Identification of structural domains in protein C involved in its interaction with thrombin-thrombomodulin on the surface of endothelial cells.
The structural domains of protein C involved in its interaction with thrombin-thrombomodulin on the endothelial cell surface have been investigated using isolated intact domains of bovine protein C produced from controlled proteolytic digests of the protein. The fragments investigated include the gamma-carboxyglutamic acid (Gla)-rich module, the two epidermal growth factor (EGF)-like modules, and a fragment consisting of the Gla and the two EGF-like modules. The effects of these fragments on the catalytic efficiency (Km and Vmax) of activation of protein C by the endothelial cell surface thrombin-thrombomodulin complex (IIa-TM) have been evaluated in vitro using a stirred microcarrier cell culture of bovine aortic endothelial cells and purified proteins. Neither the Gla nor the two EGF-like modules alone had any discernible effect on protein C activation. The intact Gla-EGF fragment, however, inhibited protein C activation. The results are consistent with a rapid equilibrium competitive inhibition model, in which the Gla-EGF fragment competes with protein C for binding to IIa-TM, and indicate that the Gla-EGF fragment alone accounts for most of the binding energy of intact protein C for IIa-TM. In addition, a requirement for the Gla residues of protein C for binding is implied by the observation that heat-decarboxylated Gla-EGF fragment was not an inhibitor of protein C activation. In addition, chloromethyl ketone-inactivated activated protein C was found to bind to IIa-TM with the same affinity as protein C, suggesting that the changes which occur in protein C upon activation do not affect that part of the protein responsible for binding to IIa-TM, that is the Gla-EGF region. The Gla-EGF region from factor X also weakly inhibited the IIa-TM activation of protein C.
Hogg
P J
PJ
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Ohlin
A K
AK
Stenflo
J
J
D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism*
D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D015055:1-Carboxyglutamic Acid; D066246:ErbB Receptors; D013917:Thrombin