Diffusion and Reaction on Cells Membranes: Surface Assembly of FVIIA (Factor VIIA) with TF (Tissue Factor) is Facilitated by FX (Factor X)

Abstract

Assembly of circulating fVIIa with the trans-membrane protein TF on procoagulant cells triggers blood coagulation by activating fX to fXa. Theoretical diffusion-reaction models and numerical simulations that incorporate surface-transfer steps for each reactant predict that the order sequence at which reactants enter the solution-phase influences subsequent surface-reaction kinetics. Here we test this prediction experimentally using live TF-bearing cells and pure fVIIa and fX.Generation of fXa from fX is measured after adding fVIIa and fX either sequentially (at a 5 min interval) or simultaneously to Vero cells anchored onto spherical microcarriers in batch and flow reactors, at concentration ratios fX/fVIIa > 100 .Surface TF by immunoassay is ∼ 25 fmol/cm2 ; MRT (mean reaction times) are calculated from the initial distribution of reaction times, i.e. while fXa generated is < 0.5 % of total fX added. Controls in both type reactors confirm that cells and fVIIa are indispensable for detectable fX activation. When reactants are added sequentially, MRT are shorter in both reactors if fVIIa rather than fX is added first, in agreement with theoretical predictions. However, if reactants are added simultaneously, in batch-reactors MRT agree with model's predictions while in flow reactors MRT are shorter than predicted. Further, MRT are also shortened in batch reactors if fVIIa is mixed with fX before adding the cells (to simulate dead-times of flow systems).Together, data is consistent with transient reaction steps where interactions between fVIIa and fX/fXa in solution facilitate subsequent surface transfer of fVIIa and/or its functional assembly with TF. These mechanisms may relate to stabilization of optimal fVIIa binding conformations and/or local changes in membrane geometry induced by coupled insertion of the two Gla-domains.