{"doi":"10.1016/j.jtha.2025.08.023","title":"Significance of the amino acid at position 2561 in the C4 domain of von Willebrand factor","abstract":"BACKGROUND: The von Willebrand factor (VWF) Phe2561Tyr variant has been previously shown to exhibit gain-of-function-like activity and increase the risk of repeated myocardial infarction in patients aged <55 years. It was hypothesized that altered stem dynamics enhanced the responsiveness of the molecule to shear stress. OBJECTIVES: In this study, we investigated the evolutionary significance of the amino acid at position 2561 and functional impacts of variants at this site. METHODS: Variants at 2561 identified by protein sequence alignments were generated by mutagenesis and expressed in HEK293T cells and function investigated using static and flow-based assays and biophysical methods. RESULTS: Phe2561 was heavily conserved in mammals; however, nonmammals occupied the site with tyrosine, threonine, or serine. Recombinant expression of Phe2561Tyr/Thr/Ser showed no effects on expression, multimers, collagen or glycoprotein IIbIIIa binding, but Ser2561 and Thr2561 demonstrated enhanced binding to gain-of-function glycoprotein Ibα. While none of the variants, including Tyr2561, enhanced VWF-mediated platelet capture to collagen at 1500/s or 5000/s, the Tyr2561, Ser2561, and Thr2561 variants exhibited enhanced formation of rolling VWF-platelet aggregates, with Ser2561 demonstrating the greatest gain-of-function activity. Optical tweezer pulling experiments using VWF-D'CK dimers revealed altered unfolding properties of the Tyr, Thr, and Ser2561 protein and atomic force microscopy imaging of VWF dimeric stems demonstrated that Tyr, Thr, and Ser2561 favored a more open stem state, suggesting this is responsible for the enhanced response to shear stress. CONCLUSION: This study indicates that the amino acid at 2561 is crucial for modulating VWF function by helping to mediate stem dynamics.","journal":"Journal of Thrombosis and Haemostasis","year":2025,"id":528795,"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":0.9611,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":434455,"name":"Yi Liu","orcid":"0000-0001-9748-0650","position":1,"is_corresponding":false},{"id":1407085,"name":"Fan Gong","orcid":"0000-0003-4328-4018","position":2,"is_corresponding":false},{"id":713725,"name":"Alain Chion","orcid":null,"position":3,"is_corresponding":false},{"id":397060,"name":"Stephen Rothery","orcid":"0000-0002-7464-7603","position":4,"is_corresponding":false},{"id":383004,"name":"Xiao Hui Zhang","orcid":"0000-0002-8778-595X","position":5,"is_corresponding":false},{"id":1407086,"name":"Golzar Mobayen","orcid":"0009-0007-3847-5035","position":6,"is_corresponding":false},{"id":1407538,"name":"Thomas A J McKinnon","orcid":null,"position":0,"is_corresponding":true}],"reference_count":42,"raw_metadata":null,"created_at":"2026-07-19T02:50:52.565868Z","pmid":"40915573","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":[]}