A Mouse Model of the Protease-Activated Receptor 4 Pro310Leu Variant has Reduced Platelet Reactivity
Document Type
Article
Publication Date
6-1-2024
Manuscript Version
vor
Abstract
Background: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. Objectives: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo 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. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbβ3 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. Results: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbβ3 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. Conclusion: PAR4-322L 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.
Keywords
animal model, blood platelets, protease-activated receptor 4, single nucleotide polymorphisms, thrombin receptor
Publication Title
Journal of Thrombosis and Haemostasis
Grant
R15HL145573
Rights
© 2024 The Authors. Published by Elsevier Inc. on behalf of International Society on Thrombosis and Haemostasis. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Xu Han, Elizabeth A. Knauss, Maria de la Fuente, Wei Li, Ronald A. Conlon, David F. LePage, Weihong Jiang, Stephanie A. Renna, Steven E. McKenzie, Marvin T. Nieman, A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity, Journal of Thrombosis and Haemostasis, Volume 22, Issue 6, 2024, Pages 1715-1726, ISSN 1538-7836, https://doi.org/10.1016/j.jtha.2024.03.004..