Factor VII autoactivation proceeds via interaction of distinct protease-cofactor and zymogen-cofactor complexes. Implications of a two-dimensional enzyme kinetic mechanism.

P.F Neuenschwander,M.M Fiore,J.H Morrissey

doi:10.1016/s0021-9258(20)80565-1

Abstract

Tissue factor (TF), an integral membrane protein, enhances the feedback activation of factor VII by factor VIIa (factor VII autoactivation). We found that, in contrast to the other known membrane-dependent coagulation activation reactions, TF-dependent factor VII autoactivation occurred preferentially on neutral phospholipid vesicles relative to negatively charged vesicles containing phosphatidylserine. This reaction was best described by a novel mechanism in which the enzyme and substrate are each bound to separate cofactor (TF) molecules. This unusual mechanism of substrate presentation to a membrane-bound protease predicts that the reaction rate will be directly dependent on the surface density, and hence lateral diffusion, of factor VII.TF and factor VIIa.TF complexes, obeying obligatorily two-dimensional enzyme kinetics. This prediction was confirmed, yielding a two-dimensional second-order rate constant (k2D) of 4.9 (+/- 0.8) x 10(6) m2 mol-1 s-1. Since intact cells normally sequester acidic phospholipids away from the outer leaflet of the plasma membrane, this reaction mechanism should permit factor VII autoactivation to predominate on unactivated/undamaged cell surfaces when other clotting reactions are dormant.

Highlights

If both enzyme and substrate are bound to Tissue factor (TF), and fVII autoactivation occurs in two dimensions on the plane of the phospholipid membrane, one can rewrite Equation 1in two-dimensional units of surface density of fVII.TF and fVII autoactivation in which the enzyme (fVIIa).TF complexes,yielding Equation 3
Models i and iipredict that fVII autoactivation will be a function of the bulk molar concentrationof fVII + fVIIa (Equation1).Model iii predicts that fVIIautoactivation will be dependent on the density of the fVII.TF and fVIIa.TcoFmplexes in thephospholipid membrane (Equation 3) and independent of the bulk molar concentrations of fVII + fVIIa when constant densityis maintained
The Effect of Varying IfVZZ + fVZZal on fVZZ Autoactivation -The effect of varying [fVII + fVIIa] on fVII autoactivation wasexamined by varying [fVII + fVIIa1 from 15 to 75 nM, keeping [TF] = [fVII + fVIIa1 at a constant TF density in the membrane

Summary

Introduction

If both enzyme and substrate are bound to TF, and fVII autoactivation occurs in two dimensions on the plane of the phospholipid membrane, one can rewrite Equation 1in two-dimensional units of surface density of fVII.TF and fVIIa.TF complexes (mol m-2),yielding Equation 3. KzD is a two-dimensional second-order rate constant in mz mol" s-I, DTFVII(ias)the total density of fVII.TF plus fVIIa.TF on the surface, in mol m-2, and DTFVIIIDTmIias the ratio of the density of substrate to enzyme (bound to TF) a t time = 0 and t.

Results

Conclusion