Knauss, E. A. | bioRxiv Science

bioRxiv2024

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

BackgroundHypercoagulation 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. ObjectiveThe 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). MethodsVenous 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. ResultsPAR4P/L and PAR4L/L had reduced incidence and size of venous clots at 48 hours. PAR4P/L and PAR4L/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. ConclusionsThe 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 PAR4s role in VTE highlighting a key role for PAR4 signaling.

physiology↗

bioRxiv2023

A Mouse Model of the Protease Activated Receptor 4 (PAR4) Pro310Leu Variant has Reduced Platelet Reactivity

BackgroundProtease activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated a single nucleotide polymorphism in extracellular loop 3 (ECL3), PAR4-P310L (rs2227376) leads to a hypo-reactive receptor. ObjectivesThe goal of this study was to determine how the hypo-reactive PAR4 variant in ECL3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). MethodsA point mutation was introduced into the PAR4 gene, F2rl3, via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by IIb{beta}3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. ResultsPAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or IIb{beta}3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. ConclusionsPAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts. EssentialsO_LIA mouse model was created to represent the PAR4-P310L sequence variant. C_LIO_LIPAR4-P322L leads to reduced platelet reactivity in response to PAR4-activation peptide and thrombin, while the ADP and GPVI signaling pathways were unaffected. C_LIO_LIThe PAR4-P322L mutation decreases time to occlusion in a mouse model of arterial thrombosis. C_LIO_LIThe PAR4-P322L mouse model provides a foundation to further explore the role of PAR4 in hemostasis and thrombosis. C_LI

physiology↗