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pubmed-article:8053894pubmed:abstractTextThe effects of phospholipid vesicles and their fatty acid compositions on the acceleration of Protein C activation by thrombin-thrombomodulin was studied in vitro. Four main phospholipid fractions were prepared from cultured human umbilical vein endothelial cells, and purified thrombomodulin from human placenta was reconstituted into vesicles consisting of phosphatidylcholine (PtdCho) alone, PtdCho plus phosphatidylethanolamine (PtdEtn), PtdCho plus phosphatidylserine (PtdSer) and PtdCho plus PtdIns (1:1, w/w in each case). Vesicles of PtdCho, PtdIns/PtdCho, PtdSer/PtdCho and PtdEtn/PtdCho increased thrombin-thrombomodulin-catalysed protein C activation by 1.2-, 1.9-, 4.3- and 8.4-fold respectively compared with that in the absence of phospholipid. This Protein C activation was not affected by distearoyl PtdEtn/distearoyl PtdCho, whereas it was markedly increased with increasing content of unsaturated fatty acid in PtdEtn. The thrombin-dependent Protein C activation by thrombomodulin reconstituted into dilinolenoyl PtdEtn/distearoyl PtdCho was 14.6 times that by thrombomodulin reconstituted into distearoyl PtdEtn/distearoyl PtdCho, as a result of a decrease in the dissociation constant (Kd) for thrombin and the Michaelis constant (Km) for Protein C of thrombomodulin. Binding of Protein C to PtdEtn/PtdCho fixed to a microwell plate required the presence of CaCl2 and increased with increasing degree of unsaturation of fatty acid in PtdEtn. As PtdEtn appeared on the outside of the plasma membrane in cultured human umbilical vein endothelial cells after thrombin stimulation, it was presumed that Protein C activation could be elevated by PtdEtn at the outer surface of the plasma membrane via an increased affinity between thrombomodulin, thrombin and Protein C, resulting from both increased formation of the thrombin-thrombomodulin complex via a conformational change in thrombomodulin and increased binding of Protein C to the membrane phospholipid in a Ca(2+)-dependent manner.lld:pubmed
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