Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood

Abstract

See also Lindahl TL, Macwan AS, Ramstr€ om S. PAR4 is more important than PAR1 for the thrombin-induced procoagulant effect on platelets.This issue, pp 1639-41. EssentialsThe platelet thrombin receptor, PAR4, is an emerging anti-thrombotic drug target. We examined the anti-platelet & anti-thrombotic effects of PAR4 inhibition in human blood. PAR4 inhibition impaired platelet procoagulant activity in isolated cells and during thrombosis. Our study shows PAR4 is required for platelet procoagulant function & thrombosis in human blood.Summary. Background: Thrombin-induced platelet activation is important for arterial thrombosis. Thrombin activates human platelets predominantly via proteaseactivated receptor (PAR)1 and PAR4. PAR1 has higher affinity for thrombin, and the first PAR1 antagonist, vorapaxar, was recently approved for use as an antiplatelet agent. However, vorapaxar is contraindicated in a significant number of patients, owing to adverse bleeding events. Consequently, there is renewed interest in the role of platelet PAR4 in the setting of thrombus formation. Objectives: To determine the specific antiplatelet effects of inhibiting PAR4 function during thrombus formation in human whole blood. Methods and Results: We developed a rabbit polyclonal antibody against the thrombin cleavage site of PAR4, and showed it to be a highly specific inhibitor of PAR4-mediated platelet function. This function-blocking anti-PAR4 antibody was used to probe for PAR4-dependent platelet functions in human isolated platelets in the absence and presence of concomitant PAR1 inhibition. The anti-PAR4 antibody alone was sufficient to abolish the sustained elevation of cytosolic calcium level and consequent phosphatidylserine exposure induced by thrombin, but did not significantly inhibit integrin a IIb b 3 activation, a-granule secretion, or aggregation. In accord with these in vitro experiments on isolated platelets, selective inhibition of PAR4, but not of PAR1, impaired thrombin activity (fluorescence resonance energy transfer-based thrombin sensor) and fibrin formation (anti-fibrin antibody) in an ex vivo whole blood flow thrombosis assay. Conclusions: These findings demonstrate that PAR4 is required for platelet procoagulant function during thrombus formation in human blood, and suggest PAR4 inhibition as a potential target for the prevention of arterial thrombosis.

Drugs targeting protease-activated receptor-4 improve the anti-thrombotic therapeutic window

“…In proof-of-concept work that supports previous studies (8)(9)(10)(11), the team at BMS first used function-blocking anti-PAR4 antibodies in a guinea pig model to show in vivo antithrombotic efficacy and relative safety of selective PAR4 blockade. To shift to the highly desired small molecule approach, they then embarked on an impressive drug discovery program.…”

“…This prolonged calcium signal mediates later-stage platelet activation events, such as the platelet procoagulant response involving phosphatidylserine exposure on the platelet membrane and consequent assembly of coagulation factors leading to thrombin generation and fibrin formation. Indeed, selective inhibition of PAR4 but not PAR1 significantly inhibits thrombin activity and fibrin deposition in human thrombi ex vivo (8). One explanation for the improved therapeutic window of BMS-986120 reported by Wong et al is that PAR4 inhibition is blocking platelet function at a distinct time and place to all existing approaches.…”

“…As a result, there has been much renewed interest in targeting the 'second' platelet thrombin receptor, PAR4, for antithrombotic therapy. Although previous studies have rationalised PAR4 as a viable antithrombotic target (8)(9)(10)(11), the work by Wong and colleagues expands on this to describe the development of a potent and specific small molecule PAR4 antagonist with a markedly improved therapeutic window over one standard antiplatelet drug (clopidogrel) in a preclinical model.…”

Counting and Tracking: Development and Use of New Methods for Detailed Analysis of Thrombus Formation

“…PAR1 has been an emerging target for anti-thrombotic inhibition with new potential drug candidates (55). The idea behind inhibiting the PAR receptors is that it would reduce the thrombin-induced platelet activation without inhibiting the other roles of thrombin, mainly coagulation (21).…”

“…The drug has not been approved in Europe. Vorapaxar was evaluated on top of dual antiplatelet therapy and for some patient groups led to an increased risk of serious bleeding, while it had a significant positive effect in the secondary prevention in others (21,47,55).…”