Interference of blood-coagulation vitamin K-dependent proteins in the activation of human protein C. Involvement of the 4-carboxyglutamic acid domain in two distinct interactions with the thrombin-thrombomodulin complex and with phospholipids.
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.
Freyssinet
J M
JM
Unité 311 INSERM, Service d'Hémostase et de Thrombose, Centre Régional de Transfusion Sanguine, Strasbourg, France.
Beretz
A
A
Klein-Soyer
C
C
Gauchy
J
J
Schuhler
S
S
Cazenave
J P
JP
D003864:Depression, Chemical; D004730:Endothelium, Vascular / Q000378:metabolism; D005170:Factor X / Q000494:pharmacology*; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism; D011486:Protein C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011498:Protein Precursors / Q000494:pharmacology*; D011516:Prothrombin / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000378:metabolism; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism
D010446:Peptide Fragments; D010743:Phospholipids; D011486:Protein C; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C029733:prothrombin fragment 1; D011516:Prothrombin; D005170:Factor X; D013917:Thrombin