{"doi":"10.1101/2023.12.01.569075","title":"A Mouse Model of the Protease Activated Receptor 4 (PAR4) Pro310Leu Variant has Reduced Platelet Reactivity","abstract":"Abstract Background Protease 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. Objectives The 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). 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. Conclusions 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. Essentials A mouse model was created to represent the PAR4-P310L sequence variant. PAR4-P322L leads to reduced platelet reactivity in response to PAR4-activation peptide and thrombin, while the ADP and GPVI signaling pathways were unaffected. The PAR4-P322L mutation decreases time to occlusion in a mouse model of arterial thrombosis. The PAR4-P322L mouse model provides a foundation to further explore the role of PAR4 in hemostasis and thrombosis.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":397827,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9525,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":866201,"name":"Elizabeth A. Knauss","orcid":"0000-0002-6422-1844","position":1,"is_corresponding":false},{"id":573985,"name":"Maria de la Fuente","orcid":"0000-0002-0249-9728","position":2,"is_corresponding":false},{"id":385323,"name":"Wei Li","orcid":"0000-0002-7973-6242","position":3,"is_corresponding":false},{"id":262239,"name":"Ronald A. Conlon","orcid":"0009-0000-8601-8129","position":4,"is_corresponding":false},{"id":301465,"name":"David F. LePage","orcid":"0009-0008-3820-0645","position":5,"is_corresponding":false},{"id":327115,"name":"Weihong Jiang","orcid":"0000-0001-8369-9694","position":6,"is_corresponding":false},{"id":1173625,"name":"Stephanie A. Renna","orcid":"0000-0003-4056-6773","position":7,"is_corresponding":false},{"id":492802,"name":"Steven E. McKenzie","orcid":"0000-0003-2654-8560","position":8,"is_corresponding":false},{"id":295123,"name":"Marvin T. Nieman","orcid":"0000-0003-2602-023X","position":9,"is_corresponding":false},{"id":322853,"name":"Han Xu","orcid":"0000-0002-1977-1046","position":0,"is_corresponding":true}],"reference_count":45,"raw_metadata":null,"created_at":"2026-07-19T01:19:39.497368Z","pmid":"38077081","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}