PB0404 - A Mouse Model of the PAR4 Sequence Variant P310L Shows Decreased Platelet Reactivity and Thrombosis

Background: Protease activated receptors 1 and 4 (PAR1, PAR4) on platelets mediate thrombin signaling and are vital for thrombus development. Hypercoagulation and thrombin generation are major risk factors for thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine (PS) exposure, and subsequent thrombin generation. A single-nucleotide polymorphism (SNP, rs2227376) leads to an amino acid change in extracellular loop 3 (ECL3) of PAR4, which decreases PAR4 activation (PAR4-P310L). This SNP is also associated with a lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis.

Aims: We aim to determine the mechanism by which PAR4-P310L (rs2227376) reduces the risk of VTE using mice with a homologous mutation (PAR4-P322L).

Methods: Ex vivo platelet function was determined using flow cytometry and aggregometry. Arterial and venous thrombosis was examined using the ferric-chloride induced carotid artery injury and the inferior vena cava (IVC) stasis models, respectively.

Results: Platelets from heterozygous (PAR4-322P/L) and homozygous (PAR4-322L/L) mice had decreased thrombin-mediated aggregation and decreased PAR4 reactivity compared to wild type. These mice also had a prolonged time to occlusion in arterial thrombosis. In venous thrombosis, wild-type mice developed clotted IVCs that averaged 31 mg after 48 hours, while global PAR4-knockout mice averaged 21mg (p < 0.01), PAR4-322P/L 23mg (p < 0.01), and PAR4-322L/L 28mg. We next examined the endogenous thrombin potential in the mutant PAR4 mice. The total thrombin and peak thrombin generation were decreased in platelet rich plasma from PAR4-322P/L and PAR4-322L/L mice.

Conclusion(s): Our new mouse model shows that the polymorphism in ECL3, PAR4-322L, decreases platelet reactivity and thrombosis. We also show PAR4-322L leads to less thrombin generation. This decreased ability to generate thrombin offers a mechanism for PAR4’s role in VTE. Our findings highlight a key role of PAR4 reactivity in platelet activation and procoagulant activity in thrombosis.