The effects of shear rate on the enzymatic activity of the tissue factor-factor VIIa complex

A novel enzyme reactor for studying phospholipid-dependent reactions was used to explore the effects of flow on tissue factor (TF)-initiated coagulation. Capillary tubes (0.27 mm i.d.) were coated with a phospholipid bilayer containing TF, a transmembrane protein that is an essential cofactor for factor VII. Production of factor Xa exiting the tube was monitored with time during perfusion of the capillary with factor X (50 to 1500 nM) in the presence of factor VIIa (10 nM). Steady-state production of factor Xa as a function of [FX] was determined by chromogenic assay (Spectrozyme Xa) for a range of wall shear rates (25 to 3000 sec-1). Diffusion was found to play a major limiting role in FXa production for TF:30% phosphatidylserine (PS)/70% phosphatidylcholine (PC) surfaces. In contrast, TF/PC surfaces slowed the reaction sufficiently to enter a kinetically controlled regime where shear fluid had little effect on Km. In contrast with classical enzyme kinetic theory there was a three-fold increase in Vmax as shear increased from 25 to 300 sec-1. This finding implies a direct effect of shear on the kinetics of factor X activation by TF/FVIIa. The perfusion system is simple to use and offers the potential for studying the role of flow on a wide variety of enzymatic reactions related to coagulation.