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J. Biol. Chem., Vol. 276, Issue 11, 7827-7835, March 16, 2001

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Surface-dependent Coagulation Enzymes
FLOW KINETICS OF FACTOR Xa GENERATION ON LIVE CELL MEMBRANES^*

Maria P. McGee^§ and Tom Chou^¶

From the  Department of Medicine, Wake-Forest University School of Medicine, Winston-Salem, North Carolina 27157 and the ^¶ Department of Biomathematics, UCLA, Los Angeles, California 90095-1766

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 cm² expressing tissue factor (TF) at >25 fmol/cm². 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/cm², 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/cm². 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.

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				M. P. McGee, H. A. Levivne, and S. Serna Identification of Mechanisms Mediating Coagulation Factor Penetration into the Glycocalyx: Mathematical Modeling of Transfer to TF (Tissue Factor) Sites. Blood (ASH Annual Meeting Abstracts), November 16, 2006; 108(11): 4019 - 4019. [Abstract] [PDF]