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. 1986 Aug 15;238(1):151-7.
doi: 10.1042/bj2380151.

The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex

The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex

J M Freyssinet et al. Biochem J. .

Abstract

Human thrombomodulin, an endothelial-cell-membrane glycoprotein, has been purified from placenta by Triton X-100 extraction and by affinity chromatography on concanavalin A-Sepharose and thrombin-Sepharose. It has been characterized by its ability to promote the activation of human protein C by human alpha-thrombin in the presence of Ca2+ and fulfilled the requirements of a cofactor. Reconstitution of thrombomodulin into phospholipid vesicles containing anionic phospholipids resulted in an increased rate of activation of protein C. Cardiolipin and vesicles containing phosphatidylcholine/phosphatidylserine (1:1, w/w) were the most effective. The apparent Km of the thrombin-thrombomodulin complex for protein C was 2 microM. It was not changed in the presence of phospholipid, whereas the Vmax. could be apparently increased up to 3.2-fold depending on the phospholipid and on its concentration, the catalytic-centre activity reaching 15.7 mol of activated protein C formed/min per mol of thrombin. Above their optimal concentrations, phospholipids inhibited the amidolytic activity of activated protein C. Phospholipids had no effect on the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues. These results show that the positive effect of anionic phospholipids in the activation of protein C by the thrombin-thrombomodulin complex involves a Ca2+-dependent interaction between protein C and phospholipids. They suggest that the enhancement of thrombomodulin activity by such phospholipids may be of functional significance.

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