Contribution of A1 subunit residue Q316 in thrombin-activated factor VIII to A2 subunit dissociation.
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.
Aflac Cancer Center and Blood Disorders Service, Children's Healthcare of Atlanta, and the Department of Pediatrics, Emory University, Atlanta, Georgia 30322, USA.
Ernest T
ET
Parker
Pete
P
Lollar
D000595:Amino Acid Sequence; D015944:Factor VIIIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000494:pharmacology
D021122:Protein Subunits; D015944:Factor VIIIa; D013917:Thrombin