BACKGROUNDHuman platelet activation and aggregation is a complex process. To date, many therapies have been developed targeting proteins that mediate this process to prevent unwanted activation. However, the current standard of care for acute coronary syndromes still has limitations, including bleeding risk.OBJECTIVETo evaluate the protease-activated receptor 4 (PAR4) anionic cluster as a viable antiplatelet target by using a polyclonal antibody (CAN12).METHODSWe used western blotting, aggregation and secretion ex vivo to evaluate the ability of CAN12 to interact with PAR4 and inhibit platelet activation. The effects of CAN12 in vivo were evaluated with the Rose Bengal arterial thrombosis model and two models of hemostasis.RESULTSCAN12 was able to interact with human PAR4 and delay PAR4 cleavage. In addition, CAN12 inhibited thrombin-induced human platelet aggregation and secretion in a dose-dependent manner. The specificity of CAN12 was agonist-dependent. In vivo, we determined that CAN12 was able to inhibit arterial thrombosis, and, using two independent methods, we found that CAN12 did not influence hemostasis.CONCLUSIONTargeting the extracellular anionic cluster on PAR4 is a viable novel strategy as an antiplatelet therapy.