title: Surface-dependent coagulation enzymes. Flow kinetics of factor Xa generation on live cell membranes.
abstract: The initial surface reactions of the extrinsic coagulation pathway on live cell membranes were examined under flow conditions. Generation of activated coagulation factor X (fXa) was measured on spherical monolayers of epithelial cells with a total surface area of 41-47 cm(2) expressing tissue factor (TF) at >25 fmol/cm(2). Concentrations of reactants and product were monitored as a function of time with radiolabeled proteins and a chromogenic substrate at resolutions of 2-8 s. At physiological concentrations of fVIIa and fX, the reaction rate was 3.05 +/- 0.75 fmol fXa/s/cm(2), independent of flux, and 10 times slower than that expected for collision-limited reactions. Rates were also independent of surface fVIIa concentrations within the range 0.6-25 fmol/cm(2). The transit time of fX activated on the reaction chamber was prolonged relative to transit times of nonreacting tracers or preformed fXa. Membrane reactions were modeled using a set of nonlinear kinetic equations and a lagged normal density curve to track the expected surface concentration of reactants for various hypothetical reaction mechanisms. The experimental results were theoretically predicted only when the models used a slow intermediate reaction step, consistent with surface diffusion. These results provide evidence that the transfer of substrate within the membrane is rate-limiting in the kinetic mechanisms leading to initiation of blood coagulation by the TF pathway.
authors: Department of Medicine, Wake-Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. mmcgee@wfubmc.edu
authors: M P
authors: MP
authors: McGee
authors: T
authors: T
authors: Chou
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005170:Factor X / Q000096:biosynthesis; D015951:Factor Xa / Q000096:biosynthesis*; D007700:Kinetics; D014709:Vero Cells
chemical_list: D005170:Factor X; D015951:Factor Xa