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

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.

SUBMITTER: Freyssinet JM

PROVIDER: S-EPMC1147109 | biostudies-other | 1986 Aug

REPOSITORIES: biostudies-other

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Project description:Human protein C is the precursor of a serine proteinase in plasma which contains nine 4-carboxyglutamic acid residues and functions as a potent anticoagulant. It is activated by thrombin in the presence of an essential endothelial-cell-membrane glycoprotein cofactor, thrombomodulin. In a purified human system, vitamin K-dependent proteins such as factor X, prothrombin and prothrombin fragment 1 were able to inhibit protein C activation by the thrombin-thrombomodulin complex, using either detergent-solubilized thrombomodulin or thrombomodulin reconstituted into vesicles consisting of phosphatidylcholine and phosphatidylserine (1:1, w/w). Factors VII and IX and protein S were much less efficient. Prothrombin fragment 1 behaved as a non-competitive inhibitor with apparent Ki values of 4 microM in the absence, and of 2-2.5 microM in the presence, of phospholipids. Heat decarboxylation of fragment 1 abolished its ability to interfere in protein C activation, and high phospholipid concentrations could attenuate its inhibitory effect and were responsible for a gradual loss of the non-competitive character. Fragment 1 also inhibited the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues, without any influence from phospholipids. At high thrombin concentrations, with respect to thrombomodulin, the inhibitory effect of fragment 1 was diminished. Fragment 1, at 3.8 microM, inhibited by 50% the activation of protein C (0.1 or 0.3 microM) by thrombin. These results suggest that the 4-carboxyglutamic acid domain of vitamin K-dependent proteins can act as a modulator of the protein C anticoagulant pathway through two distinct types of interaction. The functional 4-carboxyglutamic acid domain would be necessary to allow the enhancement of protein C activation in the presence of anionic phospholipids and it could recognize a phospholipid-independent binding site on the thrombin-thrombomodulin complex.

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

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