  The initial surface reactions of the extrinsic coagulation pathway on live
cell membranes were examined under flow conditions. Generation of fXa
(activated coagulation factor X) was measured on spherical monolayers of
epithelial cells with a total surface area of 41-47 cm$^{2}$ expressing
TF(tissue factor) 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 $3.05 \pm 0.75$ 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 tissue factor pathway.
