title: Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation.
abstract: Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.
authors.affiliation: Department of Biochemistry, University of New Hampshire, Durham 03824.
authors.forename: E A
authors.initials: EA
authors.lastname: Luckow
authors.forename: D A
authors.initials: DA
authors.lastname: Lyons
authors.forename: T M
authors.initials: TM
authors.lastname: Ridgeway
authors.forename: C T
authors.initials: CT
authors.lastname: Esmon
authors.forename: T M
authors.initials: TM
authors.lastname: Laue
mesh_terms: D000818:Animals; D002417:Cattle; D004792:Enzyme Precursors / Q000378:metabolism*; D005165:Factor V / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D004792:Enzyme Precursors; D011486:Protein C; D005165:Factor V; D011516:Prothrombin; C012007:prethrombins