Contribution of A1 subunit residue Q316 in thrombin-activated factor VIII to A2 subunit dissociation

Abstract

Blood coagulation factor VIII (fVIII) is activated by thrombin to form an A1/A2/A3-C1-C2 heterotrimer, which functions as a cofactor for factor IXa during intrinsic pathway factor X activation. Human thrombin-activated fVIII (fVIIIa) decays rapidly because of first-order dissociation of the A2 subunit, which may function to regulate the coagulation mechanism. The three fVIII A domains each consist of two cupredoxin-like subdomains. Substitution of the COOH-terminal A1 subdomain of porcine fVIIIa, which decays more slowly than human fVIIIa, reduces the dissociation rate constant for fVIIIa decay. Examination of a human fVIII A1-A2-A3 homology model [Pemberton, S., et al. (1997) Blood 89, 2413-2421) revealed a possible interaction between Q316 in the FG helix of the COOH-terminal A1 subdomain and M539 in the FG helix of the NH2-terminal A2 subdomain, which are sites where human and porcine fVIII differ. Decays of purified recombinant human and porcine fVIIIa and the human fVIIIa mutants Q316H, M539L and Q316H/M539L were compared at 23 and 37 degrees C. The decay rates of the Q316H and Q316H/M539L mutants, but not the M539L mutant, were significantly slower than human fVIIIa. These results indicate that the FG helix of the COOH-terminal A1 cupredoxin-like subdomain of fVIII may be under selective pressure by the requirements of hemostatic balance.

Fig. 1. FVIII domain and subdomain structure

(A) The arrangement of the NH2- and COOH-terminal cupredoxin-like subdomains of the three A domains is shown, along with the B, C1 and C2 domains. (B) β-strand structure of cupredoxin-like molecules (37). The two β-sheets are formed by strands A, C and E and strands G, F, and D, respectively.

Fig. 2. Alignment of the A1-COOH and A2-NH2 subdomains of human and porcine fVIII

A1-COOH and A2-NH2 subdomains are defined as residues 191-336 and 373-556, respectively, using human fVIII amino acid sequence numbering (38). Residues 337-372 (ar) correspond to an acidic region that is COOH-terminal of an activated protein C recognition site at R336. The ar segment is not paralogous to the fVIII A2 or A3 domains or orthologous to factor V or ceruloplasmin. Asterisks denote identical amino acids. Residues Q316 and M539 are marked by the closed circles. A, B, C, D, E, F and G represent β-sheets in the fVIII homology model (see Fig. 1B).

Fig. 3. Decay rates of fVIIIa constructs

Decays of human fVIIIa (●), porcine fVIIIa (■) and human fVIIIa mutants Q316H (○), M539L (▼) and Q316H/M539L (□) at 23 °C (A) or 37 °C (B) were measured at a fVIIIa starting concentration of 1 nM as described in “Experimental Procedures”. The curves are weighted nonlinear least-squares fits to a single exponential decay with the fitted parameters shown in Tables II, Experiment 1.

Fig. 3. Decay rates of fVIIIa constructs

Decays of human fVIIIa (●), porcine fVIIIa (■) and human fVIIIa mutants Q316H (○), M539L (▼) and Q316H/M539L (□) at 23 °C (A) or 37 °C (B) were measured at a fVIIIa starting concentration of 1 nM as described in “Experimental Procedures”. The curves are weighted nonlinear least-squares fits to a single exponential decay with the fitted parameters shown in Tables II, Experiment 1.

Fig. 4. Model of the interface of the fVIII COOH-terminal A1 and the NH2-terminal A2 subdomains

F and G represent the β-strands flanking the FG helices in the two subdomains.

Scheme I