An Antibody Targeted To The Anionic Region Of Human Protease Activated Receptor 4 Inhibits Thrombosis Without Influencing Bleeding

Protease activated receptors (PARs) are G-protein coupled receptors which are activated by cleavage of their N-terminus by thrombin. This generates a tethered ligand which is then able to activate the corresponding receptor. Human platelets express PAR1 and PAR4, which both have crucial roles in mediating the response of platelets to injury. Our hypothesis is that PAR4 is an ideal target for new anti-platelet therapies because it is required for stable clot formation and has limited tissue distribution. We have previously determined a region on PAR4 that is required for efficient activation by thrombin. A polyclonal antibody (CAN12) targeted to this region of the PAR4 exodomain does not cross react to PAR1. Initial studies determined that CAN12 is able to block thrombin-induced human platelet aggregation with an IC₅₀ of 10 ng/ml. Control IgG does not inhibit aggregation at 2 mg/ml. In mouse platelets, CAN12 inhibits P-selectin expression and integrin activation. In the Rose-Bengal mouse model of carotid artery thrombosis, CAN12 (1 mg/kg) administered 10 minutes prior to injury was able to completely inhibit the formation of a thrombus in a dose dependent manner. The antibody delayed thrombosis to greater than 90 min; the experiment was terminated at 90 minutes. In contrast, control treatment (2 mg/kg IgG or saline) resulted in complete occlusion at ∼40 minutes. Further, the minimal dose of CAN12 required for complete inhibition of thrombosis (0.5 mg/kg) administered fifteen minutes after injury also delayed thrombosis from ∼50 minutes to ∼80 minutes. This indicates that CAN12 is able to disrupt a thrombus after it has been initiated. Preliminary evidence indicates that CAN12 is able to delay the cleavage of PAR4. Importantly, CAN12 (2 mg/kg) treatment does not increase bleeding time or blood loss in the tail clip assay compared to control IgG (2 mg/kg) treatment. There was also no significant increase in bleeding in the saphenous vein assay. The mice treated with CAN12 (2 mg/kg) had an average bleeding time of 102 seconds for 12 clot formations in 20 minutes compared to the control mice (IgG 2 mg/kg) which had an average bleeding time of 143 seconds for 11 clot formations. These data demonstrate that we are able to inhibit platelet aggregation in vitro and thrombosis in vivo without influencing bleeding time. Overall, these studies provide insight towards the development of new anti-platelet therapies and, specifically, PAR4 as an antiplatelet therapy target.