Mice with reduced protease-activated receptor 4 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 protease-activated receptor (PAR) 4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3, which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a genome wide association studies meta-analysis.

OBJECTIVES

The goal of this study was to determine the mechanism for the association of rs2227376 with a reduced risk of VTE 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 were measured using rotational thromboelastometry. Thrombin-generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry.

RESULTS

Mice heterozygous (PAR4) or homozygous (PAR4) at position 310 had reduced sizes of venous clots at 48 hours. PAR4 and PAR4 platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, in addition to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation.

CONCLUSION

The leucine allele in extracellular loop 3, PAR4-322L, leads to fewer procoagulant platelets, decreased endogenous thrombin potential, and reduced platelet-neutrophil aggregation. This decreased ability to generate thrombin and bind to neutrophils offers a mechanism for PAR4's role in VTE, highlighting a key role for PAR4 signaling.