title: Demonstration of exosite I-dependent interactions of thrombin with human factor V and factor Va involving the factor Va heavy chain: analysis by affinity chromatography employing a novel method for active-site-selective immobilization of serine proteinases.
abstract: In an essential step of blood coagulation, factor V is proteolytically processed by thrombin to generate the activated protein cofactor, factor Va, and to release the activation fragments E and C1. For the identification and characterization of sites of thrombin binding to factor V and its activation products, a new method was developed for immobilizing thrombin and other serine proteinases specifically (>/=92%) through their active sites and used in affinity chromatography studies of the interactions. Interactions of factor V with exosite I of thrombin were shown to regulate the factor V activation pathway from the 93% +/- 12% inhibition of the rate of activation correlated with specific binding of hirudin54-65 to this exosite. Chromatography of factor V on active-site-immobilized thrombin showed only a weak interaction, while the factor Va heterodimer bound specifically and with apparently higher affinity, in an interaction that was prevented by hirudin54-65. The heavy chain of subunit-dissociated factor Va bound to immobilized thrombin, while the light-chain subunit and fragment E had no detectable affinity. These results demonstrate a previously undescribed, exosite I-dependent interaction of thrombin with factor Va that occurs through the factor Va heavy chain. They support the further conclusion that similar exosite I-dependent binding of thrombin to the heavy-chain region of factor V contributes to recognition of factor V as a specific thrombin substrate and thereby regulates proteolytic activation of the protein cofactor.
authors.affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: K R
authors.initials: KR
authors.lastname: Dharmawardana
authors.forename: P E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005165:Factor V / Q000378:metabolism*; D015943:Factor Va / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000494:pharmacology; D012685:Sepharose / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D006629:Hirudins; D010446:Peptide Fragments; C091596:hirudin (54-65); D015943:Factor Va; D005165:Factor V; D012685:Sepharose; D012697:Serine Endopeptidases; D013917:Thrombin