The PAR 1 antagonist , SCH 79797 , alters platelet morphology and function indepen-dently of PARs

doi:10.1160/TH12-06-0389 Thromb Haemost 2013; 109: 164–167 Dear Sirs, Protease-activated receptors (PARs) are a family of four G protein-coupled receptors expressed on the surface of a wide variety of cells (1). PAR function has been thoroughly studied and the use of pharmacological agents and mouse genetic models have facilitated the discovery of numerous functions for these receptors over the past decade or so (for review, see [2, 3]). PAR1 is the prototypical receptor in this family and is by far the most extensively studied– in large part because PAR1 is the major thrombin receptor on human platelets and antagonists of PAR1 have been developed as novel anti-platelet agents for the prevention of arterial thrombosis (4, 5). Both atopaxar (formerly E5555) and vorapaxar (formerly SCH530348) are small, competitive, orthosteric PAR1 antagonists under clinical investigation for the prevention of arterial thrombosis (4, 5). As a result of this clinical advancement, numerous PAR1 antagonists have been developed over the last ~15 years. However, few of these exhibit sufficiently useful potency and selectivity and, of those, only one – SCH79797 (6) (a predecessor to SCH530348) – is commercially available. For these reasons, SCH79797 has become the most commonly used experimental PAR1 antagonist over the last decade, and has been used to study the role of this receptor in a number of settings, including cell proliferation (7), metastasis (8), placentation (9), steroidogenesis (10), kidney function (11), ischaemia reperfusion (12), inflammation (13), and thrombosis (14), and in cell types as broad as neurons (15), smooth muscle cells (16), endothelial cells (17), fibroblasts (18), macrophages (19), cancer cells (20), and platelets (21). Here we report that SCH79797, at concentrations required to achieve meaningful PAR1 antagonism, exerts an important off-target effect on the structure and function of platelets independently of PARs. We first confirmed that SCH79797 displayed the potency and selectivity for PAR1 inhibition as previously reported (6). SCH79797 (10 μM; 10 minutes [min] pretreatment) abolished aggregation of human washed platelets induced by a PAR1-activating peptide (PAR1-AP; 30 μM TFLLR) but had no effect on responses to any other platelet agonist examined, including a PAR4-activating peptide (PAR4-AP; 100 μM AYPGKF), collagen-related peptide (CRP) and ADP (▶ Figure 1 A). However, platelets pretreated with SCH79797 in these experiments lacked the initial shape change response (recorded as a decrease in light transmission immediately following agonist addition) always observed in untreated or vehicle(DMSO, 0.2% v/v) treated platelets in response to any agonist examined (▶ Figure 1 A). The initial shape change response is caused by activated platelets becoming more spherical and forming pseudopods. Since the loss of this response is often due to prior shape change, we next examined the morphology of SCH79797-pretreated human platelets and saw these cells appeared more spherical than vehicle-treated platelets (▶ Figure 1 B). In line with these observations, the mean platelet volume (MPV) was significantly increased in SCH79797-pretreated human platelets compared with vehicletreated platelets (9.3 ± 0.3 vs. 7.4 ± 0.1 fL; p < 0.05, t-test). A chemically distinct PAR1 antagonist, E5555 (atopaxar, 0.1 μM), also functioned as a selective inhibitor of PAR1-AP induced aggregation of human platelets (▶ Figure 1 A), but had no effect on the shape change response (▶ Figure 1 A), platelet morphology (▶ Figure 1 B), or MPV (7.4 ± 0.1 fL) when used at concentrations required to abolish PAR1-mediated platelet aggregation. We next examined whether the altered platelet morphology induced by SCH79797 affected platelet function beyond an impairment of shape change. The plasma membranes of strongly activated platelets ‘flip’ to externalise phosphatidylserine (PS) not normally exposed on the platelet surface. Since altered platelet morphology suggested changes in membrane structure and/or function, we assessed the impact of SCH79797 on PS exposure via the binding of fluorescently labelled annexin V to the platelet surface. Strikingly, human platelets treated with SCH79797, but not E5555, externalised significant amounts of PS even in the absence of agonist stimulation (▶ Figure 1 C). PS externalisation occurred rapidly: ~20% of SCH79797-treated human platelets were PS-positive after 5 min (data not shown) and ~50% were PSpositive by 15 min compared with < 5% in vehicleor E5555-treated platelets (▶ Figure 1 C). An equivalent rate and extent of PS exposure was observed in wild-type mouse platelets, which do not express PAR1, and in platelets isolated from PAR4-/mice, which lack all PARs, indicating that the observed effect occurred independently of PAR binding and was therefore not due to partial agonist activity or any other PAR-dependent event. Finally, mouse or human platelets pretreated with SCH79797, but not E5555, and then stimulated with any of CRP (▶ Figure 1 C), ADP or thrombin (not shown) almost all expressed significant levels of PS soon (≤5 min) after stimulation (▶ Figure 1 C; data shown for 15 min CRP only). Of note, and as previously reported (22, 23), none of the platelet agonists examined stimulated robust (> 20%) PS externalisation in vehicletreated platelets (▶ Figure 1 C and data not shown). All of the PAR-independent effects Letters to the Editor