Improved Inhibition of Protease-Activated Receptor 4 (PAR4) with Function-Blocking Antibodies Provides Equivalent Antithrombotic Activity in the Face of the Hyper-Reactive Thr120 PAR4 Variant

Background: Thrombin activates platelets via two protease-activated receptors (PARs), PAR1 and PAR4, and both are targets of antithrombotic drugs - a PAR1 inhibitor is approved for clinical use and PAR4 inhibitors are in trial. However, recent studies report a PAR4 variant (rs773902; encoding Thr120 instead of Ala120) is expressed in up to 80% of individuals, depending on the population, and renders the receptor more sensitive to agonists and less sensitive to antagonists. These studies suggest the effectiveness of current strategies to inhibit PAR4 may vary substantially across the population. Here, we report the generation of a novel function-blocking PAR4 antibody that provides equivalent antithrombotic activity against the Ala120 or Thr120 PAR4 variant.

Methods: We generated a panel of anti-PAR4 antibodies and examined their inhibitory activity against the Ala120 and Thr120 PAR4 variants in cultured cells, isolated platelets, and in an ex vivo whole blood thrombosis assay.

Results: Our lead function-blocking antibody bound human PAR4 with high selectivity (~16-fold over PARs1, 2 or 3) and affinity (K_D ~ 5nM). This antibody provided near-complete inhibition of thrombin cleavage of either the Ala120 or Thr120 PAR4 variant when transfected in HEK293T cells. Platelets isolated from individuals expressing the Thr120 PAR4 variant exhibited increased thrombin-induced aggregation and phosphatidylserine exposure versus those expressing the Ala120 PAR4 variant. Yet the anti-PAR4 antibody inhibited these responses with an equivalent IC₅₀ of ~ 5 µg/ml. Furthermore, this function-blocking anti-PAR4 antibody significantly impaired platelet procoagulant activity in an ex vivo whole blood thrombosis assay. Here, equivalent inhibition of thrombin generation (FRET-based thrombin substrate) and fibrin formation (anti-fibrin antibody) was observed in thrombi generated in blood from individuals expressing the Ala120 or Thr120 PAR4 variant.

Conclusion: We generated a highly selective and effective function-blocking anti-PAR4 antibody that impairs thrombin-induced PAR4 cleavage, platelet activation, and pro-thrombotic activity equivalently in the presence of the Ala120 or Thr120 PAR4 variant. These findings reveal a novel approach to PAR4 inhibition that overcomes the impaired sensitivity of existing approaches against the commonly expressed Thr120 PAR4 variant, and provide rationale for such an approach for improved antithrombotic therapy.