title: Anticoagulant effects of a synthetic peptide containing residues Thr-2253-Gln-2270 within factor VIII C2 domain that selectively inhibits factor Xa-catalysed factor VIII activation.
abstract: Factor VIII (FVIII), an essential cofactor that accelerates the generation of factor Xa (FXa) in the tenase complex, is activated by proteolytic cleavage by thrombin or FXa. A strong relationship has been reported between high levels of FVIII activity and thrombosis. We have demonstrated previously that an anti-FVIII C2 antibody (ESH8) with a Val-2248-Gly-2285 epitope inhibited FXa-catalysed FVIII activation, and that a synthetic peptide designated EP-2 (residues 2253-2270) blocked C2 domain binding to FXa. We investigated the inhibitory effect of EP-2 on FXa-catalysed FVIII activation and its anticoagulant effect in the blood coagulation system. EP-2 inhibited FXa-catalysed activation in a clotting assay in a dose-dependent manner and reduced FXa generation in a chromogenic assay using FVIII, factor X, factor IXa and phospholipid. The peptide only inhibited FVIII binding to FXa. We also tested the anticoagulant effect of EP-2 in the plasma milieu. The peptide prolonged the activated partial thromboplastin time and activated clotting time in a dose-dependent manner, but not prothrombin time. Our results indicate that EP-2 mediates the anticoagulant effect by specific inhibition of FVIII and FXa interaction in the intrinsic pathway, and that FXa-catalysed FVIII activation plays a significant role in blood clotting. The peptide may provide the basis for the development of novel anticoagulant therapy.
authors.affiliation: Department of Paediatrics, Nara Medical University, Kashihara, Nara, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Nogami
authors.forename: Midori
authors.initials: M
authors.lastname: Shima
authors.forename: Katsumi
authors.initials: K
authors.lastname: Nishiya
authors.forename: Kazuya
authors.initials: K
authors.lastname: Hosokawa
authors.forename: Evgueni L
authors.initials: EL
authors.lastname: Saenko
authors.forename: John C
authors.initials: JC
authors.lastname: Giddings
authors.forename: Ichiro
authors.initials: I
authors.lastname: Tanaka
authors.forename: Akira
authors.initials: A
authors.lastname: Yoshioka
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology*; D001760:Bleeding Time; D003864:Depression, Chemical; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005165:Factor V / Q000378:metabolism; D005169:Factor VIII / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D010314:Partial Thromboplastin Time; D011485:Protein Binding / Q000187:drug effects
chemical_list: D000925:Anticoagulants; D005165:Factor V; D005169:Factor VIII; D015951:Factor Xa