The activation of factor X by Tissue Factor(TF):VIIa on a membrane surface is one of the principal events in the initiation of coagulation. We have previously shown and presently confirm that reactions initiated by TF:VIIa do not typically result in the complete activation of substrate, but rather some intermediate level of fX activation. Excess phospholipid has complex effects on the TF:VIIa reaction, resulting in either inhibition, due to substrate depletion, or acceleration, by virtue of binding fXa. This complexity results in unavoidable ambiguities in interpretation of all previous reports. To tease apart these complexities, we have used benzamidine-sepharose that only binds fXa and not fX. We now show that the reaction product, fXa, can impede, and in fact, completely inhibit TF:VIIa activity. We monitored progress curves of fX activation by TF:VIIa in the presence and absence of benzamidine beads, which specifically partition the reaction product away from the TF:VIIa complexes. In the presence of these benzamidine beads, the activation rate of fX increased by 60% confirming that removal of fXa enhances tissue factor activity. Moreover, binding studies of fXa to TF-phospholipid surfaces indicate that the surface occupancy by fXa regulates TF:VIIa activity. Surface occupancy was evaluated by Total Internal Reflectance (on macroscopic lipid bilayers) and Fluorescence Resonance Energy Transfer (phospholipid vesicles). Our measurements of the off-rate of fXa (0.06–0.08 /s) are similar to some literature values, but differ by up to 500-fold from others. Stochastic simulations of TF:VIIa kinetics on phospholipid surfaces confirm that product leaving rates strongly regulate the observed experimental kinetics. Our approach indicates that current models of coagulation need to be altered to accommodate product surface occupancy. Indeed, if fXa occupancy is ignored, as is generally the case, the models simply cannot reflect reality.

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