title: Exosite-interactive regions in the A1 and A2 domains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain.
abstract: Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser(205) --> Ala mutant indicated a high affinity site in the A1 subunit (K(d) approximately 5 nm) that was dependent upon the Na(+)-bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K(d) approximately 100 nm) was observed for both Na(+)-bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719-740, blocked thrombin cleavage of the isolated heavy chain at Arg(372) and Arg(740) and inhibited A2 binding to thrombin Ser(205) --> Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp(392) --> Ala/Asp(394) --> Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg(740), whereas cleavage at Arg(372) was not affected. These data suggest the acidic region comprising residues 389-394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation.
authors.affiliation: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
authors.forename: Keiji
authors.initials: K
authors.lastname: Nogami
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.forename: Timothy
authors.initials: T
authors.lastname: Myles
authors.forename: Lawrence L K
authors.initials: LL
authors.lastname: Leung
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D002384:Catalysis / Q000187:drug effects; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
chemical_list: D010446:Peptide Fragments; D021122:Protein Subunits; D005169:Factor VIII; D013917:Thrombin