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