Dissecting the roles of dynamin and clathrin in platelet pinocytosis.

Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.

Application of the Cellular Thermal Shift Assay (CETSA) to validate drug target engagement in platelets.

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.

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.

Utilizing orthodontic appliances to retain and disguise a pediatric maxillary obturator throughout growth.

Unique challenges are encountered when providing an obturator for an adolescent patient. Challenges include the need to modify the obturator throughout growth, engaging the mixed dentition or partially erupted teeth with minimal undercuts, the psychosocial challenges of an actively maturing patient, and the possible need for coincidental orthodontic therapy. This clinical report describes the ongoing rehabilitation of a patient who presented at the age of 10 with a biopsy-confirmed palatal mucoepidermoid carcinoma. Incorporating orthodontic brackets for retention of innovative adjustable obturator clasps allowed for the favorable function of the obturator prosthesis and the ability to alter the prosthesis over time.

Platelet-Derived Extracellular Vesicles in Arterial Thrombosis.

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.

Human soluble CD39 displays substrate inhibition in a substrate-specific manner.

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.

Accuracy of Digital Impressions at Varying Implant Depths: An In Vitro Study.

PURPOSE: Implants placed at variable depths may vary the amount of visible scannable surface of a scan body. Intraoral scanner technology uses advanced optical principles to record the surface of the scan body to accurately capture the implant position. The purpose of this study is to investigate the effect implant placement depth has on the accuracy of digital implant impressions using an intraoral scanner. MATERIALS AND METHODS: A partially edentulous gypsum master model was fabricated to allow the positioning of a single implant analog at different depths. Four groups were created based on the planned implant depths of 7, 6, 3, and 0 mm and corresponding visibility of the scan body at 2, 3, 6, and 9 mm. The model was digitized with a laboratory scanner for the reference scan and with an intraoral scanner to generate 15 test scans per group, with a total of 60 scans. The test scans were superimposed onto the reference scan using the best fit algorithm to analyze and measure the positional (dXYZ) and angular deviation (d⍬) of the scan body using three-dimensional metrology software. Statistical analysis was performed using a one-way ANOVA and pairwise comparison was done with a Tukey-Kramer HSD test (α = 0.05). RESULTS: The one-way ANOVA of the groups for the dXYZ and dθ parameters was statistically significant (F3,56 = 11.45, p < 0.001, F3,56 = 24.04, p < 0.001). Group D (9 mm) showed the least positional deviation at 38.41 µm (95% CI 30.26; 46.56) and the least angular deviation of 0.17° (95% CI 0.12; 0.21). Group A (2 mm) showed the greatest positional deviation of 77.17 µm (95% CI 65.23; 89.11) and greatest angular deviation of 0.84° (95% CI 0.65; 1.03). The positional and angular deviation increased with increased implant depth. CONCLUSIONS: The accuracy of digital impressions is influenced by the implant depth and the amount of visibility of the scan body. The trueness and precision are highest when the implant is placed at 0 mm depth with complete visibility of the scan body and decreases with subgingival implant placement.

Protease-activated receptor antagonists prevent thrombosis when dual antiplatelet therapy is insufficient in an occlusive thrombosis microfluidic model.

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.

Endothelial inflammation and neutrophil transmigration are modulated by extracellular matrix composition in an inflammation-on-a-chip model.

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.

Q94 is not a selective modulator of proteinase-activated receptor 1 (PAR1) in platelets.

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 αIIbß3 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, αIIbß3 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.

Epigenetic Regulation of F2RL3 Associates With Myocardial Infarction and Platelet Function.

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.

Maintaining flippase activity in procoagulant platelets is a novel approach to reducing thrombin generation.

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.

Epigallocatechin gallate inhibits release of extracellular vesicles from platelets without inhibiting phosphatidylserine exposure.

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.

An "occlusive thrombosis-on-a-chip" microfluidic device for investigating the effect of anti-thrombotic drugs.

Cardiovascular disease remains one of the world's leading causes of death. Myocardial infarction (heart attack) is triggered by occlusion of coronary arteries by platelet-rich thrombi (clots). The development of new anti-platelet drugs to prevent myocardial infarction continues to be an active area of research and is dependent on accurately modelling the process of clot formation. Occlusive thrombi can be generated in vivo in a range of species, but these models are limited by variability and lack of relevance to human disease. Although in vitro models using human blood can overcome species-specific differences and improve translatability, many models do not generate occlusive thrombi. In those models that do achieve occlusion, time to occlusion is difficult to measure in an unbiased and objective manner. In this study we developed a simple and robust approach to determine occlusion time of a novel in vitro microfluidic assay. This highlighted the potential for occlusion to occur in thrombosis microfluidic devices through off-site coagulation, obscuring the effect of anti-platelet drugs. We therefore designed a novel occlusive thrombosis-on-a-chip microfluidic device that reliably generates occlusive thrombi at arterial shear rates by quenching downstream coagulation. We further validated our device and methods by using the approved anti-platelet drug, eptifibatide, recording a significant difference in the "time to occlude" in treated devices compared to control conditions. These results demonstrate that this device can be used to monitor the effect of antithrombotic drugs on time to occlude, and, for the first time, delivers this essential data in an unbiased and objective manner.

Establishing an Analogue Based In Silico Pipeline in the Pursuit of Novel Inhibitory Scaffolds against the SARS Coronavirus 2 Papain-Like Protease.

The ongoing coronavirus pandemic has been a burden on the worldwide population, with mass fatalities and devastating socioeconomic consequences. It has particularly drawn attention to the lack of approved small-molecule drugs to inhibit SARS coronaviruses. Importantly, lessons learned from the SARS outbreak of 2002-2004, caused by severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), can be applied to current drug discovery ventures. SARS-CoV-1 and SARS-CoV-2 both possess two cysteine proteases, the main protease (Mpro) and the papain-like protease (PLpro), which play a significant role in facilitating viral replication, and are important drug targets. The non-covalent inhibitor, GRL-0617, which was found to inhibit replication of SARS-CoV-1, and more recently SARS-CoV-2, is the only PLpro inhibitor co-crystallised with the recently solved SARS-CoV-2 PLpro crystal structure. Therefore, the GRL-0617 structural template and pharmacophore features are instrumental in the design and development of more potent PLpro inhibitors. In this work, we conducted scaffold hopping using GRL-0617 as a reference to screen over 339,000 ligands in the chemical space using the ChemDiv, MayBridge, and Enamine screening libraries. Twenty-four distinct scaffolds with structural and electrostatic similarity to GRL-0617 were obtained. These proceeded to molecular docking against PLpro using the AutoDock tools. Of two compounds that showed the most favourable predicted binding affinities to the target site, as well as comparable protein-ligand interactions to GRL-0617, one was chosen for further analogue-based work. Twenty-seven analogues of this compound were further docked against the PLpro, which resulted in two additional hits with promising docking profiles. Our in silico pipeline consisted of an integrative four-step approach: (1) ligand-based virtual screening (scaffold-hopping), (2) molecular docking, (3) an analogue search, and, (4) evaluation of scaffold drug-likeness, to identify promising scaffolds and eliminate those with undesirable properties. Overall, we present four novel, and lipophilic, scaffolds obtained from an exhaustive search of diverse and uncharted regions of chemical space, which may be further explored in vitro through structure-activity relationship (SAR) studies in the search for more potent inhibitors. Furthermore, these scaffolds were predicted to have fewer off-target interactions than GRL-0617. Lastly, to our knowledge, this work contains the largest ligand-based virtual screen performed against GRL-0617.

Cytosolic and mitochondrial Ca2+ signaling in procoagulant platelets.

SummaryPlatelets are the major cellular contributor to arterial thrombosis. However, activated platelets form two distinct subpopulations during thrombosis. Pro-aggregatory platelets aggregate to form the main body of the thrombus. In contrast, procoagulant platelets expose phosphatidylserine on their outer surface and promote thrombin generation. This apparently all-or-nothing segregation into subpopulations indicates that, during activation, platelets commit to becoming procoagulant or pro-aggregatory. Although the signaling pathways that control this commitment are not understood, distinct cytosolic and mitochondrial Ca2+ signals in different subpopulations are likely to be central. In this review, we discuss how these Ca2+ signals control procoagulant platelet formation and whether this process can be targeted pharmacologically to prevent arterial thrombosis.

Comparison of putative BH3 mimetics AT-101, HA14-1, sabutoclax and TW-37 with ABT-737 in platelets.

Platelet lifespan is regulated by intrinsic apoptosis. Platelet apoptosis can be triggered by BH3 mimetics that inhibit the pro-survival Bcl-2 family protein, Bcl-xL. Here, we investigated several small molecules that are reported to act as BH3 mimetics and compared their effects to the well-established BH3 mimetic, ABT-737. Platelet phosphatidylserine (PS) exposure was determined by flow cytometry. Changes in cytosolic Ca2+ signaling were detected using Cal-520. Plasma membrane integrity was determined by calcein leakage. The roles of caspases and calpain in these processes were determined using Q-VD-OPh and calpeptin, respectively. As previously reported, ABT-737 triggered PS exposure in a caspase-dependent manner and calcein loss in a caspase and calpain-dependent manner. In contrast, AT-101 and sabutoclax triggered PS exposure independently of caspases. HA14-1 also triggered PS exposure in a caspase-independent but calpain-dependent manner. There were also significant differences in the pattern and protease-dependency of cytosolic Ca2+ signaling in response to these drugs compared to ABT-737. Since there are clear differences between the action of ABT-737 and the other putative BH3 mimetics investigated here, AT-101, HA14-1 and sabutoclax cannot be considered as acting as BH3 mimetics in platelets. Furthermore, the platelet death caused by these drugs is likely to be distinct from apoptosis.

High extracellular phosphate increases platelet polyphosphate content.

Platelet-derived extracellular polyphosphate (PolyP) is a major mediator of thrombosis. PolyP is a linear chain of inorganic phosphate (Pi) and is stored in platelet dense granules. Pi enters cells from the extracellular fluid through phosphate transporters and may be stored as PolyP. Here we show that high extracellular Pi concentration in vitro increases platelet PolyP content, in a manner dependent on phosphate transporters, IP6K and V-type ATPases. The increased PolyP also enhanced PolyP-dependent coagulation in platelet-rich plasma. These data suggest a mechanistic link between hyperphosphatemia, PolyP and enhanced coagulation, which may be important in pathologies such as chronic kidney disease.

Novel Small-Molecule Scaffolds as Candidates against the SARS Coronavirus 2 Main Protease: A Fragment-Guided in Silico Approach.

The ongoing pandemic caused by the novel coronavirus has been the greatest global health crisis since the Spanish flu pandemic of 1918. Thus far, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 1 million deaths, and there is no cure or vaccine to date. The recently solved crystal structure of the SARS-CoV-2 main protease has been a major focus for drug-discovery efforts. Here, we present a fragment-guided approach using ZINCPharmer, where 17 active fragments known to bind to the catalytic centre of the SARS-CoV-2 main protease (SARS-CoV-2 Mpro) were used as pharmacophore queries to search the ZINC databases of natural compounds and natural derivatives. This search yielded 134 hits that were then subjected to multiple rounds of in silico analyses, including blind and focused docking against the 3D structure of the main protease. We scrutinised the poses, scores, and protein-ligand interactions of 15 hits and selected 7. The scaffolds of the seven hits were structurally distinct from known inhibitor scaffolds, thus indicating scaffold novelty. Our work presents several novel scaffolds as potential candidates for experimental validation against SARS-CoV-2 Mpro.

2-Aminoethoxydiphenylborate (2-APB) inhibits release of phosphatidylserine-exposing extracellular vesicles from platelets.

Activated, procoagulant platelets shed phosphatidylserine (PS)-exposing extracellular vesicles (EVs) from their surface in a Ca2+- and calpain-dependent manner. These PS-exposing EVs are prothrombotic and proinflammatory and are found at elevated levels in many cardiovascular and metabolic diseases. How PS-exposing EVs are shed is not fully understood. A clearer understanding of this process may aid the development of drugs to selectively block their release. In this study we report that 2-aminoethoxydiphenylborate (2-APB) significantly inhibits the release of PS-exposing EVs from platelets stimulated with the Ca2+ ionophore, A23187, or the pore-forming toxin, streptolysin-O. Two analogues of 2-APB, diphenylboronic anhydride (DPBA) and 3-(diphenylphosphino)-1-propylamine (DP3A), inhibited PS-exposing EV release with similar potency. Although 2-APB and DPBA weakly inhibited platelet PS exposure and calpain activity, this was not seen with DP3A despite inhibiting PS-exposing EV release. These data suggest that there is a further target of 2-APB, independent of cytosolic Ca2+ signalling, PS exposure and calpain activity, that is required for PS-exposing EV release. DP3A is likely to inhibit the same target, without these other effects. Identifying the target of 2-APB, DPBA and DP3A may provide a new way to inhibit PS-exposing EV release from activated platelets and inhibit their contribution to thrombosis and inflammation.