Abstract Number: OC 16.4
Meeting: ISTH 2021 Congress
Background: Protease activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated a single nucleotide polymorphism in ECL3, PAR4-P310L (rs2227376) leads to a hypo-reactive receptor in in vitro signaling assays. A multi-ancestry GWAS meta-analysis revealed that the Leu310 allele was associated with a 15% relative risk reduction for VTE compared to Pro310. This is consistent with the Leu310 having decreased PAR4 reactivity.
Aims: The goal of this study is to determine how the hypo-reactive PAR4 sequence variant in ECL3 impacts platelet function using a novel knock-in mouse model (PAR4-322L).
Methods: A point mutation was introduced into the PAR4 gene, F2rl3, via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Founders were characterized by miSeq analysis and confirmed by Sanger sequencing. Platelet response to PAR4-AP (AYPGKF), thrombin, ADP, and convulxin was monitored by measuring αIIbβ3 integrin activation and P-selectin surface expression using flow cytometry.
Wildtype (PAR4-322P) platelets responded to AYPGKF with an EC50 of 180μM for P-selectin surface expression or 143μM for αIIbβ3 activation. The EC50 for both markers of activation was increased ~2-fold for platelets from heterozygous mice (P/L) to 368μM or 284μM, respectively. Platelets from PAR4-322L mice required at least 1600 μM AYPGKF in both assays (Figure A and B). Compared to platelets from wildtype and heterozygous mice, the EC50 for thrombin activation was increased from 1.1 to 1.8nM for P-selectin translocation and from 1.0 to 1.7nM for αIIbβ3 activation in PAR4-322L (Figure C and D). The response to ADP and convulxin was unchanged among genotypes.
Conclusions: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and in vitro data. The PAR4-322L mouse model will provide the foundation to mechanistically determine the role of PAR4 variants in VTE.
To cite this abstract in AMA style:Han X, de la Fuente M, Knauss E, Nieman M. A Mouse Model of Protease Activated Receptor 4 (PAR4) variant rs2227376 Has Reduced Platelet Reactivity to Physiological Doses of Thrombin [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/a-mouse-model-of-protease-activated-receptor-4-par4-variant-rs2227376-has-reduced-platelet-reactivity-to-physiological-doses-of-thrombin/. Accessed May 16, 2022.
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