Activated protein C signals through the thrombin receptor PAR1 in endothelial cells

Abstract

The anti-inflammatory effects of activated protein C (APC) have lead to its recent approval for the treatment of sepsis. Although the endothelial cell protein C receptor (EPCR) plays a crucial role in APC's protective roles in septicemia, the precise signaling mechanism of the protease APC remains unclear. In fibroblast overexpression systems, we find that APC activates protease activated receptors (PAR) 1 and 2 in an EPCR-dependent manner. Human endothelial cells (HUVECs) express PAR1, PAR2 and EPCR. Stimulation of HUVECs with either APC, or specific receptor activating peptides for PAR1 or PAR2, show that all three agonists induce a very similar set of early response genes as assessed by high density microarray analysis. Only the transcript for monocyte chemo-attractant protein-1 (MCP-1) was selectively induced by APC and the PAR1 agonist, but not by the PAR2 agonist. APC-mediated MAP kinase phosphorylation and gene induction were inhibited by cleavage blocking antibodies to PAR1, demonstrating that APC signals exclusively through PAR1 in endothelial cells. MCP-1 is protective in animal models of endotoxemia, suggesting that APC may prevent lethality in sepsis by inducing MCP-1 expression through EPCR-dependent activation of endothelial cell PAR1. These data demonstrate unexpected protective functions of the major thrombin receptor PAR1 in endothelial cells.

References

Creasey AA , Chang AC , Feigen L. , Wun TC , Taylor Jr FB , Hinshaw LB Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock. J Clin Invest 1993; 91: 2850-2856.

Taylor FB , Chang AC , Peer G. , Li A. , Ezban M. , Hedner U. Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD100 Escherichia coli. Blood 1998; 91: 1609-1615.

Taylor Jr FB , Chang A. , Ruf W. et al. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock 1991; 33: 127-134.

Coughlin SR Thrombin signalling and protease-activated receptors. Nature 2000; 407: 258-264.

O'Brien PJ , Molino M. , Kahn M. , Brass LF Protease activated receptors: theme and variations. Oncogene 2001; 20: 1570-1581.

Camerer E. , Huang W. , Coughlin SR Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc Natl Acad Sci USA 2000 ; 97: 5255-5260.

Riewald M. , Ruf W. Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor. Proc Natl Acad Sci USA 2001 ; 98: 7742-7747.

Taylor FB , Stearns-Kurosawa DJ , Kurosawa S. et al. The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis. Blood 2000 ; 95: 1680-1686.

Taylor FB , Chang A. , Esmon CT , D'Angelo A. , Vigano-D'Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79: 918-925.

10.

Bernard GR , Vincent JL , Laterre PF et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699-709.

11.

Esmon CT Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB J 1995; 9: 946-955.

12.

Joyce DE , Gelbert L. , Ciaccia A. , DeHoff B. , Grinnell BW Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J Biol Chem 2001; 276: 11199-11203.

13.

Liaw PC , Mather T. , Oganesyan N. , Ferrell GL , Esmon CT Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor. J Biol Chem 2001; 276: 8364-8370.

14.

O'Brien PJ , Prevost N. , Molino M. et al. Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1. J Biol Chem 2000; 275: 13502-13509.

15.

Riewald M. , Kravchenko VV , Petrovan RJ et al. Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1. Blood 2001 ; 97: 3109-3116.

16.

Riewald M. , Petrovan RJ , Donner A. , Mueller BM , Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 2002; 296: 1880-1882.

17.

Hanson SR , Griffin JH , Harker LA , Kelly AB , Esmon CT , Gruber A. Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates. J Clin Invest 1993; 92: 2003-2012.

18.

Grinnell BW , Berg DT Surface thrombomodulin modulates thrombin receptor responses on vascular smooth muscle cells. Am J Physiol 1996; 270: H603-H609.

19.

Esmon CT , Xu J. , Gu JM et al. Endothelial protein C receptor. Thromb Haemost 1999; 82: 251-258.

20.

Faust SN , Levin M. , Harrison OB et al. Dysfunction of endothelial protein C activation in severe meningococcal sepsis. N Engl J Med 2001; 345: 408-416.

21.

Zisman DA , Kunkel SL , Strieter RM et al. MCP-1 protects mice in lethal endotoxemia. J Clin Invest 1997; 99: 2832-2836.

22.

Bone-Larson CL , Hogaboam CM , Steinhauser ML et al. Novel protective effects of stem cell factor in a murine model of acute septic peritonitis. Dependence on MCP-1 . Am J Pathol 2000; 157: 1177-1186.