zlamal et al. 2023 ). The two processes are caused by an elevated calcium level, leading to hemostasis and thrombosis(Obydennyy et al. 2016 ; Varga-Szabo et al. 2009 ), ultimately pointing to the same outcome: the arterial thrombosis. In this study, we initially established that intravenous administration of TiO 2 NPs swiftly instigates procoagulant activity within platelets by enhancing phosphatidylserine exposure and microvesicle production. Additionally, we observed prompt platelet activation and aggregation following TiO 2 NP treatment through the activation of GPIIb/IIIa and the expression of P-selectin. These findings indicate that TiO 2 NPs possess the potential to alter platelet function and promote the carotid artery thrombosis, a process that necessitates an elevated intracellular calcium level. Indeed, we also observed dysregulation of blood flow signals and arterial thrombosis along with increased PLT deposition using a carotid artery thrombosis mice model. These results provide clear evidence for the risk of PLT-related arterial thrombosis caused by TiO 2 NP treatment, and help to improve the understanding of the arterial thrombotic risk caused by TiO 2 NPs. Methods Materials TiO 2 NPs (anatase, nanopowder, < 100 nm particle size), trisodium citrate, HEPES, prostaglandin E1 (PGE1), glutaraldehyde, EDTA, EGTA, ferric chloride, urethane, clopidogrel, and bovine serum albumin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Thrombin and collagen were obtained from Calbiochem (San Diego, CA, USA) and Chrono-log (Havertown, PA, USA), respectively. Fluorescein isothiocyanate (FITC)-labeled anti-CD62P antibody (anti-CD62P-FITC Ab), FITC-labeled PAC-1 (PAC-1-FITC), and FITC-labeled annexin V (annexin V-FITC) were from BD Biosciences (San Jose, CA, USA). Fluo-4 acetoxymethyl ester (Fluo-4 AM) was obtained from Invitrogen (Carlsbad, CA, USA). Blood collection and preparation of human platelets (hPLTs) This work was supported by the Ethics Committee of the Health Service Center at Seoul National University with the approval from the Institutional Review Board of Seoul National University (IRB No. 1702/003–004, 5, March, 2019). To simplify our study design, we selected only healthy male donors aged 20–30 years, excluding risk factors for thrombosis such as gender, age, and disease, and taking any medication in the past 2 weeks. For platelet-rich-plasma (PRP) preparation, whole blood with 3.2% trisodium citrate was centrifuged at 150 × g for 15 min and platelet cell count in PRP was adjusted 3 × 10 8 cells/mL by diluting with platelet-poor-plasma (PPP) on the day of experiments. Characterization of TiO 2 NPs TiO 2 NPs were not surface treated and insoluble in water, hydrochloric acid, or nitric acid. The particles were dispersed in distilled water and sonication was performed before each experiment, using an ultrasonicator with a maximum output of 150–200 W for 15 s to prevent agglomeration. TiO 2 NPs were dried and observed with scanning electron microscope (SEM) (SU8010, Hitachi Limited, Japan) to examine the size distribution. Detailed statistical analysis (Nano measure 1.2 and OraginPro2021) of TiO 2 NPs were performed by random measurement of 100 nanoparticles in the images taken by SEM. The dynamic particle size of the nanoparticles was evaluated using a Malvern laser particle size analyzer (DLS-7000, Otsuka Electronics, Co., Osaka, Japan). The sample was weighed and dispersed in deionized water (0.1% mass fraction), sonicated for 3 min, adjusted to pH 7.4 with NaOH or HCl. The zeta potential was measured with a nanoparticle size analyzer (ZS-90, Malvern Instruments, UK). All data were repeated three times and averaged. Technical support was provided by Beijing Standard Spectrum Testing Technology Co. Measurement of PLT aggregation After incubation with TiO 2 NPs (0.5, 1, 5, 10, 25, and 100 µg/ml; 5 min, 30 min, and 60 min) at 37°C, the number of individual PLTs per microliter was calculated using optical microscopy and the degree of PLT aggregation was assessed based on the count of single cells. Data were presented as percentages of PLT aggregation. Besides, PLT aggregation induced by TiO 2 NP treatment under physiology-mimicking conditions was observed by the above method under stimulation with thrombin (0.6–0.8 U/ml), collagen (2–4 µg/ml), and shear stress at 1500 s − 1 for 3 min. Evaluation of LDH leakage Lactate dehydrogenase (LDH) leakage from PLT was measured by spectrophotometry. After incubation with TiO 2 NPs for 5 min, the supernatant obtained from the centrifuge reaction mixture was used for LDH determination (digitonin 50 µM treatment for 1 h was used as positive control). The degree of cell lysis was expressed as a percentage of total enzyme activity compared to control incubation with cleavage with digitonin. Flow cytometry analysis After PLTs were exposed with TiO 2 NPs at 37°C for 10 min, the level of P-selectin and the activation of glycoprotein GPIIb/IIIa were determined by staining with CD62P-FITC and PAC-1-FITC for 20 min, respectively. PS exposure and MV generation in PLTs were examined by staining with both annexin V-FITC and anti-CD42b-PE Ab (as PLT identifier). Intracellular calcium level was determined by pre-loading fluo-4/AM (5 µM) for 45 min. All the determinations were conducted using FACS Calibur (Becton Dickinson, USA), and Cell Quest Pro software was used to collect and analyze data from 10,000 events. A prothrombinase assay The prothrombinase assay was applied to assess procoagulant activity. Specifically, TiO 2 NP-treated PLTs were further induced into thrombin generation by adding 5 nM Xa factor, 10 nM Va factor and prothrombin. Then, chromogenic substrate S2238 (Chromogenic, Milan, Italy) was used to measure the generated thrombin after adding a stop buffer (50 mM Tris-HCl, 120 mM NaCl, 2 mM EDTA, pH 7.9). Calculation of thrombin production rate was based on the absorbance change at 405 nm from the calibration curve. Animals The experimental C57BL/6 J mice (male, 12 weeks old) were kept in a clean environment without specific pathogens and fed with SPF chow diet and distilled water. The indoor temperature was controlled at 23 ± 1 ℃, the humidity was 55–70%, and the circadian rhythm was alternated (12 h/12 h). Mice were randomly divided into two groups: control group and exposed group (TiO 2 NPs, 25 mg/kg, intravenous injection). All animal experiments were reviewed and approved by the Animal Ethics Committee of China Medical University (CMU20231000). Ex vivo assessment using blood cell analyzer Blood (30 µl) was collected from mice tail with EDTA-K 2− containing tube 1 h post TiO 2 NP injection. Then fresh blood samples were tested by an automated five-classification animal blood cell analyzer (IDEXX ProCyte Dx, Japan) for the following indicators: PLT count (performed by both impedance (PLT-I) and optical (PLT-O) method), percentage of PLT-larger cell ratio (L-PCR%), PLT crit (PLT%), mean PLT volume (MPV), mean PLT volume/PLT count (MPV/P), PLT distribution width (PDW), red blood cell count, percentage of hematocrit, hemoglobin, and white blood cell-related indexes. Ex vivo measurement of mPLT aggregation was conducted by an animal blood cell analyzer with impedance method, which only allows one single cell go through. The PLT aggregation rate was calculated by the formula: (PLT Cont -PLT TiO2NPs )/PLT Cont ×100)%. Arterial thrombosis in mice Arterial thrombosis mouse model The left common carotid artery was isolated from the left side of the trachea using 3×5 mm tin foil to isolate the surrounding tissues. Afterwards, 1×2 mm filter paper was fully submerged with 5% FeCl 3 and applied to the left common carotid artery for 15 min. Ultrasound observation The blood flow signal by the Doppler ultrasound in the mouse model was slightly adjusted based on previous study(Jing et al. 2023 ; Wang and Xu 2005 ). Mice were anesthetized with isoflurane inhalation at 3% and maintained at 2% throughout the procedure and then cleaned of hair on the neck and chest to facilitate ultrasound. AVINNO6 LAB small animal Doppler ultrasound system (VINNO Co., China) was used. The left common carotid artery was imaged and the flow signals (including velocity, flow volume, internal diameter, perfusion index, heart rate) were quantified. Thrombus pathological experiment The tissue of the common carotid artery was collected for the pathological experiment. The thrombus tissue was embedded using the frozen section embedding agent Sakura OCT (a water-soluble mixture of polyethylene glycol and polyvinyl alcohol), sectioned at 6 µm thickness with frozen sectioning machine (CM 1950, LEICA, Germany), and collected using adhesive slides to observe thrombus morphology under a general light microscope. By measuring the percentage of each blank area in the thrombus (the area that blood flow can pass through), it is artificially divided into the following groups: 0–1, 1–5, and 5–10% of the total area. Less blank area means more compact thrombus. Immunofluorescence observations for frozen sections were examined by adding α-fibronogen with Alexa Fluor® 488 and CD42b with Alexa Fluor® 568, respectively. Then, the samples were sealed with a blocking buffer containing a liquid with an anti-fluorescent cracking agent. Statistical Analysis All data are presented as the mean and standard deviation. Data were subjected to Student's t -test or two-way ANOVA followed by Duncan's multiple range test. In all cases, a P value < 0.05 was considered statistically significant. The asterisk represents significant differences from the control group (*** P <0.001; ** P <0.01; * P <0.05). The pound represents significant differences from the TiO 2 NP treatment group ( ### P <0.001; ## P <0.01; # P <0.05). Results Characterization of TiO 2 NPs and the uptake by human platelets (hPLTs) The physicochemical characterization of TiO 2 NPs was examined by SEM and DLS. The size distribution of TiO 2 NPs was in the range of 20 to 70 nm with an average diameter of 35.7 nm (Fig. 1 a), which was randomly calculated from 100 particles shown in SEM images. DLS data further showed that the average size by intensity was 148.3 nm in saline (with 10% FBS) and 197.2 nm in PBS, respectively (Fig. 1 b). The zeta potential of TiO 2 NPs was + 11.7 mV at pH 7.4 (Fig. 1 c). Interestingly, TEM observation showed that TiO 2 NPs were within hPLTs (dashed circles) or adhered to hPLT membrane (dashed box) (Fig. 1 D), indicating a possible PLT-associated outcome in response to TiO 2 NP treatment. TiO 2 NPs enhance hPLT procoagulant activity through PS exposure and MVs generation In resting PLTs, PS are commonly in the inner leaflet of the membrane. Upon stimuli, PS are externalized to the outer leaflets and microvesicles (MVs, < 1 µm) are released, both of which participate in coagulation pathway and then promote the production of thrombin from prothrombin under prothrombinase complex (Va, Xa)(Lentz 2003 ). To estimate procoagulant activity of PLTs induced by TiO 2 NPs, we treated hPLTs with TiO 2 NPs for 10 min, determined PS exposure and MVs generation using FACs, and estimated thrombin generation under prothrombinase complex (Va, Xa) (Fig. 2 a). PS exposure and MVs were significantly increased in a concentration-dependent manner after TiO 2 NP treatment (Fig. 2 b and 2 c). In parallel, with a prothrombinase assay, TiO 2 NP treatment accelerated thrombin generation in hPLTs, reflecting increased procoagulant activity (Fig. 2 d). TiO 2 NPs induce hPLT aggregation and activation via GPⅡb/Ⅲa activation and increased P-selectin level To exam whether TiO 2 NPs induce PLT aggregation, we used PRP freshly isolated from healthy volunteers and incubated hPLTs with TiO 2 NPs for 5 min, 30 min or 60 min as shown in Fig. 3 a. In consequence, TiO 2 NPs caused PLT aggregation at 5 min in vitro (Fig. 3 b). Upon 30 and 60 min exposure, TiO 2 NPs also caused PLT aggregation but did not show obvious time-dependent effects even when compared with 5 min exposure (Fig. 3 c), indicating that TiO 2 NPs rapidly exaggerate PLT aggregation which occurs within 5 min. Next, we examined the leakage of LDH from hPLTs and found that no cytotoxicity was induced by TiO 2 NP treatment (digitonin was used as a positive control) (Fig. 3 d). Glycoprotein IIb/IIIa (GPIIb/IIIa) plays a key role in the maintenance of PLT aggregation(Aliotta et al. 2021 ). In addition, P-selectin is an adhesion molecule belonging to the selectin family expressed on PLTs, and activated PLTs express high levels of P-selectin(Yeini and Satchi-Fainaro 2022 ). As shown in Fig. 3 E and 3 F, GPⅡb/Ⅲa activation and P-selectin expression were both significantly induced by TiO 2 NP treatment. TiO 2 NP-induced hPLT aggregation is exacerbated under physiology-mimicking conditions In the physiological system, the presence of physiological aggregators (e.g. thrombin and collagen) contributes to the heterogeneity in PLT responses(Aslam et al. 2013 ; Petzold et al. 2016 ; Sang et al. 2021 ). Additionally, vascular shear force plays an existential role in the physiological function of PLTs(Casa et al. 2015 ; Yagi et al. 2017 ). To assess the effect of TiO 2 NPs on PLT aggregation more accurately, we determined PLT aggregation using an in vitro experimental method with adjustable physiological simulating conditions (Fig. 4 a, ⅰ) and compared the results with those from a basic in vitro experimental method (Fig. 4 a, ⅱ). After adding thrombin or collagen in vitro , accelerated TiO 2 NP-induced PLT aggregation was found (Fig. 4 b). Similarly, after adding shear stress in vitro that simulates physiological high shear flow, enhanced PLT aggregation was observed (Fig. 4 c). More significantly, the existence of physiological aggregators (thrombin and collagen) and high shear stress reduced the minimum toxic level of TiO 2 NPs for PLT aggregation from 1 µg/mL to 0.1 µg/mL (Fig. 4 b, insert) and 0.5 µg/mL (Fig. 4 c, insert), respectively, as compared to the basic condition. These data indicate that TiO 2 NPs boost more severe PLT aggregation under physiology-mimicking conditions. Involvement of Ca 2+ in hPLT aggregation and procoagulant activity induced by TiO 2 NPs Intracellular Ca 2+ plays a key role in PLT activation, aggregation and procogulant activity(Back et al. 2022 ; Xiang et al. 2021 ; Zhu et al. 2016 ). Here, we observed a concentration-dependent increase in intracellular Ca 2+ level after TiO 2 NP treatment (Fig. 5 a). To further assess the role of Ca 2+ in PLT dysfunction, we treated hPLTs with EGTA (a chelator of Ca 2+ ) prior to TiO 2 NP treatment. In consequence, increased PS exposure (Fig. 5 b) and procoagulant activity (Fig. 5 c) induced by TiO 2 NPs were both effectively reduced by EGTA. Concurrently, TiO 2 NP-induced hPLT aggregation was markedly blocked by EGTA (Fig. 5 d). Meanwhile, elevated GPⅡb/Ⅲa activation (Fig. 5 e) and P-selectin level (Fig. 5 f) by TiO 2 NP treatment were both significantly inhibited by EGTA. TiO 2 NP treatment decreases PLT counts and increases large PLT ratio in mice To elucidate the in vivo effect of TiO 2 NPs, mice were intravenously injected with TiO 2 NPs and after 1 h, blood was collected and analyzed using a blood cell analyzer. It is clear from the data that PLT counts (Fig. 6 a) detected by the impedance method (Fig. 6 a, left) and the optical method (Fig. 6 A, right), respectively, as well as PCT% (Fig. 6 b), were significantly declined in TiO 2 NP-exposed mice. Meanwhile, P-LCR% was increased apparently in TiO 2 NP-exposed mice (Fig. 6 c). In addition to P-LCR, MPV, PDW and MPV/P were all considered as potential indicators of PLT shape and function(Azab et al. 2011 ; Gasparyan et al. 2011 ; Han et al. 2013 ). Accordingly, increases in MPV (Fig. 6 d) and MPV/P (Fig. 6 f) after TiO 2 NP treatment were observed, but there was no change in PDW (Fig. 6 e). Moreover, TiO 2 NP-exposed mice showed no significant change in red blood cells (RBC) index including RBC counts (Fig. 6 G, left), hematocrit (HCT%) (Fig. 6 g, right), hemoglobin (HGB) (Fig. 6 h), or white blood cells (WBC) index including neutrophils, lymphocytes, monocytes, eosinophils and basophils (Fig. 6 i). TiO 2 NPs trigger PLT dysfunction and locally alter arterial blood flow signals in mice To further determine whether TiO 2 NPs cause PLT aggregation and activation, we firstly did ex vivo study using PLTs isolated from mice (mPLTs) treated with TiO 2 NPs (Fig. 7 a). TiO 2 NPs resulted in increased mPLT aggregation (Fig. 7 b) and P-selectin (Fig. 7 c) level 1 h post treatment. Numerous studies have shown that PLT activation and aggregation are closely related to thrombosis(Nayak et al. 2021 ; Yeung et al. 2018 ). Therefore, we established an AT-initiating mice model 1 h after TiO 2 NP treatment, and phenotypes were determined by color doppler ultrasonography (Fig. 7 d, ⅰ). As shown in Fig. 7 e, TiO 2 NP-exposed group showed different flow signals compared with control group. Specifically, TiO 2 NP-exposed mice represented obvious accelerated arterial blood flow velocity (Fig. 7 f), increased blood flow volume (Fig. 7 g), and a slightly declined trend in the vascular resistance index (Fig. 7 i) with unconspicuous alterations in artery internal diameter (Fig. 7 h). Such doppler data indicate thrombi ahead, which is consistent with early reports on the phenomena of increased blood flow signals and thrombus formation(He et al. 2023 ). Briefly, these all point to a critical clue that TiO 2 NP treatment increases the risk of AT. TiO 2 NP treatment leads to carotid artery thrombosis with PLT deposition in mice In addition to the doppler ultrasonic detection, we conducted pathological sections of the arterial thrombus in mouse AT model (Fig. 7 d, ⅱ), and found increased thrombus formation in TiO 2 NP-exposed mice (Fig. 8 a). Besides, we noticed that thrombi formed in TiO 2 NP-exposed mice were fuller and more compact, reflecting that the blood flow became more sluggish. Hence, we further analyzed the blank area of aorta (area that blood flow can go through) as mentioned in methods, to reflect the potential unobstructed capacity of blood flow. The total counts were separated into three distinguished levels including the tiny (less than 1% of the total aorta), the moderate (1–5% of the total aorta) and the loose blank area (5–10% of the total aorta). Consistent with the increase in thrombosis, mice exposed to TiO 2 NPs showed significantly less total blank areas or blank areas at each level compared to control mice, reflecting a decrease in blood flow patency in mice with AT induced by TiO 2 NPs (Fig. 8 b). Finally, we stained the thrombi by immunofluorescence with CD42b (red color) to reflect the active role of PLTs in TiO 2 NP-induced AT. The red fluorescence indicating PLT deposition within the thrombi was increased in TiO 2 NP-exposed mice (Fig. 8 c). Discussion The growing medical application of TiO 2 NPs has sparked critical consideration of their bio-safety. This study, for the first time, reveals that TiO 2 NPs can enhance procoagulant activity in isolated human platelets by increasing exposure of PS and the generation of MVs. Additionally, exposure to TiO 2 NPs rapidly triggers platelet activation and aggregation through increased expression of P-selectin and activation of GPIIb/IIIa. Here, intracellular calcium plays a role in both processes. Furthermore, TiO 2 NPs alter blood flow and exacerbate arterial thrombus formation in mice, accompanied by increased platelet deposition, underscoring the relevance of our findings to real in vivo conditions. Nanoparticles are often used intravenously in medical use, which inevitably have unintended side effects on blood cells. Numerous studies have shown that these particles can impact macrophages(Dey et al. 2021 ), neutrophils(Yamano et al. 2022 ), and lymphocytes. In addition, our previous research revealed that TiO 2 NPs can contribute to venous thrombosis by enhancing the procoagulant activity of red blood cells(Bian et al. 2021 ). In peripheral blood, platelets also play key roles in hemostasis and thrombosis(Koupenova et al. 2018 ). In this study, using isolated human samples, we discovered that platelets are more sensitive to TiO 2 NPs than red blood cells were in our previous finding. This heightened sensitivity was evident at concentrations as low as 1 µg/mL of TiO 2 NPs (after less than 10 minutes in vitro ), which preceded the dysfunction of red blood cells, which occurred relatively high at 10 µg/mL after approximately 24 hours in vitro . The findings of this study indicate that intravenous administration of TiO 2 NPs may pose a significant risk to blood cell function and homeostasis. It is crucial for future research to further investigate the potential long-term effects of these nanoparticles on blood cell function and cardiovascular health. The reports on toxicity of NPs are always controversial due to the ability to release ionic form from metal NPs. Some studies claim that the toxicity of metal NPs is due to the metal ions released by NPs, while others argue that the toxicity of metal NPs is due to the small particle size of NPs themselves and other physicochemical properties as additional influencing factors(Bian et al. 2019 ; Ye et al. 2018 ; Zhang et al. 2020 ). For instance, zinc oxide and copper oxide nanoparticles (ZnONPs and CuONPs) are easy to release their ionic forms and their main toxicity is considered to be caused by the metal ions(Liu et al. 2016 ; Wang et al. 2016 ). However, with respect to silver nanoparticles (AgNPs), toxic effects are currently attributed into two aspects, the metal ion (Ag + ) effect and the NP effect, respectively(Poynton et al. 2012 ). Most earlier studies suggest that it is Ag + released from the AgNPs exerting a significant influence on protein regulation and the induction of cellular stress(Zhang et al. 2020 ). Different from this perspective, our previous study found that the level of Ag + released from AgNPs as detected by ICP-MS method was negligible (< 0.01%) and hardly initiated a significant response of the cell(Bian et al. 2019 ), indicating that the effect of NPs themselves rather than the ion effect is the main contributor of AgNP toxicity. A recent study has revealed that though the ions of AgNPs cause a certain degree of toxicity, but not as severe as the toxicity due to their physical characteristics as NPs(Cvjetko et al. 2017 ). Unlike those typical metal NPs possessing the capability of releasing ionic forms, the level of metal titanium ions (Ti 4+ ) dissociated from TiO 2 NPs is extremely low due to the special nature of titanium(Prokopiuk et al. 2023 ; Qin et al. 2017 ). Generally speaking, the toxic effects of TiO 2 NPs are proved to be mainly based on their unique NP properties. Physiological factors play important regulatory roles in the functional performance of PLTs(Sang et al. 2021 ). Flowing blood generates a frictional force called shear stress(Souilhol et al. 2020 ), and a common pathological symptom for myocardial infarction and ischemic stroke is thrombotic blood flow obstruction that forms at high shear rates in the arteries(Casa and Ku 2017 ). Thrombin, collagen, and ADP are currently considered to be the most potent physiological agonists of PLTs. In the present study, under physiology-mimicking conditions, we found that TiO 2 NP-induced hPLT aggregation was dramatically aggravated. Although we tried our best to simulate in vivo system by adding physiological aggregators and imposing high shear stress, and as expected, observed increased PLT aggregation, the real human body is a large complex system quite difficult to completely and perfectly simulate. Calcium signaling and its network of interactions are involved in mediating many cellular physiological functions. Studies have shown that the agonist-induced Ca 2+ level is critical for PLT activation in hemostasis and thrombosis(Varga-Szabo et al. 2009 ). Increased procoagulant activity of PLTs can be initiated by integrin αIIbβ3 (GPIIb/IIIa)/Gα13-mediated co-stimulation of outward-inward signaling and GPVI signaling, leading to intracellular Ca 2+ release above a threshold(Kaiser et al. 2022 ). In a study on procoagulant PLTs, it was mentioned that only the procoagulant PLTs showed high cytoplasmic Ca 2+ level as detected by fluorescent probes(Abbasian et al. 2020 ). In good accordance with this, our study reveal that TiO 2 NP treatment increases intracellular Ca 2+ level, which is required in induction of PLT activation, aggregation and procoagulant activity, finally leading to AT in mice. Thus, it is noteworthy that our study may provide new clues on the preventive and therapeutic strategies of AT caused by TiO 2 NP treatment. Conclusions Our study reveals a pro-thrombotic effect of TiO 2 NPs by pro-coagulant activity and activation/aggregation of PLTs. Mechanistically, TiO 2 NPs initiate PS exposure and MVs generation, ultimately leading to a heightened pro-coagulant activity. Simultaneously, these particles boost the expression of P-selectin and activate GPIIb/IIIa, thereby facilitating the activation/aggregation of platelets. This entire process involves an increase in intracellular Ca 2+ levels, making it calcium-dependent. Most importantly, we demonstrated the relevance of our findings in vivo by showing that intravenous administration of TiO 2 NPs can increase arterial thrombosis in mouse carotid arteries, emphasizing the need for caution during medical applications of TiO 2 NPs. Declarations Not applicable. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fundings This research was funded by the National Natural Science Foundation of China 82241090 (Y.X.), 82022063 (Y.X.), 82003500 (Y.B), 82211540403 (Y.B.) and Department of Science and Technology of Liaoning Province 2022JH2/20200035(Y.X.), 2022JH2/20200017 (Y.B.). Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions Yiying Bian : designed and performed most part of in vitro experiments, writing-review & editing, Funding acquisition, Supervision. Qiushuo Jin : writing original manuscript, and performed most part of animal experiments. Jinrui He and Thien Ngo : did partial animal experiments and data analysis. OK-Nam Bae, Jingbo Pi and Han Young Chung : validation, data analysis and review. Yuanyuan Xu : writing - review & editing, funding acquisition, data interpretation, and supervision. All authors reviewed the manuscript. Ethics approval With the approval from the Ethics Committee of the Health Service Center at Seoul National University, human blood was obtained from healthy male donors. All the animal protocols used in vivo experiments were approved by the Ethics Committee of the Animal Service Center at China Medical University. Data availability The data and material that support the findings of this study are available from the corresponding author upon reasonable request. References Thrombosis: a major contributor to global disease burden. 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Supplementary Files Graphicabstract.jpg Cite Share Download PDF Status: Published Journal Publication published 07 Aug, 2024 Read the published version in Cell Biology and Toxicology → Version 1 posted Editorial decision: Revision requested 03 Jun, 2024 Reviews received at journal 22 May, 2024 Reviews received at journal 16 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers invited by journal 16 May, 2024 Submission checks completed at journal 31 Mar, 2024 Editor assigned by journal 31 Mar, 2024 First submitted to journal 29 Mar, 2024 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. 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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-4187973","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285959646,"identity":"61fa59f2-b46c-4944-9cba-759f3f3434e9","order_by":0,"name":"Yiying Bian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYFACHoYDDAw2MDbxWtJI1AIEh0nQYnAj9+CBnzvOy+vOSGB88LaNQd6csJa8hIO9Z24bbruRwGw4t43BcGcDQS05Bgd4224nmN1IYJPmbWNIMDhAhJaDf9vOgbSw/yZay2HetgNgW5iJ0iJ55l3CYdm2ZMNtZx42S845J2G4gZAWvuO5hz++bbOTNzuefPDDmzIbeYK2KFxIgDEZG4CEBAH1QCDfT8jQUTAKRsEoGAUAg0hG6COOIY4AAAAASUVORK5CYII=","orcid":"","institution":"Key Laboratory of Environmental Stress and Chronic Disease Control \u0026 Prevention Ministry of Education (China Medical University)","correspondingAuthor":true,"prefix":"","firstName":"Yiying","middleName":"","lastName":"Bian","suffix":""},{"id":285959647,"identity":"3ac91177-2658-4d1a-8326-167210eff87e","order_by":1,"name":"Qiushuo Jin","email":"","orcid":"","institution":"Key Laboratory of Environmental Stress and Chronic Disease Control \u0026 Prevention Ministry of Education (China Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Qiushuo","middleName":"","lastName":"Jin","suffix":""},{"id":285959648,"identity":"7634e465-ef44-4b5d-b6ab-7605f7c7f1a2","order_by":2,"name":"Jinrui He","email":"","orcid":"","institution":"Key Laboratory of Environmental Stress and Chronic Disease Control \u0026 Prevention Ministry of Education (China Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Jinrui","middleName":"","lastName":"He","suffix":""},{"id":285959649,"identity":"0d7a722a-aee8-4042-a004-942d7945f38e","order_by":3,"name":"Thien Ngo","email":"","orcid":"","institution":"Faculty of Pharmacy, Thai Binh University of Medicine and Pharmacy, Thai Binh City 410000, Vietnam","correspondingAuthor":false,"prefix":"","firstName":"Thien","middleName":"","lastName":"Ngo","suffix":""},{"id":285959650,"identity":"49b134df-324e-4b9f-a0bd-b2644642e114","order_by":4,"name":"OK-Nam Bae","email":"","orcid":"","institution":"College of Pharmacy, Hanyang University, Ansan, Gyeonggido, 426-791, South Korea","correspondingAuthor":false,"prefix":"","firstName":"OK-Nam","middleName":"","lastName":"Bae","suffix":""},{"id":285959651,"identity":"eecdd6c7-d1d4-4e98-a093-beb5f4997f3c","order_by":5,"name":"Jingbo Pi","email":"","orcid":"","institution":"Key Laboratory of Environmental Stress and Chronic Disease Control \u0026 Prevention Ministry of Education (China Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Jingbo","middleName":"","lastName":"Pi","suffix":""},{"id":285959652,"identity":"49c0dde9-6c78-4752-8efa-7ff5927d5348","order_by":6,"name":"Han Young Chung","email":"","orcid":"","institution":"Center for Food and Bioconvergence, Seoul National University, Seoul 08826, South Korea","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"Young","lastName":"Chung","suffix":""},{"id":285959653,"identity":"87c7c997-ebc5-4eb4-902d-e6bbfe2bb9f7","order_by":7,"name":"Yuanyuan Xu","email":"","orcid":"","institution":"Key Laboratory of Liaoning Province on Toxic and Biological Effects of Arsenic (China Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-03-29 12:48:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4187973/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4187973/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10565-024-09908-y","type":"published","date":"2024-08-07T15:57:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54033137,"identity":"93844cbe-821a-4afd-9722-1f02112b3228","added_by":"auto","created_at":"2024-04-03 16:29:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17685366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of titanium dioxide nanoparticles (TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNPs) and the uptake by human platelets (hPLTs).\u003c/strong\u003e (a) SEM observation showed the size of TiO\u003csub\u003e2\u003c/sub\u003eNPs between 20 and 70 nm with an average diameter of 35.7 nm. Scale bar: 100 nm. (b) The dynamic size distribution of TiO\u003csub\u003e2\u003c/sub\u003eNPs by the intensity in saline and in PBS with a peak of distribution at 148.3 nm and 197.2 nm, respectively. (c) The zeta potential of TiO\u003csub\u003e2\u003c/sub\u003eNPs in distilled water was +11.7 mV as tested by a nanoparticle size analyzer. (d) TEM observation of uptake of control (distilled water) and TiO\u003csub\u003e2\u003c/sub\u003eNPs by hPLTs. Yellow arrowhead: hPLT; black scale bar: 500 nm; dashed circles: TiO\u003csub\u003e2\u003c/sub\u003eNPs within hPLTs; dashed box: TiO\u003csub\u003e2\u003c/sub\u003eNPs adhered to hPLT membrane surface.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/0f481a28c3388be6d9437541.jpg"},{"id":54033135,"identity":"fc8e4a39-3289-4dc7-baee-6a13e8a1529f","added_by":"auto","created_at":"2024-04-03 16:29:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16179930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNPs enhance hPLT procoagulant activity through phosphatidylserine (PS) exposure.\u003c/strong\u003e (a) A diagram of prothrombinase assay showing PS exposure of PLTs in the process of thrombin generation indicating increased procoagulant activity after TiO\u003csub\u003e2\u003c/sub\u003eNP treatment. (b-c) Isolated hPLTs were treated with various concentrations (1, 10, 25, and 100 μg/mL) of TiO\u003csub\u003e2\u003c/sub\u003eNPs at 37 °C for 10 min. Both (b) PS exposure and (c) MV generation were measured by flow cytometry. (d) The procoagulant activity was determined by thrombin generation using prothrombinase assays as mentioned in Methods. Values are mean ± SD of the independent experiments from different blood donors (n = 5 - 7). The asterisk represents significant differences from the control group (***\u003cem\u003eP\u003c/em\u003e＜0.001; **\u003cem\u003eP\u003c/em\u003e＜0.01; *\u003cem\u003eP\u003c/em\u003e＜0.05).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/9271c02f65fbef05183f4c03.jpg"},{"id":54033136,"identity":"0077adde-5e41-459d-acb7-40b5dc1eac37","added_by":"auto","created_at":"2024-04-03 16:29:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16447185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNPs induce hPLT aggregation and activation through increased GPⅡb/Ⅲa activation and P-selectin level. \u003c/strong\u003e(a) \u003cem\u003eIn vitro\u003c/em\u003e experimental method using PLTs freshly isolated from health volunteers. (b-c) Percentage of hPLT aggregation after hPLTs were treated to various concentrations (0.5, 1, 5, 10, 25, and 100 μg/ml) of TiO\u003csub\u003e2\u003c/sub\u003eNPs for (b) 5 min, (c) 30 min and 60 min. (d) LDH leakage of hPLTs induced by TiO\u003csub\u003e2\u003c/sub\u003eNPs. DIG, digitonin 50 μM treatment for 1 h, used as positive control. (e) GPIIb/IIIa activation and (f) P-selectin level were measured by flow cytometry. Values are mean ± SD of the independent experiments from different blood donors (n = 3 - 5). The asterisk represents significant differences from the control group (***\u003cem\u003eP\u003c/em\u003e＜0.001; **\u003cem\u003eP\u003c/em\u003e＜0.01; *\u003cem\u003eP\u003c/em\u003e＜0.05).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/f26702acd482e43f6aed4209.jpg"},{"id":54033140,"identity":"ca00f77b-4d73-465c-bf91-d4e114b429e4","added_by":"auto","created_at":"2024-04-03 16:29:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16812907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNP-induced hPLT aggregation is exacerbated under physiology-mimicking conditions. \u003c/strong\u003e(a) A diagram showing a basic \u003cem\u003ein vitro\u003c/em\u003e experimental method \u003cem\u003eversus\u003c/em\u003e an \u003cem\u003ein vitro\u003c/em\u003e experimental method with adjustable physiological simulating conditions. (b-c) Aggregation of hPLTs after adding thrombin (0.6-0.8 U/mL), collagen (2-4 μg/mL) or shear stress (1500 s\u003csup\u003e−1\u003c/sup\u003e for 3 min) under various concentrations (0.1, 0.5, 1, and 10 μg/ml) of TiO\u003csub\u003e2\u003c/sub\u003eNPs. Values are mean ± SD of the independent experiments from different blood donors (n = 3 - 5). The asterisk represents significant differences from the no stimuli or no shear stress group (***\u003cem\u003eP\u003c/em\u003e＜0.001; *\u003cem\u003eP\u003c/em\u003e＜0.05).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/bde41b633131fb4498a63ddd.jpg"},{"id":54033138,"identity":"c5eb6015-85ab-4bf8-bd5e-45e02769f2f9","added_by":"auto","created_at":"2024-04-03 16:29:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":15477894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInvolvement of Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e in procoagulant activity, activation and aggregation of hPLT induced by TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNPs. \u003c/strong\u003e(a) Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e level in hPLTs was measured by adding Fluo-4 AM after hPLTs were treated with 100 μg/mL TiO\u003csub\u003e2\u003c/sub\u003eNPs for 10 min. (b) PS exposure, (c) procoagulant activity, (d) hPLT aggregation, (e) GPIIb/IIIa activation, and (f) P-selectin level were performed by preloading a calcium chelating agent (EGTA) prior to TiO\u003csub\u003e2\u003c/sub\u003eNP treatment. Values are mean ± SD of the independent experiments from different blood donors (n = 3 - 6). The asterisk represents significant differences from the control group (***\u003cem\u003eP\u003c/em\u003e＜0.001; **\u003cem\u003eP\u003c/em\u003e＜0.01; *\u003cem\u003eP\u003c/em\u003e＜0.05). The pound represents significant differences from the TiO\u003csub\u003e2\u003c/sub\u003eNP treatment group (\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.001; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/318a64b8659694f99927a7b9.jpg"},{"id":54033143,"identity":"16dc609b-db0d-4e23-8573-60c6a1b1857b","added_by":"auto","created_at":"2024-04-03 16:29:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16061494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNP treatment results in decreased PLT counts and increased large PLT ratio in mice. \u003c/strong\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs were intravenously injected into mice, and 1 h later, blood was collected from mice for hemocyte analysis. (a) PLT counts by impedance method (PLT-I), PLT counts by optical method (PLT-O), (b) percentage of plateletcrit (PCT%), (c) percentage of large PLTs (P-LCR%), (d) mean PLT volume by impedance (MPV-I) and by optical (MPV-O), (e) PLT volume distribution width (PDW), and (f) mean PLT volume/PLT count (MPV/P). At the same time, red blood cell-related indexes, such as (g) red blood cell count, percentage of hematocrit, (h) hemoglobin, and (i) white blood cell-related indexes, including white blood cell count and classification percentage were tested separately. Values are mean ± SD of the independent experiments from mice (n = 4 - 6). The asterisk represents significant differences from the control group (***\u003cem\u003eP\u003c/em\u003e＜0.001; *\u003cem\u003eP\u003c/em\u003e＜0.05)\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/386b06689d25449fc307106c.jpg"},{"id":54033142,"identity":"81c61976-ca47-468b-8309-c985ce2f8712","added_by":"auto","created_at":"2024-04-03 16:29:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":17713953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNPs trigger PLT dysfunction\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand locally accelerate arterial blood flow signals in mice. \u003c/strong\u003e(a-c) A diagram of \u003cem\u003eex vivo\u003c/em\u003e study: 1 h after TiO\u003csub\u003e2\u003c/sub\u003eNP intravenous injection, (b) percentage of PLT aggregation was calculated by the impedance method and (c) P-selectin was measured using flow cytometry. (d) A diagram of the FeCl\u003csub\u003e3\u003c/sub\u003e-initiated AT mouse model. (e-i) Blood flow signals in the common carotid artery of mice were measured using small animal doppler color ultrasound. (f) Blood flow velocity, (g) blood flow volume, (h) internal diameter of the common carotid artery, and (i) vascular resistance index, were detected and quantified. Values are mean ± SD of the independent experiments from mice (n = 3 - 5). The asterisk represents significant differences from the control group (**\u003cem\u003eP\u003c/em\u003e＜0.01; *\u003cem\u003eP\u003c/em\u003e＜0.05).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/7e0311848abb9c6fcfffcf52.jpg"},{"id":54033141,"identity":"c1e46848-76dc-4d25-a1f1-468f214820f5","added_by":"auto","created_at":"2024-04-03 16:29:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3020509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNP treatment leads to PLT-involved AT in mice. \u003c/strong\u003e(a) Immunofluorescence staining of arterial thrombi was obtained using AT mouse model and (b) the blank area of aorta (area that blood flow can go through) was quantified as described in Methods. (c) The pathological composition of the thrombus was observed using immunofluorescence staining for PLTs (red, CD42b), α-fibrinogen (green) and DAPI (blue), respectively. Scale bar: 100 μm. Thrombus were obtained from independent mouse samples (n = 3).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/4d58936a268cce48cda63807.jpg"},{"id":62298341,"identity":"29be5142-46df-4456-a9e1-9c5d9885a4df","added_by":"auto","created_at":"2024-08-12 16:12:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":120389915,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/bb452f5c-2ed4-437c-a4ee-87550b940eeb.pdf"},{"id":54033134,"identity":"802bd55c-52bd-47de-be4c-2d949b9c6500","added_by":"auto","created_at":"2024-04-03 16:29:39","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3768011,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4187973/v1/644dbd06dd54e94496d1470f.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBiomedical application of TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe titanium dioxide nanoparticles (TiO\u003csub\u003e2\u003c/sub\u003eNPs) that are smaller than 100 nm are widely utilized in biomedical fields such as photoimaging, drug delivery and biological analysis due to their antimicrobial properties, photocatalytic activity, excellent biocompatibility, and corrosion resistance(Najahi-Missaoui et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao and Castranova \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Often, intravenous injection is the norm, allowing the distribution of these nanoparticles to each organ via the bloodstream for therapeutic purposes. However, this process frequently leads to unexpected organ toxicity, including liver, kidney, and brain injuries(Shi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Given the significance of blood cells as the primary and essential contact component in the blood circulation process, it is noteworthy that toxicological research on these cells remains relatively scarce. Hence, the potential health risk of TiO\u003csub\u003e2\u003c/sub\u003eNP treatment, particularly from a perspective focusing on blood cells, is becoming a large concern(Lee et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Naserzadeh et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Qi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shakeel et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe thrombosis, which is responsible for one in four deaths(2014), can be primarily categorized into venous, arterial, and microvascular thrombosis(Brandtner et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Petri \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our previous study have demonstrated that TiO\u003csub\u003e2\u003c/sub\u003eNPs have the ability to initiate procoagulant activity in red blood cells, ultimately culminating in the development of venous thrombosis(Bian et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although some reports have proposed that TiO\u003csub\u003e2\u003c/sub\u003eNPs can target platelets and cause microclots in microcirculation, their effects appear to be primarily restricted to platelet aggregation \u003cem\u003ein vitro\u003c/em\u003e(Haberl et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Nonetheless, in-depth thorough investigation of the underlying mechanism and the presence of other forms of platelet dysfunction still remain unexplored.\u003c/p\u003e \u003cp\u003eRecently, the activation and aggregation of platelets, as indicated by an elevated expression of the integrin αIIbβ3 (GPIIb/IIIa complex, CD41/CD61) and the transmembrane protein P-selectin within the alpha granules, have been strongly linked to thrombotic disorders(Huang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Qiao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yeini and Satchi-Fainaro \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, recent studies have indicated that the pro-coagulant activity of platelets (PLTs) plays a crucial role in the development of thrombosis, which is primarily attributed to the level of phosphatidylserine (PS) exposed on the outer membrane surface and the release of microvesicles (MVs)(Pang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zlamal et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The two processes are caused by an elevated calcium level, leading to hemostasis and thrombosis(Obydennyy et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Varga-Szabo et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), ultimately pointing to the same outcome: the arterial thrombosis.\u003c/p\u003e \u003cp\u003eIn this study, we initially established that intravenous administration of TiO\u003csub\u003e2\u003c/sub\u003eNPs swiftly instigates procoagulant activity within platelets by enhancing phosphatidylserine exposure and microvesicle production. Additionally, we observed prompt platelet activation and aggregation following TiO\u003csub\u003e2\u003c/sub\u003eNP treatment through the activation of GPIIb/IIIa and the expression of P-selectin. These findings indicate that TiO\u003csub\u003e2\u003c/sub\u003eNPs possess the potential to alter platelet function and promote the carotid artery thrombosis, a process that necessitates an elevated intracellular calcium level. Indeed, we also observed dysregulation of blood flow signals and arterial thrombosis along with increased PLT deposition using a carotid artery thrombosis mice model. These results provide clear evidence for the risk of PLT-related arterial thrombosis caused by TiO\u003csub\u003e2\u003c/sub\u003eNP treatment, and help to improve the understanding of the arterial thrombotic risk caused by TiO\u003csub\u003e2\u003c/sub\u003eNPs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs (anatase, nanopowder, \u0026lt; 100 nm particle size), trisodium citrate, HEPES, prostaglandin E1 (PGE1), glutaraldehyde, EDTA, EGTA, ferric chloride, urethane, clopidogrel, and bovine serum albumin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Thrombin and collagen were obtained from Calbiochem (San Diego, CA, USA) and Chrono-log (Havertown, PA, USA), respectively. Fluorescein isothiocyanate (FITC)-labeled anti-CD62P antibody (anti-CD62P-FITC Ab), FITC-labeled PAC-1 (PAC-1-FITC), and FITC-labeled annexin V (annexin V-FITC) were from BD Biosciences (San Jose, CA, USA). Fluo-4 acetoxymethyl ester (Fluo-4 AM) was obtained from Invitrogen (Carlsbad, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBlood collection and preparation of human platelets (hPLTs)\u003c/h2\u003e \u003cp\u003e This work was supported by the Ethics Committee of the Health Service Center at Seoul National University with the approval from the Institutional Review Board of Seoul National University (IRB No. 1702/003\u0026ndash;004, 5, March, 2019). To simplify our study design, we selected only healthy male donors aged 20\u0026ndash;30 years, excluding risk factors for thrombosis such as gender, age, and disease, and taking any medication in the past 2 weeks. For platelet-rich-plasma (PRP) preparation, whole blood with 3.2% trisodium citrate was centrifuged at 150 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min and platelet cell count in PRP was adjusted 3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells/mL by diluting with platelet-poor-plasma (PPP) on the day of experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of TiO\u003csub\u003e2\u003c/sub\u003eNPs\u003c/h2\u003e \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs were not surface treated and insoluble in water, hydrochloric acid, or nitric acid. The particles were dispersed in distilled water and sonication was performed before each experiment, using an ultrasonicator with a maximum output of 150\u0026ndash;200 W for 15 s to prevent agglomeration. TiO\u003csub\u003e2\u003c/sub\u003eNPs were dried and observed with scanning electron microscope (SEM) (SU8010, Hitachi Limited, Japan) to examine the size distribution. Detailed statistical analysis (Nano measure 1.2 and OraginPro2021) of TiO\u003csub\u003e2\u003c/sub\u003eNPs were performed by random measurement of 100 nanoparticles in the images taken by SEM. The dynamic particle size of the nanoparticles was evaluated using a Malvern laser particle size analyzer (DLS-7000, Otsuka Electronics, Co., Osaka, Japan). The sample was weighed and dispersed in deionized water (0.1% mass fraction), sonicated for 3 min, adjusted to pH 7.4 with NaOH or HCl. The zeta potential was measured with a nanoparticle size analyzer (ZS-90, Malvern Instruments, UK). All data were repeated three times and averaged. Technical support was provided by Beijing Standard Spectrum Testing Technology Co.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of PLT aggregation\u003c/h2\u003e \u003cp\u003eAfter incubation with TiO\u003csub\u003e2\u003c/sub\u003eNPs (0.5, 1, 5, 10, 25, and 100 \u0026micro;g/ml; 5 min, 30 min, and 60 min) at 37\u0026deg;C, the number of individual PLTs per microliter was calculated using optical microscopy and the degree of PLT aggregation was assessed based on the count of single cells. Data were presented as percentages of PLT aggregation. Besides, PLT aggregation induced by TiO\u003csub\u003e2\u003c/sub\u003eNP treatment under physiology-mimicking conditions was observed by the above method under stimulation with thrombin (0.6\u0026ndash;0.8 U/ml), collagen (2\u0026ndash;4 \u0026micro;g/ml), and shear stress at 1500 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 3 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of LDH leakage\u003c/h2\u003e \u003cp\u003eLactate dehydrogenase (LDH) leakage from PLT was measured by spectrophotometry. After incubation with TiO\u003csub\u003e2\u003c/sub\u003eNPs for 5 min, the supernatant obtained from the centrifuge reaction mixture was used for LDH determination (digitonin 50 \u0026micro;M treatment for 1 h was used as positive control). The degree of cell lysis was expressed as a percentage of total enzyme activity compared to control incubation with cleavage with digitonin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eAfter PLTs were exposed with TiO\u003csub\u003e2\u003c/sub\u003eNPs at 37\u0026deg;C for 10 min, the level of P-selectin and the activation of glycoprotein GPIIb/IIIa were determined by staining with CD62P-FITC and PAC-1-FITC for 20 min, respectively. PS exposure and MV generation in PLTs were examined by staining with both annexin V-FITC and anti-CD42b-PE Ab (as PLT identifier). Intracellular calcium level was determined by pre-loading fluo-4/AM (5 \u0026micro;M) for 45 min. All the determinations were conducted using FACS Calibur (Becton Dickinson, USA), and Cell Quest Pro software was used to collect and analyze data from 10,000 events.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eA prothrombinase assay\u003c/h2\u003e \u003cp\u003eThe prothrombinase assay was applied to assess procoagulant activity. Specifically, TiO\u003csub\u003e2\u003c/sub\u003eNP-treated PLTs were further induced into thrombin generation by adding 5 nM Xa factor, 10 nM Va factor and prothrombin. Then, chromogenic substrate S2238 (Chromogenic, Milan, Italy) was used to measure the generated thrombin after adding a stop buffer (50 mM Tris-HCl, 120 mM NaCl, 2 mM EDTA, pH 7.9). Calculation of thrombin production rate was based on the absorbance change at 405 nm from the calibration curve.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThe experimental C57BL/6 J mice (male, 12 weeks old) were kept in a clean environment without specific pathogens and fed with SPF chow diet and distilled water. The indoor temperature was controlled at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃, the humidity was 55\u0026ndash;70%, and the circadian rhythm was alternated (12 h/12 h). Mice were randomly divided into two groups: control group and exposed group (TiO\u003csub\u003e2\u003c/sub\u003eNPs, 25 mg/kg, intravenous injection). All animal experiments were reviewed and approved by the Animal Ethics Committee of China Medical University (CMU20231000).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eassessment using blood cell analyzer\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBlood (30 \u0026micro;l) was collected from mice tail with EDTA-K\u003csub\u003e2\u0026minus;\u003c/sub\u003econtaining tube 1 h post TiO\u003csub\u003e2\u003c/sub\u003eNP injection. Then fresh blood samples were tested by an automated five-classification animal blood cell analyzer (IDEXX ProCyte Dx, Japan) for the following indicators: PLT count (performed by both impedance (PLT-I) and optical (PLT-O) method), percentage of PLT-larger cell ratio (L-PCR%), PLT crit (PLT%), mean PLT volume (MPV), mean PLT volume/PLT count (MPV/P), PLT distribution width (PDW), red blood cell count, percentage of hematocrit, hemoglobin, and white blood cell-related indexes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEx vivo\u003c/em\u003e measurement of mPLT aggregation was conducted by an animal blood cell analyzer with impedance method, which only allows one single cell go through. The PLT aggregation rate was calculated by the formula: (PLT\u003csub\u003eCont\u003c/sub\u003e-PLT\u003csub\u003eTiO2NPs\u003c/sub\u003e)/PLT\u003csub\u003eCont\u003c/sub\u003e \u0026times;100)%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eArterial thrombosis in mice\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eArterial thrombosis mouse model\u003c/strong\u003e \u003cp\u003eThe left common carotid artery was isolated from the left side of the trachea using 3\u0026times;5 mm tin foil to isolate the surrounding tissues. Afterwards, 1\u0026times;2 mm filter paper was fully submerged with 5% FeCl\u003csub\u003e3\u003c/sub\u003e and applied to the left common carotid artery for 15 min.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eUltrasound observation\u003c/strong\u003e \u003cp\u003eThe blood flow signal by the Doppler ultrasound in the mouse model was slightly adjusted based on previous study(Jing et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang and Xu \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Mice were anesthetized with isoflurane inhalation at 3% and maintained at 2% throughout the procedure and then cleaned of hair on the neck and chest to facilitate ultrasound. AVINNO6 LAB small animal Doppler ultrasound system (VINNO Co., China) was used. The left common carotid artery was imaged and the flow signals (including velocity, flow volume, internal diameter, perfusion index, heart rate) were quantified.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThrombus pathological experiment\u003c/h2\u003e \u003cp\u003eThe tissue of the common carotid artery was collected for the pathological experiment. The thrombus tissue was embedded using the frozen section embedding agent Sakura OCT (a water-soluble mixture of polyethylene glycol and polyvinyl alcohol), sectioned at 6 \u0026micro;m thickness with frozen sectioning machine (CM 1950, LEICA, Germany), and collected using adhesive slides to observe thrombus morphology under a general light microscope. By measuring the percentage of each blank area in the thrombus (the area that blood flow can pass through), it is artificially divided into the following groups: 0\u0026ndash;1, 1\u0026ndash;5, and 5\u0026ndash;10% of the total area. Less blank area means more compact thrombus.\u003c/p\u003e \u003cp\u003eImmunofluorescence observations for frozen sections were examined by adding α-fibronogen with Alexa Fluor\u0026reg; 488 and CD42b with Alexa Fluor\u0026reg; 568, respectively. Then, the samples were sealed with a blocking buffer containing a liquid with an anti-fluorescent cracking agent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean and standard deviation. Data were subjected to Student's \u003cem\u003et\u003c/em\u003e-test or two-way ANOVA followed by Duncan's multiple range test. In all cases, a \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. The asterisk represents significant differences from the control group (***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The pound represents significant differences from the TiO\u003csub\u003e2\u003c/sub\u003eNP treatment group (\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of TiO\u003csub\u003e2\u003c/sub\u003eNPs and the uptake by human platelets (hPLTs)\u003c/h2\u003e \u003cp\u003eThe physicochemical characterization of TiO\u003csub\u003e2\u003c/sub\u003eNPs was examined by SEM and DLS. The size distribution of TiO\u003csub\u003e2\u003c/sub\u003eNPs was in the range of 20 to 70 nm with an average diameter of 35.7 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), which was randomly calculated from 100 particles shown in SEM images. DLS data further showed that the average size by intensity was 148.3 nm in saline (with 10% FBS) and 197.2 nm in PBS, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The zeta potential of TiO\u003csub\u003e2\u003c/sub\u003eNPs was +\u0026thinsp;11.7 mV at pH 7.4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Interestingly, TEM observation showed that TiO\u003csub\u003e2\u003c/sub\u003eNPs were within hPLTs (dashed circles) or adhered to hPLT membrane (dashed box) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating a possible PLT-associated outcome in response to TiO\u003csub\u003e2\u003c/sub\u003eNP treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs enhance hPLT procoagulant activity through PS exposure and MVs generation\u003c/h2\u003e \u003cp\u003eIn resting PLTs, PS are commonly in the inner leaflet of the membrane. Upon stimuli, PS are externalized to the outer leaflets and microvesicles (MVs, \u0026lt; 1 \u0026micro;m) are released, both of which participate in coagulation pathway and then promote the production of thrombin from prothrombin under prothrombinase complex (Va, Xa)(Lentz \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). To estimate procoagulant activity of PLTs induced by TiO\u003csub\u003e2\u003c/sub\u003eNPs, we treated hPLTs with TiO\u003csub\u003e2\u003c/sub\u003eNPs for 10 min, determined PS exposure and MVs generation using FACs, and estimated thrombin generation under prothrombinase complex (Va, Xa) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). PS exposure and MVs were significantly increased in a concentration-dependent manner after TiO\u003csub\u003e2\u003c/sub\u003eNP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In parallel, with a prothrombinase assay, TiO\u003csub\u003e2\u003c/sub\u003eNP treatment accelerated thrombin generation in hPLTs, reflecting increased procoagulant activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs induce hPLT aggregation and activation via GPⅡb/Ⅲa activation and increased P-selectin level\u003c/h2\u003e \u003cp\u003eTo exam whether TiO\u003csub\u003e2\u003c/sub\u003eNPs induce PLT aggregation, we used PRP freshly isolated from healthy volunteers and incubated hPLTs with TiO\u003csub\u003e2\u003c/sub\u003eNPs for 5 min, 30 min or 60 min as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. In consequence, TiO\u003csub\u003e2\u003c/sub\u003eNPs caused PLT aggregation at 5 min \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Upon 30 and 60 min exposure, TiO\u003csub\u003e2\u003c/sub\u003eNPs also caused PLT aggregation but did not show obvious time-dependent effects even when compared with 5 min exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), indicating that TiO\u003csub\u003e2\u003c/sub\u003eNPs rapidly exaggerate PLT aggregation which occurs within 5 min. Next, we examined the leakage of LDH from hPLTs and found that no cytotoxicity was induced by TiO\u003csub\u003e2\u003c/sub\u003eNP treatment (digitonin was used as a positive control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Glycoprotein IIb/IIIa (GPIIb/IIIa) plays a key role in the maintenance of PLT aggregation(Aliotta et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, P-selectin is an adhesion molecule belonging to the selectin family expressed on PLTs, and activated PLTs express high levels of P-selectin(Yeini and Satchi-Fainaro \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, GPⅡb/Ⅲa activation and P-selectin expression were both significantly induced by TiO\u003csub\u003e2\u003c/sub\u003eNP treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTiO\u003csub\u003e2\u003c/sub\u003eNP-induced hPLT aggregation is exacerbated under physiology-mimicking conditions\u003c/h2\u003e \u003cp\u003eIn the physiological system, the presence of physiological aggregators (e.g. thrombin and collagen) contributes to the heterogeneity in PLT responses(Aslam et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Petzold et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, vascular shear force plays an existential role in the physiological function of PLTs(Casa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yagi et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). To assess the effect of TiO\u003csub\u003e2\u003c/sub\u003eNPs on PLT aggregation more accurately, we determined PLT aggregation using an \u003cem\u003ein vitro\u003c/em\u003e experimental method with adjustable physiological simulating conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, ⅰ) and compared the results with those from a basic \u003cem\u003ein vitro\u003c/em\u003e experimental method (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, ⅱ). After adding thrombin or collagen \u003cem\u003ein vitro\u003c/em\u003e, accelerated TiO\u003csub\u003e2\u003c/sub\u003eNP-induced PLT aggregation was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Similarly, after adding shear stress \u003cem\u003ein vitro\u003c/em\u003e that simulates physiological high shear flow, enhanced PLT aggregation was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). More significantly, the existence of physiological aggregators (thrombin and collagen) and high shear stress reduced the minimum toxic level of TiO\u003csub\u003e2\u003c/sub\u003eNPs for PLT aggregation from 1 \u0026micro;g/mL to 0.1 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, insert) and 0.5 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, insert), respectively, as compared to the basic condition. These data indicate that TiO\u003csub\u003e2\u003c/sub\u003eNPs boost more severe PLT aggregation under physiology-mimicking conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eInvolvement of Ca\u003csup\u003e2+\u003c/sup\u003e in hPLT aggregation and procoagulant activity induced by TiO\u003csub\u003e2\u003c/sub\u003eNPs\u003c/h2\u003e \u003cp\u003eIntracellular Ca\u003csup\u003e2+\u003c/sup\u003e plays a key role in PLT activation, aggregation and procogulant activity(Back et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xiang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Here, we observed a concentration-dependent increase in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e level after TiO\u003csub\u003e2\u003c/sub\u003eNP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). To further assess the role of Ca\u003csup\u003e2+\u003c/sup\u003e in PLT dysfunction, we treated hPLTs with EGTA (a chelator of Ca\u003csup\u003e2+\u003c/sup\u003e) prior to TiO\u003csub\u003e2\u003c/sub\u003eNP treatment. In consequence, increased PS exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and procoagulant activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) induced by TiO\u003csub\u003e2\u003c/sub\u003eNPs were both effectively reduced by EGTA. Concurrently, TiO\u003csub\u003e2\u003c/sub\u003eNP-induced hPLT aggregation was markedly blocked by EGTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Meanwhile, elevated GPⅡb/Ⅲa activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) and P-selectin level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) by TiO\u003csub\u003e2\u003c/sub\u003eNP treatment were both significantly inhibited by EGTA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTiO\u003csub\u003e2\u003c/sub\u003eNP treatment decreases PLT counts and increases large PLT ratio in mice\u003c/h2\u003e \u003cp\u003eTo elucidate the \u003cem\u003ein vivo\u003c/em\u003e effect of TiO\u003csub\u003e2\u003c/sub\u003eNPs, mice were intravenously injected with TiO\u003csub\u003e2\u003c/sub\u003eNPs and after 1 h, blood was collected and analyzed using a blood cell analyzer. It is clear from the data that PLT counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) detected by the impedance method (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, left) and the optical method (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, right), respectively, as well as PCT% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), were significantly declined in TiO\u003csub\u003e2\u003c/sub\u003eNP-exposed mice. Meanwhile, P-LCR% was increased apparently in TiO\u003csub\u003e2\u003c/sub\u003eNP-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). In addition to P-LCR, MPV, PDW and MPV/P were all considered as potential indicators of PLT shape and function(Azab et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Gasparyan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Accordingly, increases in MPV (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed) and MPV/P (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef) after TiO\u003csub\u003e2\u003c/sub\u003eNP treatment were observed, but there was no change in PDW (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eMoreover, TiO\u003csub\u003e2\u003c/sub\u003eNP-exposed mice showed no significant change in red blood cells (RBC) index including RBC counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, left), hematocrit (HCT%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, right), hemoglobin (HGB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh), or white blood cells (WBC) index including neutrophils, lymphocytes, monocytes, eosinophils and basophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs trigger PLT dysfunction and locally alter arterial blood flow signals in mice\u003c/h2\u003e \u003cp\u003eTo further determine whether TiO\u003csub\u003e2\u003c/sub\u003eNPs cause PLT aggregation and activation, we firstly did \u003cem\u003eex vivo\u003c/em\u003e study using PLTs isolated from mice (mPLTs) treated with TiO\u003csub\u003e2\u003c/sub\u003eNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). TiO\u003csub\u003e2\u003c/sub\u003eNPs resulted in increased mPLT aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) and P-selectin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) level 1 h post treatment. Numerous studies have shown that PLT activation and aggregation are closely related to thrombosis(Nayak et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yeung et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, we established an AT-initiating mice model 1 h after TiO\u003csub\u003e2\u003c/sub\u003eNP treatment, and phenotypes were determined by color doppler ultrasonography (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, ⅰ). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, TiO\u003csub\u003e2\u003c/sub\u003eNP-exposed group showed different flow signals compared with control group. Specifically, TiO\u003csub\u003e2\u003c/sub\u003eNP-exposed mice represented obvious accelerated arterial blood flow velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef), increased blood flow volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg), and a slightly declined trend in the vascular resistance index (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei) with unconspicuous alterations in artery internal diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). Such doppler data indicate thrombi ahead, which is consistent with early reports on the phenomena of increased blood flow signals and thrombus formation(He et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Briefly, these all point to a critical clue that TiO\u003csub\u003e2\u003c/sub\u003eNP treatment increases the risk of AT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTiO\u003csub\u003e2\u003c/sub\u003eNP treatment leads to carotid artery thrombosis with PLT deposition in mice\u003c/h2\u003e \u003cp\u003eIn addition to the doppler ultrasonic detection, we conducted pathological sections of the arterial thrombus in mouse AT model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, ⅱ), and found increased thrombus formation in TiO\u003csub\u003e2\u003c/sub\u003eNP-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Besides, we noticed that thrombi formed in TiO\u003csub\u003e2\u003c/sub\u003eNP-exposed mice were fuller and more compact, reflecting that the blood flow became more sluggish. Hence, we further analyzed the blank area of aorta (area that blood flow can go through) as mentioned in methods, to reflect the potential unobstructed capacity of blood flow. The total counts were separated into three distinguished levels including the tiny (less than 1% of the total aorta), the moderate (1\u0026ndash;5% of the total aorta) and the loose blank area (5\u0026ndash;10% of the total aorta). Consistent with the increase in thrombosis, mice exposed to TiO\u003csub\u003e2\u003c/sub\u003eNPs showed significantly less total blank areas or blank areas at each level compared to control mice, reflecting a decrease in blood flow patency in mice with AT induced by TiO\u003csub\u003e2\u003c/sub\u003eNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Finally, we stained the thrombi by immunofluorescence with CD42b (red color) to reflect the active role of PLTs in TiO\u003csub\u003e2\u003c/sub\u003eNP-induced AT. The red fluorescence indicating PLT deposition within the thrombi was increased in TiO\u003csub\u003e2\u003c/sub\u003eNP-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe growing medical application of TiO\u003csub\u003e2\u003c/sub\u003eNPs has sparked critical consideration of their bio-safety. This study, for the first time, reveals that TiO\u003csub\u003e2\u003c/sub\u003eNPs can enhance procoagulant activity in isolated human platelets by increasing exposure of PS and the generation of MVs. Additionally, exposure to TiO\u003csub\u003e2\u003c/sub\u003eNPs rapidly triggers platelet activation and aggregation through increased expression of P-selectin and activation of GPIIb/IIIa. Here, intracellular calcium plays a role in both processes. Furthermore, TiO\u003csub\u003e2\u003c/sub\u003eNPs alter blood flow and exacerbate arterial thrombus formation in mice, accompanied by increased platelet deposition, underscoring the relevance of our findings to real \u003cem\u003ein vivo\u003c/em\u003e conditions.\u003c/p\u003e \u003cp\u003eNanoparticles are often used intravenously in medical use, which inevitably have unintended side effects on blood cells. Numerous studies have shown that these particles can impact macrophages(Dey et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), neutrophils(Yamano et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and lymphocytes. In addition, our previous research revealed that TiO\u003csub\u003e2\u003c/sub\u003eNPs can contribute to venous thrombosis by enhancing the procoagulant activity of red blood cells(Bian et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In peripheral blood, platelets also play key roles in hemostasis and thrombosis(Koupenova et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, using isolated human samples, we discovered that platelets are more sensitive to TiO\u003csub\u003e2\u003c/sub\u003eNPs than red blood cells were in our previous finding. This heightened sensitivity was evident at concentrations as low as 1 \u0026micro;g/mL of TiO\u003csub\u003e2\u003c/sub\u003eNPs (after less than 10 minutes \u003cem\u003ein vitro\u003c/em\u003e), which preceded the dysfunction of red blood cells, which occurred relatively high at 10 \u0026micro;g/mL after approximately 24 hours \u003cem\u003ein vitro\u003c/em\u003e. The findings of this study indicate that intravenous administration of TiO\u003csub\u003e2\u003c/sub\u003eNPs may pose a significant risk to blood cell function and homeostasis. It is crucial for future research to further investigate the potential long-term effects of these nanoparticles on blood cell function and cardiovascular health.\u003c/p\u003e \u003cp\u003eThe reports on toxicity of NPs are always controversial due to the ability to release ionic form from metal NPs. Some studies claim that the toxicity of metal NPs is due to the metal ions released by NPs, while others argue that the toxicity of metal NPs is due to the small particle size of NPs themselves and other physicochemical properties as additional influencing factors(Bian et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ye et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For instance, zinc oxide and copper oxide nanoparticles (ZnONPs and CuONPs) are easy to release their ionic forms and their main toxicity is considered to be caused by the metal ions(Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, with respect to silver nanoparticles (AgNPs), toxic effects are currently attributed into two aspects, the metal ion (Ag\u003csup\u003e+\u003c/sup\u003e) effect and the NP effect, respectively(Poynton et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Most earlier studies suggest that it is Ag\u003csup\u003e+\u003c/sup\u003e released from the AgNPs exerting a significant influence on protein regulation and the induction of cellular stress(Zhang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Different from this perspective, our previous study found that the level of Ag\u003csup\u003e+\u003c/sup\u003e released from AgNPs as detected by ICP-MS method was negligible (\u0026lt;\u0026thinsp;0.01%) and hardly initiated a significant response of the cell(Bian et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), indicating that the effect of NPs themselves rather than the ion effect is the main contributor of AgNP toxicity. A recent study has revealed that though the ions of AgNPs cause a certain degree of toxicity, but not as severe as the toxicity due to their physical characteristics as NPs(Cvjetko et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Unlike those typical metal NPs possessing the capability of releasing ionic forms, the level of metal titanium ions (Ti\u003csup\u003e4+\u003c/sup\u003e) dissociated from TiO\u003csub\u003e2\u003c/sub\u003eNPs is extremely low due to the special nature of titanium(Prokopiuk et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Generally speaking, the toxic effects of TiO\u003csub\u003e2\u003c/sub\u003eNPs are proved to be mainly based on their unique NP properties.\u003c/p\u003e \u003cp\u003ePhysiological factors play important regulatory roles in the functional performance of PLTs(Sang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Flowing blood generates a frictional force called shear stress(Souilhol et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and a common pathological symptom for myocardial infarction and ischemic stroke is thrombotic blood flow obstruction that forms at high shear rates in the arteries(Casa and Ku \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thrombin, collagen, and ADP are currently considered to be the most potent physiological agonists of PLTs. In the present study, under physiology-mimicking conditions, we found that TiO\u003csub\u003e2\u003c/sub\u003eNP-induced hPLT aggregation was dramatically aggravated. Although we tried our best to simulate \u003cem\u003ein vivo\u003c/em\u003e system by adding physiological aggregators and imposing high shear stress, and as expected, observed increased PLT aggregation, the real human body is a large complex system quite difficult to completely and perfectly simulate.\u003c/p\u003e \u003cp\u003eCalcium signaling and its network of interactions are involved in mediating many cellular physiological functions. Studies have shown that the agonist-induced Ca\u003csup\u003e2+\u003c/sup\u003e level is critical for PLT activation in hemostasis and thrombosis(Varga-Szabo et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Increased procoagulant activity of PLTs can be initiated by integrin αIIbβ3 (GPIIb/IIIa)/Gα13-mediated co-stimulation of outward-inward signaling and GPVI signaling, leading to intracellular Ca\u003csup\u003e2+\u003c/sup\u003e release above a threshold(Kaiser et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In a study on procoagulant PLTs, it was mentioned that only the procoagulant PLTs showed high cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e level as detected by fluorescent probes(Abbasian et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In good accordance with this, our study reveal that TiO\u003csub\u003e2\u003c/sub\u003eNP treatment increases intracellular Ca\u003csup\u003e2+\u003c/sup\u003e level, which is required in induction of PLT activation, aggregation and procoagulant activity, finally leading to AT in mice. Thus, it is noteworthy that our study may provide new clues on the preventive and therapeutic strategies of AT caused by TiO\u003csub\u003e2\u003c/sub\u003eNP treatment.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study reveals a pro-thrombotic effect of TiO\u003csub\u003e2\u003c/sub\u003eNPs by pro-coagulant activity and activation/aggregation of PLTs. Mechanistically, TiO\u003csub\u003e2\u003c/sub\u003eNPs initiate PS exposure and MVs generation, ultimately leading to a heightened pro-coagulant activity. Simultaneously, these particles boost the expression of P-selectin and activate GPIIb/IIIa, thereby facilitating the activation/aggregation of platelets. This entire process involves an increase in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels, making it calcium-dependent. Most importantly, we demonstrated the relevance of our findings in vivo by showing that intravenous administration of TiO\u003csub\u003e2\u003c/sub\u003eNPs can increase arterial thrombosis in mouse carotid arteries, emphasizing the need for caution during medical applications of TiO\u003csub\u003e2\u003c/sub\u003eNPs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eNot applicable. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;This research was funded by the National Natural Science Foundation of China\u0026nbsp;82241090 (Y.X.), 82022063 (Y.X.), 82003500 (Y.B), 82211540403 (Y.B.) and Department of Science and Technology of Liaoning Province 2022JH2/20200035(Y.X.), 2022JH2/20200017 (Y.B.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYiying Bian\u003c/strong\u003e: designed and performed most part of \u003cem\u003ein vitro\u003c/em\u003e experiments, writing-review \u0026amp; editing, Funding acquisition, Supervision. \u003cstrong\u003eQiushuo Jin\u003c/strong\u003e: writing original manuscript, and performed most part of animal experiments. \u003cstrong\u003eJinrui He and Thien Ngo\u003c/strong\u003e: did partial animal experiments and data analysis. \u003cstrong\u003eOK-Nam Bae, Jingbo Pi and Han Young Chung\u003c/strong\u003e: validation, data analysis and review. \u003cstrong\u003eYuanyuan Xu\u003c/strong\u003e: writing - review \u0026amp; editing, funding acquisition, data interpretation, and supervision. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith the approval from the Ethics Committee of the Health Service Center at Seoul National University, human blood was obtained from healthy male donors. All the animal protocols used \u003cem\u003ein vivo\u003c/em\u003e experiments were approved by the Ethics Committee of the Animal Service Center at China Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and material that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eThrombosis: a major contributor to global disease burden. 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Journal of toxicology and environmental health Part B, Critical reviews. 2011;14:593-632. https://doi.org/10.1080/10937404.2011.615113\u003c/li\u003e\n\u003cli\u003eZhu W, Gregory JC, Org E, Buffa JA, Gupta N, Wang Z, Li L, Fu X, Wu Y, Mehrabian M, Sartor RB, McIntyre TM, Silverstein RL, Tang WHW, DiDonato JA, Brown JM, Lusis AJ, Hazen SL. Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk. Cell. 2016;165:111-24. https://doi.org/10.1016/j.cell.2016.02.011\u003c/li\u003e\n\u003cli\u003eZlamal J, Singh A, Weich K, Jaffal H, Uzun G, Pelzl L, Althaus K, Bakchoul T. Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia. Haematologica. 2023;108:2690-702. https://doi.org/10.3324/haematol.2022.282275\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-biology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbto","sideBox":"Learn more about [Cell Biology and Toxicology](https://www.springer.com/journal/10565)","snPcode":"10565","submissionUrl":"https://submission.nature.com/new-submission/10565/3","title":"Cell Biology and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Titanium dioxide nanoparticles (TiO2NPs), Platelet (PLT), Calcium, Arterial thrombosis (AT)","lastPublishedDoi":"10.21203/rs.3.rs-4187973/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4187973/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTitanium dioxide nanoparticles (TiO\u003csub\u003e2\u003c/sub\u003eNPs) are widely used in medical application. However, the relevant health risk has not been completely assessed, the potential of inducing arterial thrombosis (AT) in particular.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAlterations in platelet function and susceptibility to arterial thrombosis induced by TiO\u003csub\u003e2\u003c/sub\u003eNPs were examined using peripheral blood samples from healthy adult males and an \u003cem\u003ein vivo\u003c/em\u003e mouse model, respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, using human platelets (hPLTs) freshly isolated from health volunteers, we demonstrated TiO\u003csub\u003e2\u003c/sub\u003eNP treatment triggered the procoagulant activity of hPLTs through phosphatidylserine exposure and microvesicles generation. In addition, TiO\u003csub\u003e2\u003c/sub\u003eNP treatment increased the levels of glycoprotein IIb/IIIa and P-selectin leading to aggregation and activation of hPLTs, which were aggravated by providing physiology-mimicking conditions, including introduction of thrombin, collagen, and high shear stress. Interestingly, intracellular calcium levels in hPLTs were increased upon TiO\u003csub\u003e2\u003c/sub\u003eNP treatment, which were crucial in TiO\u003csub\u003e2\u003c/sub\u003eNP-induced hPLT procoagulant activity, activation and aggregation. Moreover, using mice \u003cem\u003ein vivo\u003c/em\u003e models, we further confirmed that TiO\u003csub\u003e2\u003c/sub\u003eNP treatment a reduction in mouse platelet (mPLT) counts, disrupted blood flow, and exacerbated carotid arterial thrombosis with enhanced deposition of mPLT.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTogether, our study provides evidence for an ignored health risk caused by TiO\u003csub\u003e2\u003c/sub\u003eNPs, specifically TiO\u003csub\u003e2\u003c/sub\u003eNP treatment augments procoagulant activity, activation and aggregation of PLTs via calcium-dependent mechanism and thus increases the risk of AT.\u003c/p\u003e","manuscriptTitle":"Biomedical application of TiO2NPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-03 16:29:34","doi":"10.21203/rs.3.rs-4187973/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-03T06:01:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-22T12:11:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-16T15:02:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97128278417838711481529007644892336839","date":"2024-05-16T09:54:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74640583733427859969203655008134593709","date":"2024-05-16T09:42:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92281780616748417448612895943024968699","date":"2024-05-16T09:17:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99503623771833138528340846352644292305","date":"2024-05-16T09:07:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247601146778399520886138766228321102949","date":"2024-05-16T09:05:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-16T08:51:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-01T01:57:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-01T01:57:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Biology and Toxicology","date":"2024-03-29T12:47:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-biology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbto","sideBox":"Learn more about [Cell Biology and Toxicology](https://www.springer.com/journal/10565)","snPcode":"10565","submissionUrl":"https://submission.nature.com/new-submission/10565/3","title":"Cell Biology and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7d61d0d5-ee59-4e93-9f02-6d0abc348af3","owner":[],"postedDate":"April 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T16:02:10+00:00","versionOfRecord":{"articleIdentity":"rs-4187973","link":"https://doi.org/10.1007/s10565-024-09908-y","journal":{"identity":"cell-biology-and-toxicology","isVorOnly":false,"title":"Cell Biology and Toxicology"},"publishedOn":"2024-08-07 15:57:31","publishedOnDateReadable":"August 7th, 2024"},"versionCreatedAt":"2024-04-03 16:29:34","video":"","vorDoi":"10.1007/s10565-024-09908-y","vorDoiUrl":"https://doi.org/10.1007/s10565-024-09908-y","workflowStages":[]},"version":"v1","identity":"rs-4187973","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4187973","identity":"rs-4187973","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}