Lipid mobility in the assembly and expression of the activity of the prothrombinase complex.

Abstract

A phospholipid or membrane surface is a required component of the prothrombinase complex, yet little is known about the influence of the lipid on the assembly and expression of this complex. Vesicles composed of synthetic phospholipids were used to investigate the effects of membrane "fluidity" on the prothrombinase complex. All vesicle types studied were capable of supporting the prothrombinase reaction which in each case was characterized by a similar apparent Km. The binding constants for the interaction of Factor Va and prothrombin with synthetic phospholipid vesicles were not significantly affected by temperature. The rate of thrombin production, however, increased with increasing temperature. The fluidity of the vesicles was assessed by measuring the fluorescence lifetimes, steady state anisotropies, and differential phase fluorometry of diphenylhexatriene embedded in the vesicles. No correlation was observed between the fluidity of the vesicles and the steady-state rate of thrombin production, even when the enzymatic activity was monitored below and above the phase transition temperature of the lipid vesicles. A distinct correlation, however, was found between the fluidity of the vesicle and the time required to reach the maximum rate of thrombin production (pre-steady-state interval). We believe that this "lag" time corresponds to the time required for the assembly of the prothrombinase complex. Thus, although lipid fluidity does affect the assembly of the prothrombinase complex, after the complex is assembled, this property has little effect on the catalytic process itself.