Platelet activation is a key driver of in arterial thrombosis and myocardial infarction. Despite current anti-platelet therapies, mortality and morbidity from myocardial infarction remain high. Alternative anti-platelet therapies are needed that can reduce the risk of myocardial infarction without further increasing bleeding risk.Activated platelets rapidly diverge into distinct subpopulations. ‘Procoagulant’ platelets form through a mitochondria-dependent pathway. These procoagulant platelets expose phosphatidylserine (PS), which localises and amplifies the coagulation cascade leading to a large increase in thrombin generation and stable thrombosis. Procoagulant platelet formation may be a new target for anti-platelet therapies.A key regulatory step controlling progression to procoagulant platelets is opening of the mitochondrial permeability transition pore (mPTP). OSCP acts as a regulatory node for mPTP activity. Since mPTP opening regulates procoagulant platelet formation, we hypothesised that OSCP might also be a key regulatory node in platelets.OSCP is a target of the immunomodulatory benzodiazepine, Bz-423. In this study, we show that Bz-423 increased procoagulant platelet formation. This was associated with mitochondrial hyperpolarisation prior to platelet activation, and increased mPTP opening after activation. Cellular thermal shift assays (CETSA) showed that both Bz-423 and a structurally similar benzodiazepine, 4-chloro-dizaepam, bind OSCP in platelets. Despite this, only Bz-423 increased procoagulant platelet formation. Our findings indicate that Bz-423 promotes procoagulant platelet progression through a target distinct from OSCP.

Forkhead box protein P1 (FOXP1), a transcription factor enriched in the neocortex, is associated with autism spectrum disorders (ASDs) and FOXP1 syndrome. Emx1Cre/+;Foxp1fl/fl conditional deletion (Foxp1 conditional knockout [cKO]) in the mouse cortex leads to overall reduced cortex thickness, alterations in cortical lamination, and changes in the relative thickness of cortical layers. However, the developmental and cell-type-specific mechanisms underlying these changes remained unclear. We find that Foxp1 deletion results in accelerated pseudo-age during early neurogenesis, increased cell cycle exit during late neurogenesis, altered gene expression and chromatin accessibility, and selective migration deficits in a subset of upper-layer neurons. These data explain the postnatal differences observed in cortical layers and relative cortical thickness. We also highlight genes regulated by FOXP1 and their enrichment with high-confidence ASD or synaptic genes. Together, these results underscore a network of neurodevelopmental-disorder-related genes that may serve as potential modulatory targets for postnatal modification relevant to ASDs and FOXP1 syndrome.

Ca2+-release activated Ca2+ (CRAC) channels mediate the store-operated Ca2+ entry (SOCE) that is used almost by all cells to replenish depleted intracellular Ca2+ stores. Aberrant CRAC channel functions have been implicated in various diseases including autoimmune diseases and as such these channels and the resultant pathway (SOCE) have been intensively studied by academic and pharmaceutical scientists over decades. To date, several small molecule inhibitors and few antibodies have been developed against these ion channels. Some small molecule inhibitors of the CRAC channels, namely BTP2, Synta66, Pyr6 and GSK-7975A, are widely used as tool compounds in the field but their mode of action remains unclear. In this study, we used whole cell electrophysiology supported by in silico modelling to propose likely binding site and binding mode of these four popular CRAC channel inhibitors. ### Competing Interest Statement The authors have declared no competing interest.

Small molecule drugs play a major role in the study of human platelets. Effective action of a drug requires it to bind to one or more targets within the platelet (target engagement). However, although in vitro assays with isolated proteins can be used to determine drug affinity to these targets, additional factors affect target engagement and its consequences in an intact platelet, including plasma membrane permeability, intracellular metabolism or compartmentalization, and level of target expression. Mechanistic interpretation of the effect of drugs on platelet activity requires comprehensive investigation of drug binding in the proper cellular context, i.e. in intact platelets. The Cellular Thermal Shift Assay (CETSA) is a valuable method to investigate target engagement within complex cellular environments. The assay is based on the principle that drug binding to a target protein increases that protein's thermal stability. In this technical report, we describe the application of CETSA to platelets. We highlight CETSA as a quick and informative technique for confirming the direct binding of drugs to platelet protein targets, providing a platform for understanding the mechanism of action of drugs in platelets, and which will be a valuable tool for investigating platelet signaling and function. Plain Language Summary Platelets control blood clotting in health and disease. Small molecule drugs are often used to study human platelets. Here, describe how Cellular Thermal Shift Assay (CETSA) can be used in platelets to investigate the binding between these drugs and their targets inside platelets. This technique can be used to increase our understanding of how existing and future drugs work in platelets.

The immune system identifies and destroys invading pathogens. However, chronic inflammation and excessive tissue damage can result if inflammation is not resolved or if the immune system misidentifies a threat. Therefore, the immune system must be tightly controlled. However, the complex cell-cell and cell-microenvironment interactions that regulate immune cell recruitment are challenging to recapitulate in 2D in vitro models, and poorly representative of human physiology in in vivo animal models. Organ-on-a-chip technology has the potential to overcome these limitations and provide powerful pre-clinical models. In this study, we developed a vessel-on-a-chip model to investigate the role of platelets in inflammation and inflammatory hemostasis. Endothelial vessels cultured in the OrganoPlate exhibited physiological barrier function to macromolecules and red blood cells (RBCs), which was enhanced by platelets. Cytokine stimulation increased vessel permeability and induced neutrophil transmigration. Leakage of RBCs occurred at sites of inflammation and/or neutrophil transmigration, depending on the ECM composition. Addition of platelets prevented RBC leakage at these sites, whilst simultaneously increasing permeability and neutrophil transmigration, demonstrating the multifaceted role of platelets in inflammatory hemostasis. In angiogenic neo-vessels, platelets played a protective role, preventing leakage of both small molecules and RBCs. Together, our model successfully recapitulated the modulation of endothelial barrier function and inflammation by platelets, and demonstrated the feasibility of investigating complex cellular interactions in in vitro models.

BACKGROUND AND PURPOSE:Abdominal pain is a leading cause of morbidity for people living with gastrointestinal disease. Whereas the transient receptor potential vanilloid 4 (TRPV4) ion channel has been implicated in the pathogenesis of abdominal pain, the relative paucity of TRPV4 expression in colon-projecting sensory neurons suggests that non-neuronal cells may contribute to TRPV4-mediated nociceptor stimulation. EXPERIMENTAL APPROACH:Changes in murine colonic afferent activity were examined using ex vivo electrophysiology in tissues with the gut mucosa present or removed. ATP and glutamate release were measured by bioluminescence assays from human colon organoid cultures and mouse colon. Dorsal root ganglion sensory neuron activity was evaluated by Ca2+ imaging when cultured alone or co-cultured with colonic mucosa. KEY RESULTS:Bath application of TRPV4 agonist GSK1016790A elicited a robust increase in murine colonic afferent activity, which was abolished by removing the gut mucosa. GSK1016790A promoted ATP and glutamate release from human colon organoid cultures and mouse colon. Inhibition of ATP degradation in mouse colon enhanced the afferent response to GSK1016790A. Pretreatment with purinoceptor or glutamate receptor antagonists attenuated and abolished the response to GSK1016790A when given alone or in combination, respectively. Sensory neurons co-cultured with colonic mucosal cells produced a marked increase in intracellular Ca2+ to GSK1016790A compared with neurons cultured alone. CONCLUSION AND IMPLICATIONS:Our data indicate that mucosal release of ATP and glutamate is responsible for the stimulation of colonic afferents following TRPV4 activation. These findings highlight an opportunity to target the gut mucosa for the development of new visceral analgesics.

PurposeTo evaluate visuo-cognitive sequelae following blast-induced traumatic brain injury in a rat model.MethodsRats were randomly assigned to one of four groups depending on the intensity/quantity of a blast received in a blast chamber: sham (no blast), low intensity (22 psi), medium intensity (26 psi), or three medium intensity blasts (26 psi × 3). After recovery, all subjects were given visual discrimination tasks of increasing complexity, until mastery. After behavioral training, visual function was assessed via spectral-domain optical coherence tomography and pattern electroretinogram, and the extent of retinal damage was quantified via immunohistochemistry of retinal ganglion cells.ResultsNone of the measures assessing visual function revealed significant differences as a function of blast intensity/quantity. Behavioral training did not disclose short-term effects of blast in general motivation or the development of anticipatory responding. No differences in general learning ability and the number of perseverative errors were observed. However, behavioral training found effects of blast in attentional function; relative to controls, subjects that received blasts were faster in learning to attend to informative (over non-informative) cues in the most difficult visual discrimination task.ConclusionBlast exposure in rats resulted in increased attention following blast, with no appreciable deficits in visual function. These results are contrary to what is often reported for human clinical populations; as such, more research bridging methodological differences is necessary.

Blood platelets are necessary for normal haemostasis but also form life-threatening arterial thrombi when atherosclerotic plaques rupture. Activated platelets release many extracellular vesicles during thrombosis. Phosphatidylserine-exposing microparticles promote coagulation. Small exosomes released during granule secretion deliver cargoes including microRNAs to cells throughout the cardiovascular system. Here, we discuss the mechanisms by which platelets release these extracellular vesicles, together with the possibility of inhibiting this release as an antithrombotic strategy.

The purpose of this study was to examine the effect of a blast exposure generated from a shock tube on retinal ganglion cell (RGC) function and structure. Mice were exposed to one of three blast conditions using a shock tube; a single blast wave of 20 PSI, a single blast wave of 30 PSI, or three blast waves of 30 PSI given on three consecutive days with a one-day inter-blast interval. The structure and function of the retina were analyzed using the pattern electroretinogram (PERG), the optomotor reflex (OMR), and optical coherence tomography (OCT). The in vivo parameters were examined at baseline, and then again 1-week, 4-weeks, and 16-weeks following blast exposure. The number of surviving RGCs was quantified at the end of the study. Analysis of mice receiving a 20 PSI injury showed decreased PERG and OMR responses 16-weeks post blast, without evidence of changed retinal thickness or RGC death. Mice subjected to a 30 PSI injury showed decreased PERG responses 4 weeks and 16 weeks after injury, without changes in the retinal thickness or RGC density. Mice subjected to 30 PSI X 3 blast exposures had PERG deficits 1-week and 4-weeks post exposure. There was also significant change in retinal thickness 1-week and 16-weeks post blast exposure. Mice receiving 30 PSI X 3 blast injuries had regional loss of RGCs in the central retina, but not in the mid-peripheral or peripheral retina. Overall, this study has shown that increasing the number of blast exposures and the intensity leads to earlier functional loss of RGCs. We have also shown regional RGC loss only when using the highest blast intensity and number of blast injuries.

Long-range glutamatergic inputs from the cortex and thalamus are critical for motor and cognitive processing in the striatum. Transcription factors that orchestrate the development of these inputs are largely unknown. We investigated the role of a transcription factor and high-risk autism-associated gene, FOXP1, in the development of glutamatergic inputs onto spiny projection neurons (SPNs) in the striatum. We find that FOXP1 robustly drives the strengthening and maturation of glutamatergic input onto dopamine receptor 2-expressing SPNs (D2 SPNs) but has a comparatively milder effect on D1 SPNs. This process is cell-autonomous and is likely mediated through postnatal FOXP1 function at the postsynapse. We identified postsynaptic FOXP1-regulated transcripts as potential candidates for mediating these effects. Postnatal reinstatement of FOXP1 rescues electrophysiological deficits, reverses gene expression alterations resulting from embryonic deletion, and mitigates behavioral phenotypes. These results provide support for a possible therapeutic approach for individuals with FOXP1 syndrome.

Core circadian-related proteins such as the transcription factor CLOCK are ubiquitously expressed and important for regulating molecular pathways underlying circadian rhythms. Previous work has suggested that CLOCK has evolved human neocortex-specific gene regulation and therefore may have extra-circadian functions. To test this in vivo, we generated a mouse model that recapitulates human cortical expression of CLOCK. The CLOCK humanized (HU) mice show enhanced cognitive flexibility, which is associated with the alteration in spatiotemporal expression of CLOCK. Cell type specific genomic profiling of HU mice identified upregulated genes related to dendritic growth and spine formation in excitatory neurons. Consistent with this result, we found that excitatory neurons in HU mice have increased complexity of dendritic branching and spine density, as well as a greater frequency of excitatory postsynaptic currents, suggesting an increase in neural connectivity. In contrast, CLOCK knockout in human induced pluripotent stem cell-induced neurons showed reduced complexity of dendrites and lower density of presynaptic puncta. Together, our data demonstrate that CLOCK evolved extra-circadian gains of function via altered spatiotemporal gene expression and these functions may underlie human brain specializations.

CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD1) metabolizes extracellular ATP and ADP to AMP. AMP is subsequently metabolized by CD79 to adenosine. CD39 activity is therefore a key regulator of purinergic signalling in cancer, thrombosis, and autoimmune diseases. In this study we demonstrate that soluble, recombinant CD39 shows substrate inhibition with ADP or ATP as the substrate. Although CD39 activity initially increased with increasing substrate concentration, at high concentrations of ATP or ADP, CD39 activity was markedly reduced. Although the reaction product, AMP, inhibits CD39 activity, insufficient AMP was generated under our conditions to account for the substrate inhibition seen. In contrast, inhibition was not seen with UDP or UTP as substrates. 2-methylthio-ADP also showed no substrate inhibition, indicating the nucleotide base is an important determinant of substrate inhibition. Molecular dynamics simulations revealed that ADP can undergo conformational rearrangements within the CD39 active site that were not seen with UDP or 2-methylthio-ADP. Appreciating the existence of substrate inhibition of CD39 will help the interpretation of studies of CD39 activity, including investigations into drugs that modulate CD39 activity.

Thrombin is a potent platelet activator, acting through proteinase-activated receptors -1 and -4 (PAR1 and PAR4). Of these, PAR-1 is activated more rapidly and by lower thrombin concentrations. Consequently, PAR-1 has been extensively investigated as a target for anti-platelet drugs to prevent myocardial infarction. Q94 has been reported to act as an allosteric modulator of PAR1, potently and selectively inhibiting PAR1-Gαq coupling in multiple cell lines, but its effects on human platelet activation have not been previously studied. Platelet Ca2+ signaling, integrin αIIbβ3 activation and α-granule secretion were monitored following stimulation by a PAR1-activating peptide (PAR1-AP). Although Q94 inhibited these responses, its potency was low compared to other PAR1 antagonists. In addition, αIIbβ3 activation and α-granule secretion in response to other platelet activators were also inhibited with similar potency. Finally, in endothelial cells, Q94 did not inhibit PAR1-dependent Ca2+ signaling. Our data suggest that Q94 may have PAR1-independent off-target effects in platelets, precluding its use as a selective PAR1 allosteric modulator.

Purpose: The purpose of this study was to examine the role of the immune system and its influence on chronic retinal ganglion cell (RGC) dysfunction following blast -mediated traumatic brain injury (bTBI). Methods: C57BL/6J and B6.129S7-Rag1(tm1Mom)/J (Rag-( /-)) mice were exposed to one blast injury of 140 kPa. A separate cohort of C57BL/6J mice was exposed to sham-blast. Four weeks following bTBI mice were euthanized, and splenocytes were collected. Adoptive transfer (AT) of splenocytes into naive C57BL/6J recipient mice was accomplished via tail vein injection. Three groups of mice were analyzed: those receiving AT of splenocytes from C57BL/6J mice exposed to blast (AT-TBI), those receiving AT of splenocytes from C57BL/6J mice exposed to sham (AT-Sham), and those receiving AT of splenocytes from Rag(-/-)mice exposed to blast (AT-Rag(-/-)). The visual function of recipient mice was analyzed with the pattern electroretinogram (PERG), and the optomotor response (OMR). The structure of the retina was evaluated using optical coherence tomography (OCT), and histologically using BRN3A-antibody staining. Results: Analysis of the PERG showed a decreased amplitude two months post-AT that persisted for the duration of the study in AT-TBI mice. We also observed a significant decrease in the retinal thickness of AT-TBI mice two months post-AT compared to sham, but not at four or six months post-AT. The OMR response was significantly decreased in AT-TBI mice 5-and 6-months post-AT. BRN3A staining showed a loss of RGCs in AT-TBI and AT -Rag(-/-)mice. Conclusion: These results suggest that the immune system contributes to chronic RGC dysfunction following bTBI.

Inflammatory diseases are often characterised by excessive neutrophil infiltration from the blood stream to the site of inflammation, which damages healthy tissue and prevents resolution of inflammation. Development of anti-inflammatory drugs is hindered by lack of in vitro and in vivo models which accurately represent the disease microenvironment. In this study, we used the OrganoPlate to develop a humanized 3D in vitro inflammation-on-a-chip model to recapitulate neutrophil transmigration across the endothelium and subsequent migration through the extracellular matrix (ECM). Human umbilical vein endothelial cells formed confluent vessels against collagen I and geltrex mix, a mix of basement membrane extract and collagen I. TNF-α-stimulation of vessels upregulated inflammatory cytokine expression and promoted neutrophil transmigration. Intriguingly, major differences were found depending on the composition of the ECM. Neutrophils transmigrated in higher number and further in geltrex mix than collagen I, and did not require an N -formyl-methionyl-leucyl-phenylalanine (fMLP) gradient for transmigration. Inhibition of neutrophil proteases inhibited neutrophil transmigration on geltrex mix, but not collagen I. These findings highlight the important role of the ECM in determining cell phenotype and response to inhibitors. Future work could adapt the ECM composition for individual diseases, producing accurate models for drug development.

Background: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. Methods: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. Results: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2-2.4, P=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. Conclusions: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

Background Platelet activation and arterial thrombosis on a ruptured atherosclerotic plaque is a major cause of myocardial infarction. Dual antiplatelet therapy (DAPT), the combination of platelet aggregation inhibitors, aspirin and a P2Y12 antagonist, is used to prevent arterial thrombosis. However, many people continue to have arterial thrombosis and myocardial infarction despite DAPT, indicating that additional therapies are required where DAPT is insufficient. Objectives To determine whether antagonists of protease-activated receptors (PARs) can prevent occlusive thrombosis under conditions where DAPT is insufficient. Methods We used human whole blood in a microfluidic model of occlusive thrombosis to compare conditions under which DAPT is effective to those under which DAPT was not. Cangrelor (a P2Y12 antagonist) and aspirin were used to mimic DAPT. We then investigated whether the PAR1 antagonist vorapaxar or the PAR4 antagonist BMS 986120, alone or in combination with DAPT, prevented occlusive thrombosis. Results and Conclusions A ruptured plaque exposes collagen fibers and is often rich in tissue factor, triggering activation of platelets and coagulation. Occlusive thrombi formed on type I collagen in the presence or absence of tissue factor (TF). However, although DAPT prevented occlusive thrombosis in the absence of TF, DAPT had little effect when TF was also present. Under these conditions, PAR antagonism was also ineffective. However, occlusive thrombosis was prevented by combining DAPT with PAR antagonism. These data demonstrate that PAR antagonists may be a useful addition to DAPT in some patients and further demonstrate the utility of in vitro models of occlusive thrombosis.

The purpose of this study was to examine the expression of glial-derived neurotrophic factor (GDNF), the GDNF receptors GFRα1 and GFRα2, ciliary neurotrophic factor (CNTF), and the CNTF receptor CNTFRα in normal and glaucomatous human tissue.Human retinas were collected from 8 donors that had been clinically diagnosed and treated for glaucoma, and also from 9 healthy control donors. Immunohistochemical analysis for each trophic factor and receptor was performed. The percent of each retinal section labeled with each antibody was quantified for the total retinal thickness, and separately for the retinal ganglion cell (RGC) complex + retinal nerve fiber layer (RNFL). The expression of each protein was correlated with measures of the subject's ocular histories.The percentage area immunopositive for GFRα2 was significantly decreased in the total retinal thickness containing all retinal layers and in the combined RGC complex + RNFL in glaucomatous eyes in both the peripapillary region and more peripheral retinal locations. We also observed a decrease in GFRα1 expression in the peripapillary RGC Complex + RNFL in glaucoma patients compared to healthy control patients. We also observed a relationship between GDNF and its receptors with several outcomes obtained from the medical record. No differences in CNTF or CNTFR labeling were observed.Decreases in GDNF receptor expression in glaucomatous tissue may limit the potential for neuroprotective therapy by supplementation with GDNF.

Background During thrombosis, procoagulant platelets expose phosphatidylserine (PS), which enhances local thrombin generation. Reducing platelet PS exposure could be a novel anti-thrombotic approach. PS is confined to the inner leaflet of the plasma membrane in unstimulated platelets by ATP-dependent "flippase" activity. Ca2+ ionophores trigger all platelets to expose a high level of PS by activating a scramblase protein and inactivating the flippase. Although R5421 was previously shown to reduce Ca2+ ionophore-induced PS exposure, its mechanism of action is unknown. Objectives To determine the mechanism by which R5421 reduces platelet PS exposure. Methods Washed human platelets were stimulated with the Ca2+ ionophore, A23187, to induce procoagulant platelet formation while bypassing proximal receptor signalling. Platelets PS exposure was detected using annexin V or lactadherin in flow cytometry. NBD (7-nitro-2-1,3-benzoxadiazol-4-yl)-PS was used to assess scramblase and flippase activity. Thrombin generation was monitored using a fluorogenic substrate. Results and conclusions R5421 reduced the extent of A23187-stimulated platelet PS exposure, as demonstrated with annexin V or lactadherin binding. R5421 also maintained flippase activity in procoagulant platelets. Although R5421 appeared to inhibit scramblase activity in procoagulant platelets, it did not once the flippase had been inhibited, demonstrating that scramblase activity is not directly inhibited. Furthermore, R5421 inhibited the contribution of A23187-stimulated platelets to thrombin generation. Together these data demonstrate that R5421 reduces the extent of PS exposure in procoagulant platelets by maintaining flippase activity. Maintaining flippase activity in procoagulant platelets is a novel and effective approach to reducing thrombin generation.

Arterial thrombosis triggers myocardial infarction and is a leading cause of death worldwide. Procoagulant platelets, a subpopulation of activated platelets that expose phosphatidylserine (PS), promote coagulation and occlusive thrombosis. Procoagulant platelets may therefore be a therapeutic target. PS exposure in procoagulant platelets requires TMEM16F, a phospholipid scramblase. Epigallocatechin gallate (EGCG) has been reported to inhibit TMEM16F but this has been challenged. We investigated whether EGCG inhibits PS exposure in procoagulant platelets. PS exposure is often measured using fluorophore-conjugated annexin V. EGCG quenched annexin V-FITC fluorescence, which gives the appearance of inhibition of PS exposure. However, EGCG did not quench annexin V-APC fluorescence. Using this fluorophore, we show that EGCG does not inhibit annexin V binding to procoagulant platelets. We confirmed this by using NBD-labelled PS to monitor PS scrambling. EGCG did not quench NBD fluorescence and did not inhibit PS scrambling. Procoagulant platelets also release PS-exposing extracellular vesicles (EVs) that further propagate coagulation. Surprisingly, EGCG inhibited EV release. This inhibition required the gallate group of EGCG. In conclusion, EGCG does not inhibit PS exposure in procoagulant platelets but does inhibit the EV release. Future investigation of this inhibition may help us further understand how EVs are released by procoagulant platelets.