Kinetics of the interaction between the human factor VIIIa subunits: effects of pH, ionic strength, Ca2+ concentration, heparin, and activated protein C-catalyzed proteolysis.
Coagulation factor VIIIa consists of a heterotrimer in which the A2 subunit is bound to the A1/A3C1C2 dimer. The dissociation of this complex causes the spontaneous and reversible decay of factor VIIIa activity. In order to characterize the kinetics and affinity of the interaction between A2 and A1/A3C1C2, as well as the influence of different parameters on the interaction, the subunits were chromatographically separated and reassembled in a BIAcore instrument (Pharmacia Biosensor). In the binding experiments, A2 was free in solution, whereas A1/A3C1C2 was immobilized on the dextran surface by direct coupling or captured on an immobilized monoclonal anti-C2 antibody. At our chosen standard condition (pH = 6.0, I = 0.12, and [Ca2+] = 2 mM), the association rate constant, dissociation rate constant, and resulting equilibrium dissociation constant were ca. 1.4 x 10(4) M-1s-1, 2.1 x 10(-4)s-1, and 16 nM, respectively. Increasing the ionic strength or Ca2+ concentration resulted in both slower association and faster dissociation. At 0.3 M NaCl or 25 mM Ca2+, the dissociation constant was > 1 microM. This implies that electrostatic forces involved in the interaction contribute at least one-fourth of the total binding energy. Increasing pH caused a similar effect, yielding a dissociation constant of ca. 0.9 microM at pH 7.5. In those cases where the equilibrium dissociation constants had been determined from solution phase experiments [Fay, P. J., & Smudzin, T. M. (1992) J. Biol. Chem. 267, 13246-13250; Lollar, P., Parker, E. T., & Fay, P. J. (1992) J. Biol. Chem. 267, 23652-23657], these constants agreed well with our results.(ABSTRACT TRUNCATED AT 250 WORDS)
Persson
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Department of Coagulation Research, Novo Nordisk A/S, Gentofte, Denmark.
Ezban
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M
Shymko
R M
RM
D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D015944:Factor VIIIa / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009994:Osmolar Concentration; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*
D011486:Protein C; D015944:Factor VIIIa; D006493:Heparin; D002118:Calcium