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J. Biol. Chem., Vol. 281, Issue 29, 20077-20084, July 21, 2006

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Protective Signaling by Activated Protein C Is Mechanistically Linked to Protein C Activation on Endothelial Cells^*

Clemens Feistritzer, Reto A. Schuepbach, Laurent O. Mosnier, Leslie A. Bush^¶, Enrico Di Cera^¶, John H. Griffin, and Matthias Riewald¹

From the Department of Immunology and Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the ^¶Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.

Received for publication, January 18, 2006 , and in revised form, May 2, 2006.

^* This work was supported by National Institutes of Health Grants HL 73318 (to M. R.), HL52246 (to J. H. G.), and HL49413, HL58141, and HL73813 (to E. D. C.); Junior Faculty (to M. R.) and Basic Research (to L. O. M.) Scholar Awards from the American Society of Hematology; an Austrian fellowship; Erwin Schrödinger-Auslandsstipendium J2413-B13 (to C. F.); and a grant from the Swiss National Science Foundation PBBBE-108544 (to R. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: The Scripps Research Inst., Dept. of Immunology, SP30-3040, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8226; Fax: 858-784-7276; E-mail: riewald{at}scripps.edu.

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