Enhancement of thrombin-thrombomodulin-catalysed protein C activation by phosphatidylethanolamine containing unsaturated fatty acids: possible physiological significance of phosphatidylethanolamine in anticoagulant activity of thrombomodulin

Abstract

The 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.

Expression of Thrombomodulin in Atherosclerotic Lesions and Mitogenic Activity of Recombinant Thrombomodulin in Vascular Smooth Muscle Cells

“…The anticoagulant activity of TM depends on the local cell-membrane status. 29,30 TM serves as a regulator in maintaining the fluidity of circulating blood; accordingly, TM downregulation on damaged endothelial cells potentiates the prothrombotic properties of the vascular wall, promotes coagulation, and contributes to thrombosis.…”

Acceleration of Thrombomodulin Gene Transcription by Retinoic Acid

“…24 h after the addition of culture medium, t-RA (10 M in 0.1% dimethyl sulfoxide (Me 2 SO), final concentration) or 9C-RA (10 M in 0.1% Me 2 SO, final concentration) was added to the DNA-transfected cells for 24 h. The cells were recovered and sonicated, and the resulting supernatants were used for determinations of CAT and ␤-galactosidase activities. HUVE cells were cultured in Dulbecco's modified Eagle's medium containing 20% FCS without endothelial cell growth supplement and heparin, and secondary cultures on collagen-coated dishes were used for experiments (11,36).…”

Oxidized phospholipids in oxidized low-density lipoprotein down-regulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RARβ-RXRα heterodimers and Sp1 and Sp3 to their binding sequences in the TM promoter

“…The cofactor activity of TM isolated from human placenta is markedly enhanced by reconstitution into vesicles consisting of phosphatidylcholine and acidic phospholipids, such as phosphatidylserine and phosphatidylethanolamine, so that optimal antithrombotic TM activity depends on the local cell membrane status. 6,7 The cDNA sequence of human TM encodes for 575 amino acids. 8,9 The 5Ј-flanking region of the human TM gene contains a TATA box, a CAAT box, and several conserved transcription factor binding elements such as a retinoic acid receptor responsive element (RARE) and stimulatory protein 1 (Sp1) binding element.…”