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.

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.

Rapid kinetics of changes in oxygen consumption rate in thrombin-stimulated platelets measured by high-resolution respirometry.

Platelet activation plays a key role in normal haemostasis and pathological thrombosis. Platelet activation is rapid; within minutes of stimulation, platelets generate bioactive phospholipids, secrete their granule contents, activate integrins and aggregate together to form a haemostatic plug. These events are dependent on ATP synthesis. Mitochondrial function in platelets from healthy volunteers and patients with a range of diseases indicate an important role for oxygen consumption in oxidative phosphorylation in normal and pathological function. Platelets also consume oxygen during oxidation reactions, such as cyclooxygenase-dependent thromboxane A2 synthesis. In this study, we used high-resolution respirometry to investigate rapid changes in oxygen consumption during platelet activation. We demonstrated a rapid, transient increase in oxygen consumption rate within minutes of platelet stimulation by the physiological activator, thrombin. This was partly inhibited by aspirin and by oligomycin. This shows that high resolution respirometry can provide information regarding rapid and dynamic changes in oxygen consumption during platelet activation.

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.

Supramaximal calcium signaling triggers procoagulant platelet formation.

Procoagulant platelets promote thrombin generation during thrombosis. Platelets become procoagulant in an all-or-nothing manner. We investigated how distinct Ca2+ signaling between platelet subpopulations commits some platelets to become procoagulant, using the high-affinity Ca2+ indicator Fluo-4, which may become saturated during platelet stimulation, or low-affinity Fluo-5N, which reports only very high cytosolic Ca2+ concentrations. All activated platelets had high Fluo-4 fluorescence. However, in Fluo-5N-loaded platelets, only the procoagulant platelets had high fluorescence, indicating very high cytosolic Ca2+. This finding indicates a novel, "supramaximal" Ca2+ signal in procoagulant platelets (ie, much higher than normally considered maximal). Supramaximal Ca2+ signaling and the percentage of procoagulant platelets were inhibited by cyclosporin A, a mitochondrial permeability transition pore blocker, and Ru360, an inhibitor of the mitochondrial Ca2+ uniporter, with no effect on Fluo-4 fluorescence. In contrast, Synta-66, an Orai1 blocker, reduced Fluo-4 fluorescence but did not directly inhibit generation of the supramaximal Ca2+ signal. Our findings show a distinct pattern of Ca2+ signaling in procoagulant platelets and provide a new framework to interpret the role of platelet signaling pathways in procoagulant platelets. This requires reassessment of the role of different Ca2+ channels and may provide new targets to prevent formation of procoagulant platelets and limit thrombosis.