Modulation of tissue factor and tissue factor pathway inhibitor-1 by neutrophil proteases

• Published in: Thrombosis and haemostasis Vol. 100; no. 6; p. 1068
• Main Authors: Steppich, Birgit A, Seitz, Isabell, Busch, Gabi, Stein, Andreas, Ott, Ilka
• Format: Journal Article
• Language: English
• Published: Germany 01.12.2008
• Subjects: Blood Coagulation; Cathepsin G; Cathepsins - metabolism; Cells, Cultured; Coculture Techniques; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Flow Cytometry; Humans; Leukocyte Elastase - metabolism; Lipoproteins - genetics; Lipoproteins - metabolism; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; Myeloblastin - metabolism; Neutrophil Activation; Neutrophils - enzymology; Peptide Fragments - pharmacology; Receptor, PAR-1 - agonists; Receptor, PAR-1 - metabolism; Receptor, PAR-2 - metabolism; RNA Interference; RNA, Messenger - metabolism; RNA, Small Interfering - metabolism; Serine Endopeptidases - metabolism; Thromboplastin - metabolism; Time Factors
• DOI: 10.1160/th08-05-0293
• ISSN: 0340-6245

During systemic inflammation, neutrophil activation is accompanied by endothelial cell damage and hypercoagulability. Activated neutrophils release serine proteases that participate in tissue injury. We sought to investigate the effects of neutrophil proteases on proinflammatory and procoagulant changes in endothelial cells. The effects of elastase (HNE), cathepsin G (CG), and proteinase 3 (PR3) on expression of tissue factor (TF) and tissue factor pathway inhibitor-1 (TFPI) were examined in human umbilical vein endothelial cells. Flow cytometry demonstrated that these proteases proteolytically degraded endothelial cell-bound TFPI. TFPI mRNA expression was reduced by HNE and CG. PR3, but not HNE or CG, increased surface expression of TF and TF mRNA. Yet, increased TF expression did not enhance TF activity suggesting induction of encrypted TF. Using antibodies and siRNA to inhibit and silence PAR-1 and PAR-2, we observed that PR3 upregulation of TF is at least in part mediated by PAR-1. Although CG and HNE cleaved PAR-1, antibody reactivity to the PAR-1 hirudin-like sequence demonstrated inactivating cleavage, accounting for the selective ability of PR3 to induce PAR-1-mediated procoagulant effects. This was supported by induction of p42/44 MAPK by PR3. In conclusion, PR3 degradation of TFPI increases the procoagulant activity of endothelial cells. Release of PR3 after neutrophil activation may represent an important step in neutrophil-mediated vascular injury.