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

Han, X.; Knauss, E. A.; de la Fuente, M.; Li, W.; Conlon, R. A.; LePage, D. F.; Jiang, W.; Renna, S. A.; McKenzie, S. E.; Nieman, M. T.

doi:10.1101/2023.12.01.569075

bioRxiv · 10.1101/2023.12.01.569075

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

Han, X.·Knauss, E. A.·de la Fuente, M.·Li, W.·Conlon, R. A.·LePage, D. F.·Jiang, W.·Renna, S. A.·McKenzie, S. E.·Nieman, M. T.

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Abstract

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

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Han, X., Knauss, E. A., de la Fuente, M., Li, W., Conlon, R. A., LePage, D. F., Jiang, W., Renna, S. A., McKenzie, S. E., Nieman, M. T.. 2023-12-01. A Mouse Model of the Protease Activated Receptor 4 (PAR4) Pro310Leu Variant has Reduced Platelet Reactivity. https://doi.org/10.1101/2023.12.01.569075

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