Role of the membrane surface in the activation of human coagulation factor X.
Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS) or phosphatidylcholine alone (PC). Factor X activation was evaluated by discontinuous measurements of the amidolytic activity of the product, factor Xa, or continuously monitored using the fluorescent serine protease inhibitor 4-aminobenzamidine. The results of both techniques were verified by direct physical measurements of zymogen activation using SDS-polyacrylamide gel electrophoresis. The rate of factor X activation with PC vesicles was less than 5% of that observed with PCPS vesicles. Since factor X does not bind to vesicles containing only PC, these data suggested an important role for the substrate-membrane interaction in the catalytic cycle. The importance of the substrate-membrane interaction in the activation process was investigated by using membrane-binding proteins to compete with the substrate for combining sites on PCPS vesicles. Prothrombin fragment 1 was an inhibitor of factor X activation. The dependence of inhibition by fragment 1 on PCPS and factor X was consistent with a significant reduction in initial velocity due to the displacement of factor X from the membrane surface. The inhibition data also suggested that the membrane-bound pool of factor X was the preferred substrate for the human extrinsic Xase complex. The influence of PCPS concentrations on the rate of factor X activation was systematically investigated. Increasing concentrations of PCPS resulted in a modest change in the Km,app and a dramatic change in the Vmax,app for the reaction. The initial velocity data could be globally analyzed according to the preferential utilization of membrane-bound factor X with the intrinsic kinetic constants: Km approximately equal to 1 microM and kcat = 37 s-1 at saturating PCPS. In addition, the equilibrium parameters for the factor X-membrane interaction inferred from these studies were in excellent agreement with the directly determined values. Collectively, the data suggest that the substrate-membrane interaction must precede catalysis for the efficient activation of human factor X by the extrinsic Xase complex.
Krishnaswamy
S
S
Department of Biochemistry, University of Vermont, Burlington 05405.
Field
K A
KA
Edgington
T S
TS
Morrissey
J H
JH
Mann
K G
KG
D000818:Animals; D002417:Cattle; D015942:Factor VIIa / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D008565:Membrane Proteins / Q000378:metabolism; D008567:Membranes, Artificial; D008962:Models, Theoretical; D010446:Peptide Fragments / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology*; D011498:Protein Precursors / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism; D012542:Scattering, Radiation; D013925:Thromboplastin / Q000378:metabolism*; D013997:Time Factors
D008081:Liposomes; D008565:Membrane Proteins; D008567:Membranes, Artificial; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011498:Protein Precursors; D015943:Factor Va; C029733:prothrombin fragment 1; D011516:Prothrombin; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa