{"doi":"10.17615/8hph-9805","title":"Heparin Promotes Proteolytic Inactivation by Thrombin of a Reactive Site Mutant (L444R) of Recombinant Heparin Cofactor II","abstract":"A heparin cofactor II (HCII) mutant with an Arg substituted for Leu444 at the P1 position (L444R-rHCII) was previously found to have altered proteinase specificity (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 5623-5628). The present study characterizes the effect of glycosaminoglycans on the substrate versus inhibitor activity of L444R-rHCII. Heparin increased the stoichiometry of inhibition of L444R-rHCII with alpha-thrombin (compared with minus glycosaminoglycan) but decreased it with R93A,R97A,R101A-thrombin, a mutant thrombin that does not bind glycosaminoglycans. Dermatan sulfate decreased the stoichiometry of inhibition of L444R-rHCII with both proteinases. SDS-polyacrylamide gel electrophoresis showed no proteolysis of L444R-rHCII when incubated with R93A,R97A,R101A-thrombin in the absence or the presence of glycosaminoglycan or with alpha-thrombin and dermatan sulfate. In contrast, greater than 75% of the L444R-rHCII was converted to a lower molecular weight form when incubated with alpha-thrombin/heparin. A time course of alpha-thrombin inhibition by L444R-rHCII/heparin showed a rapid but transient inhibition with approximately 80% of the alpha-thrombin activity being regained after 6 h of incubation. In contrast, all other combinations of inhibitor, proteinase, and glycosaminoglycan resulted in complete and sustained inhibition of the proteinase. Heparin fragments of 8-20 polysaccharides in length rapidly accelerated L444R-rHCII inhibition of both alpha-thrombin and R93A,R97A,R101A-thrombin. After extended incubations, R93A,R97A,R101A-thrombin was completely inhibited by L444R-rHCII with all the heparin fragments, but approximately 30-50% of alpha-thrombin activity remained with fragments long enough to bridge HCII-thrombin. These results collectively indicate that ternary complex formation, mediated by heparin, increases L444R-rHCII inactivation by alpha-thrombin.","journal":"UNC Libraries","year":2021,"id":230186,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.953,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":836016,"name":"Annemieke Willemze","orcid":"0000-0001-9348-8479","position":1,"is_corresponding":false},{"id":836428,"name":"A Ciaccia","orcid":null,"position":2,"is_corresponding":false},{"id":592338,"name":"Frank Church","orcid":"0000-0002-4467-7828","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-18T23:55:05.844766Z","pmid":null,"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":[]}