Hypotension and inflammatory cytokine gene expression triggered by factor Xa–nitric oxide signaling

Hypotension and inflammatory cytokine gene expression triggered by factor Xa–nitric oxide signaling

Departments of ^*Pharmacology and ^‡Pathology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536; ^¶Dipartimento di Farmacologia Sperimentale, Universita’ degli Studi di Napoli “Federico II”, 80131 Naples, Italy; and ^‖Department of Pharmacology, Chiron Corporation, Emeryville, CA 94608

Edited by Salvador Moncada, University of London, London, United Kingdom, and approved February 12, 1998 (received for review November 3, 1997)

Abstract

The signaling pathway initiated by factor Xa on vascular endothelial cells was investigated. Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1–2.5 μg/ml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective. A factor Xa inter-epidermal growth factor synthetic peptide L⁸³FTRKL⁸⁸(G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective. Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor l-N ^G-nitroarginine methyl ester (l-NAME) but not by d-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55–3.1 ng/ml) in a reaction inhibited by l-NAME and by the inter-epidermal growth factor peptide Leu⁸³–Leu⁸⁸ but unaffected by hirudin. Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, unaffected by subsequent addition of tissue factor pathway inhibitor. These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and cytokine gene expression. This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo.

Footnotes

↵ † A.P. and P.P. contributed equally to this work.
↵ § Permanent address: Consorzio Mario Negri Sud, S. Maria Imbaro, Italy 66030.
↵ ** To whom reprint requests should be addressed at: Yale University School of Medicine, BCMM 436, 295 Congress Ave., New Haven, CT 06536. e-mail: william.sessa{at}yale.edu.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: EPR-1, effector cell protease receptor-1; HUVEC, human umbilical vein endothelial cells; l-NAME, l-N ^G-nitroarginine methyl ester; TAP, tick anticoagulant peptide; EGF, epidermal growth factor; Ach, acetylcholine; IL, interleukin; TFPI, tissue factor pathway inhibitor.