Thrombin inhibits nuclear factor κB and RhoA pathways in cytokine-stimulated vascular endothelial cells when EPCR is occupied by protein C

Summary

The occupancy of endothelial protein C receptor (EPCR) by protein C switches the protease activated receptor 1 (PAR-1)-dependent signalling specificity of thrombin from a permeability enhancing to a barrier protective response in vascular endothelial cells. In this study, the modulatory effects of thrombin and thrombin receptor agonist peptides (TRAP) on tumour necrosis factor (TNF)-α-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A were evaluated by monitoring the expression of cell surface adhesion molecules (VCAM-1, ICAM-1 and E-selectin), adhesion of freshly isolated neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factor-κB (NF-κB) pathway. The analysis of results indicate that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither PAR-1 agonist in-fluenced the proinflammatory effects of TNF-α in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin, thus leading to thrombin inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNF-α-activated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-κB pathways in response to TNF-α in cells pre-treated with protein C-S195A. Based on these results we conclude that when EPCR is ligated by protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses, thus leading to activation of Rac1 and inhibition of RhoA and NF-κB signalling cascades in vascular endothelial cells.

Keywords

Thrombin - EPCR - PAR-1 - protein C - cell adhesion - NF-κB - Rho

^* Current address: Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-Industry, Daegu Haany University, South Korea.

• References

• 1 Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Ann Rev Biochem 1988; 57: 915-956.
• 2 Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 1-5.
• 3 Bode W, Mayr I, Baumann U. et al. The refined 1.9 Å crystal structure of human α-thrombin: interaction with D-Phe-Pro-Arg chlorometheylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 8: 3467-3475.
• 4 Dang QD, Vindigni A, Di Cera E. An allosteric switch controls the procoagulant and anticoagulant activities of thrombin. Proc Natl Acad Sci USA 1995; 92: 5977-5981.
• 5 Coughlin SR. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost 2005; 3: 1800-1814.
• 6 Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood 2005; 106: 2605-2612.
• 7 Ritchie E, Saka M, MacKenzie C. et al. Cytokine upregulation of proteinase-activated receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells. Br J Pharmacol 2007; 150: 1044-1054.
• 8 Bae J-S, Yang L, Manithody C. et al. The ligand occupancy of endothelial protein C receptor switches the PAR-1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. Blood 2007; 110: 3909-3916.
• 9 McLaughlin JN, Patterson MM, Malik AB. Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization. Proc Natl Acad Sci USA 2007; 104: 5662-5667.
• 10 Feistritzer C, Schuepbach RA, Mosnier LO. et al. Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells. J Biol Chem 2006; 281: 20077-20084.
• 11 Garcia JGN, Pavalko FM, Patterson CE. Vascular endothelial cell activation and permeability responses to thrombin. Blood Coag Fibrinol 1995; 6: 609-626.
• 12 Finigan JH, Dudek SM, Singleton PA. et al. Activated protein C mediates novel lung endothelial barrier enhancement. J Biol Chem 2005; 280: 17286-17293.
• 13 Ruf W, Dorfleutner A, Riewald M. Specificity of coagulation factor signaling. J Thromb Haemost 2003; 1: 1495-1503.
• 14 Joyce DE, Grinnell BW. Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-kB. Crit Care Med 2002; 30: S288-293.
• 15 Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood 2005; 105: 3178-3184.
• 16 Joyce DE, Gelbert L, Ciaccia A. et al. Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J Biol Chem 2001; 276: 11199-11203.
• 17 Mosnier LO, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
• 18 Bernard GR, Vincent JL, Laterre PF. et al. Recombinant human protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Eng J Med 2001; 344: 699-709.
• 19 Ludeman MJ, Kataoka H, Srinivasan Y. et al. PAR1 cleavage and signaling in response to activated protein C and thrombin. J Biol Chem 2005; 280: 13122-13128.
• 20 Esmon CT. Is APC activation of endothelial cell PAR1 important in severe sepsis?: No. J Thromb Haemost 2005; 3: 1910-1911.
• 21 Ruf W. Is APC activation of endothelial cell PAR1 important in severe sepsis?: Yes. J Thromb Haemost 2005; 3: 1912-1914.
• 22 Bae J-S, Yang L, Rezaie AR. Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells. Proc Natl Acad Sci USA 2007; 104: 2867-2872.
• 23 Bae J-S, Yang L, Manithody C. et al. Engineering a disulfide bond to stabilize the calcium binding loop of activated protein C eliminates its anticoagulant but not protective signaling properties. J Biol Chem 2007; 282: 9251-9259.
• 24 Akeson AL, Woods CW. A fluorometric assay for the quantitation of cell adherence to endothelial cells. J Immunol Methods 1993; 163: 181-185.
• 25 Reid T, Bathoorn A, Ahmadian MR. et al. Identification and characterization of hPEM-2, a guanine nucleotide exchange factor specific for Cdc42. J Biol Chem 1999; 274: 33587-33593.
• 26 Bae J-S, Rezaie AR. Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand. Thromb Haemost 2008; 100: 101-109.
• 27 McLaughlin JN, Shen L, Holinstat M. et al. Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1. J Biol Chem 2005; 280: 25048-25059.
• 28 Blackhart BD, Emilsson K, Nguyen D. et al. Ligand cross-reactivity within the protease-activated receptor family. J Biol Chem 1996; 271: 16466-16471.
• 29 Taylor Jr FB, Chang A, Esmon CT. et al. Protein C prevents the coagulopathic and lethal effects of E coli infusion in the baboon. J Clin Invest 1987; 79: 918-925.
• 30 Bae J-S, Yang L, Rezaie AR. lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells. J Thromb Haemost 2008; 6: 954-961.
• 31 Ye J, Rezaie AR, Esmon CT. Glycosaminoglycan contributions to both protein C activation and thrombin inhibition involve a common arginine-rich site in thrombin that includes residues arginine 93, 97, and 101. J Biol Chem 1994; 269: 17965-17970.
• 32 Ishii K, Gerszten R, Zheng YW. et al. Determination of thrombin receptor cleavage. J Biol Chem 1995; 270: 16435-16440.
• 33 Jacques SL, LeMasurier M, Sheridan PJ. et al. Substrate-assisted catalysis of the PAR1 thrombin receptor. J Biol Chem 2000; 275: 40671-40678.
• 34 Nieman MT, Schmaier AH. Interaction of thrombin with PAR1 and PAR4 at the thrombin cleavage site. Biochemistry 2007; 46: 8603-8610.
• 35 Busch C, Cancilla P, DeBault L. et al. Use of endothelium cultured on microcarriers as a model for the microcirculation. Lab Invest 1982; 47: 498-504.
• 36 Esmon CT. Cell mediated events that control blood coagulation and vascular injury. Ann Rev Cell Biol 1993; 9: 1-26.