Mice with Reduced PAR4 Reactivity show Decreased Venous Thrombosis and Platelet Procoagulant Activity

Background Hypercoagulation and thrombin generation are major risk factors for venous thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single-nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3 (P310L), which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis. Objective The goal of this study is to determine the mechanism for the association of rs2227376 with reduced risk for VTE in using mice with a homologous mutation (PAR4-P322L). Methods Venous thrombosis was examined using our recently generated PAR4-P322L mice in the inferior vena cava stasis and stenosis models. Coagulation and clot stability was measured using rotational thromboelastometry (ROTEM). Thrombin generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry. Results PAR4 P/L and PAR4 L/L had reduced incidence and size of venous clots at 48 hours. PAR4 P/L and PAR4 L/L platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, which led to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation. Conclusions The leucine allele in extracellular loop 3, PAR4-322L leads to fewer procoagulant platelets and decreased endogenous thrombin potential. This decreased ability to generate thrombin offers a mechanism for PAR4’s role in VTE highlighting a key role for PAR4 signaling.