Protease nexin-1 interacts with thrombomodulin and modulates its anticoagulant effect.
The endothelial cell membrane glycoprotein thrombomodulin (TM) plays a critical role in the regulation of coagulation. TM is an essential cofactor in protein C activation by thrombin, and a direct inhibitor of thrombin-induced platelet activation and fibrinogen clotting. Protease nexin-1 (PN-1) is a serpin synthesized and secreted by a variety of cells including endothelial cells. PN-1 bound to the cell surface through interactions with glycosaminoglycans, is an efficient inhibitor of thrombin and controls thrombin-induced cell responses. An investigation of the interaction of PN-1 with TM using purified proteins and cultured human aortic endothelial cells was performed. Purified PN-1 was observed to bind to purified TM in a concentration-dependent manner. Double immunofluorescence studies indicated that PN-1 and TM were colocalized at the endothelial cell surface from which they were coprecipitated. Pretreatment of the cells with chondroitinase ABC greatly decreased the amount of the PN-1 associated to TM at the cell surface demonstrating the involvement of the TM chondroitin-sulfate chain in the formation of complexes. The inhibitory activity of the PN-1/TM complexes on the catalytic activity of thrombin, and on thrombin-induced fibrinogen clotting, was markedly enhanced when compared with the inhibitory activity of each partner. PN-1-overexpressing human aortic endothelial cells and PN-1-underexpressing human aortic endothelial cells exhibited respectively a significantly reduced ability and enhanced capacity to activate protein C. Furthermore, PN-1 decreased the cofactor activity of TM on thrombin activable fibrinolysis inhibitor activation by thrombin. These data show for the first time that PN-1 forms complexes with TM and modulates its anticoagulant activity.
Bouton
Marie-Christine
MC
Unité INSERM U698, CHU Xavier Bichat, Paris, France. mcbouton@bichat.inserm.fr
Venisse
Laurence
L
Richard
Benjamin
B
Pouzet
Cécile
C
Arocas
Véronique
V
Jandrot-Perrus
Martine
M
D016564:Amyloid beta-Protein Precursor / Q000378:metabolism / Q000502:physiology*; D001777:Blood Coagulation / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology; D006801:Humans; D058255:Protease Nexins; D011485:Protein Binding / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism / Q000502:physiology*; D058258:Serpin E2; D018180:Thrombomodulin / Q000378:metabolism* / Q000502:physiology
D016564:Amyloid beta-Protein Precursor; D058255:Protease Nexins; D011956:Receptors, Cell Surface; C545486:SERPINE2 protein, human; D058258:Serpin E2; D018180:Thrombomodulin