Abstract
The optimally efficient production of thrombin by the prothrombinase complex relies on suitable positioning of its component factors and substrate on phosphatidylserine-containing lipid membranes. The presence of oxidatively damaged phospholipids in a membrane disrupts the normal architecture of a lipid bilayer and might therefore be expected to interfere with prothrombinase activity. To investigate this possibility, we prepared phosphatidylserine-containing lipid vesicles containing oxidized arachidonoyl lipids, and we examined their ability to accelerate thrombin production by prothrombinase. Oxidized arachidonoyl chains caused dose-dependent increases in prothrombinase activity up to 6-fold greater than control values. These increases were completely attenuated by the presence of alpha-tocopherol, gamma-tocopherol, or ascorbate. Over the course of a 300-min oxidation, the ability of arachidonoyl lipids to accelerate prothrombinase peaked at 60 min and then declined to base-line levels. These results suggest that instead of being impeded by oxidative membrane damage, prothrombinase activity is enhanced by one or more products of nonenzymatic lipid oxidation.
Highlights
Thrombin production is controlled in vivo by a complex system of cascade and feedback mechanisms
The addition of SAPC to vesicles otherwise composed of DMPC and DMPS increased prothrombinase complex (PTase) activity in a dose-dependent manner (Fig. 1)
The activity of PTase on pure SAPC vesicles was more than 10-fold lower than that on DMPC/ DMPS vesicles. This merely shows that SAPC by itself, in the absence of DMPS, is not able to support a high level of PTase activity
Summary
The fatty acyl chains of membrane lipids have long been regarded as having little or no effect on the catalytic activity of coagulation factor complexes [9], more recent studies have reported that unsaturated acyl chains do increase the intrinsic kcat for PTase, compared with saturated acyl chains [10]. Taken together with the susceptibility of unsaturated acyl chains to oxidative damage, and the production of potent oxidizing agents by platelets and other cells capable of supporting PTase activity, these observations suggest that unsaturated and oxidized fatty acyl chains on PTase activity may have significant effects on thrombin production by PTase To facilitate their detection and characterization of these effects, these investigations have been conducted in a chemically defined in vitro system consisting of synthetic lipid vesicles and purified human coagulation factors
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