title: Factor V C2 domain contains a major thrombin-binding site responsible for thrombin-catalyzed factor V activation.
abstract: Factor (F)V is converted into its active form, FVa, by limited proteolysis. Thrombin-catalyzed activation of FV is essential for its full cofactor activation. Previously, we reported that thrombin was bound to the C2 domain in the light chain of FVIII. As FV has a similar domain structure to FVIII, we focused on the FV C2 domain as a possible binding region for thrombin. Kinetic parameters, measured by surface plasmon resonance, revealed that the K(d) values of anhydro-thrombin for FV, FVa, and the FV C2 domain were 66, 240, and 670 nmol L(-1), respectively. FV activation was increased by approximately 9-fold by the addition of thrombin. In the presence of the FV C2 domain, this increase of the FV activation was inhibited. However, FV activation was not inhibited by the addition of the FVIII C2 domain. FV was cleaved into a 105-kDa heavy chain and a 71/74-kDa light chain by thrombin-catalyzed proteolysis at Arg709, Arg1018 and Arg1545. In the presence of the FV C2 domain, the cleavage was inhibited at all sites. Proteolysis was not affected by the addition of the FVIII C2 domain. These results indicated that the FV C2 domain contains a major binding site for thrombin and that this domain is necessary for the proteolysis at all cleavage sites. Furthermore, the present results also suggested that thrombin has an independent binding site for FV different from that for FVIII.
authors.affiliation: Department of Pediatrics, Nara Medical University, Kashihara, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Suzuki
authors.forename: M
authors.initials: M
authors.lastname: Shima
authors.forename: K
authors.initials: K
authors.lastname: Nogami
authors.forename: Y
authors.initials: Y
authors.lastname: Sakurai
authors.forename: K
authors.initials: K
authors.lastname: Nishiya
authors.forename: E L
authors.initials: EL
authors.lastname: Saenko
authors.forename: I
authors.initials: I
authors.lastname: Tanaka
authors.forename: A
authors.initials: A
authors.lastname: Yoshioka
mesh_terms: D001667:Binding, Competitive; D005165:Factor V / Q000737:chemistry* / Q000378:metabolism*; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D015943:Factor Va / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000378:metabolism*
chemical_list: D015943:Factor Va; D005165:Factor V; D005169:Factor VIII; D013917:Thrombin