Human Umbilical Vein Smooth Muscle Cells as a Model to Study Thrombin Generation and Function: Effect of Thrombin Inhibitors

Summary

The aim of the present work was to study how human umbilical vein smooth muscle cells (HUVSMC) can initiate the coagulation process and to investigate the responses of these cells to thrombin. Exposure of HUVSMC to recalcified human plasma led to a time-dependent production of thrombin, measured both as amidolytic activity and as release of fibrinopeptide A. Thrombin activity was dose-dependently reduced by an anti-human tissue factor antibody (76 ± 3% at 10 Μg/ml) and by inhibitors like heparin, rec-hirudin, hirulog-1, Napap and hiru-norm, a novel hirudin-like thrombin inhibitor (IC₅₀ = 2 ± 0.4, 8 ± 1, 130 ± 22, 199 ± 29 and 68 ± 8nM, respectively). The release of fibrinopeptide A was similarly prevented (IC₅₀ = 14 ± 1,132 ± 25 and 50 ± 8 nM for rec-hirudin, Napap and hirunorm, respectively). Exogenously added thrombin increased thymidine incorporation into HUVSMC to 240 ± 30% of basal (EC₅₀ = 0.49 ± 0.09 nM) and thrombin inhibitors blocked this effect (IC₅₀ = 10 ± 3, 37 ± 17, 343 ± 165 and 1402 ± 758 nM for rec-hirudin, hirunorm, Napap and hirulog-1, respectively). Also recalcified human plasma was mitogenic for HUVSMC and its effect was mainly due to endogenously generated thrombin, as shown by the use of thrombin inhibitors. In conclusion, HUVSMC are capable of initiating the extrinsic coagulation cascade, leading to the formation of thrombin which promotes clotting and stimulates DNA synthesis. Thrombin inhibitors prevent both coagula-tive and cellular effects of thrombin.

• References

• 1 Davi EW. Biochemical and molecular aspects of the coagulation cascade. Thromb Haemost 1995; 74: 1-6
• 2 Edgington TS, Mackman N, Brand K, Ruf W. The structural biology of expression and function of tissue factor. Thromb Haemost 1991; 66: 67-79
• 3 Ruf W, Edgington TS. Structural biology of tissue factor, the initiator of thrombogenesis in vivo. FASEB 1994; 8: 385-390
• 4 Taubman MB, Marmur JD, Rosenfield CL, Guha A, Nichtberger S, Nemerson Y. Agonist-mediated tissue factor expression in cultured vascular smooth muscle cells. J Clin Invest 1993; 91: 547-552
• 5 Parolari A, Antona C, Colli S, Mussoni L. Serum and thrombin induce tissue factor expression in human smooth muscle cells. Thromb Haemost 1995; 73: 1182
• 6 Ogletree ML, Natarajan S, Seiler SM. Thrombin receptors as drug discovery targets. Perspectives in Drug Discovery and Design 1993; 1: 527-536
• 7 De Caterina R, Sicari R. Cellular effects of thrombin: pharmacology of the receptor(s) in various cell types and possible development of receptor antagonists. Pharmacol Res 1993; 27: 1-19
• 8 Coughlin SR, Vu TKH, Hung DT, Wheaton VI. Characterization of a functional thrombin receptor, issues and opportunities. J Clin Invest 1992; 89: 351-355
• 9 Fager G. Thrombin and proliferation of vascular smooth muscle cells. Circ Res 1995; 77: 645-650
• 10 Tesfamariam B. Distinct receptors and signalling pathways in a-thrombin and thrombin receptor peptide-induced vascular contractions. Circ Res 1994; 74: 930-936
• 11 Baron A, Loirand G, Pacaud P, Mironneau C, Mironneau J. Dual effect of thrombin on voltage-dependent Ca2+ channels of portal vein smooth muscle cells. Circ Res 1993; 72: 1317-1325
• 12 Wojta J, Gallichio M, Zoellner H, Hufnagl P, Last K, Filonzi EL, Binder BR, Hamilton JA, Mcgrath K. Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells. Thromb Haemost 1993; 70: 469-474
• 13 Vu TKH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-1068
• 14 Zhong C, Hayzer DJ, Corson MA, Runge MS. Molecular cloning of the rat vascular smooth muscle thrombin receptor. J Biol Chem 1992; 267: 16975-16979
• 15 Rasmussen UB, Vouret-Craviari V, Jallat S, Schlessinger Y, Pages G, Pavirani A, Lecocq JP, Pouyssegur J, Obberghen-Schilling E. cDNA cloning and expression of a hamster a-thrombin receptor coupled to Ca2+ mobilization. FEBS Lett 1991; 288: 123-128
• 16 Grand RJA, Tumell AS, Grabham PW. Cellular consequences of thrombin-receptor activation. Biochem J 1996; 313: 353-368
• 17 McNamara CA, Sarembock IJ, Gimple LW, Fenton IIJW, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993; 91: 94-98
• 18 Hung DT, Vu TK, Nelken NA, Coughlin SR. Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor. J Cell Biol 1992; 116: 827-832
• 19 Hung DT, Vu TK, Wheaton VI, Ishii K, Coughlin SR. Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation. J Clin Invest 1992; 89: 1350-1353
• 20 Joseph S, MacDermot J. The N-terminal thrombin receptor fragment SFLLRN, but not catalytically inactive thrombin-derived agonists, activate U937 human monocytic cells: evidence for receptor hydrolysis in thrombin-dependent signalling. Biochem J 1993; 290: 571-577
• 21 Stiernberg J, Redin WR, Warner WS, Carney DH. The role of thrombin and thrombin receptor activating peptide (TRAP-508) in initiation of tissue repair. Thromb Haemost 1993; 70: 158-162
• 22 Jenkins AL, Howells GL, Scott E, Lebonniec BF, Curtis MA, Stone SR. The response to thrombin of human neutrophiles: Evidence for two novel receptors. J Cell Sci 1995; 108: 3059-3066
• 23 Kaiser B, Hauptmann J. Pharmacology of synthetic thrombin inhibitors of the tripeptide type. Cardiovasc Drug Rev 1992; 10: 71-87
• 24 Tapparelli C, Mettemich R, Ehrhardt C, Cook NS. Synthetic low-molecular weight thrombin inhibitors: molecular design and pharmacological profile. TIPS 1993; 14: 366-376
• 25 Deutsch E, Rao AK, Colman RW. Selective thrombin inhibitors: the next generation of anticoagulants. J Am Coll Cardiol 1993; 22: 1089-1092
• 26 Lefkovits J, Topol EJ. Direct thrombin inhibitors in cardiovascular medicine. Circulation 1994; 90: 1522-1536
• 27 Chiu AT, Mousa SA, Pease LJ, Roscoe WA, Bozarth JM, Reilly TM, Smith RD, Timmermans PBMWM. Inhibition of the thrombin-platelet reactions by Dup754. Biochem Biophys Res Commun 1991; 179: 1500-1508
• 28 Bacher P, Walenga JM, Iqbal O, Bajusz S, Breddin K, Fareed F. The antithrombotic and anticoagulant effects of a synthetic tripeptide and recombinant hirudin in various animal models. Thromb Res 1993; 71: 251-263
• 29 Kirchhofer D, Tschopp TB, Hadvary P, Baumgartner HR. Endothelial cells stimulated with tumor necrosis factor-a express varying amounts of tissue factor resulting in inhomogeneous fibrin deposition in a native blood flow system, effects of thrombin inhibitors. J Clin Invest 1994; 93: 2073-2083
• 30 Herbert JM, Lamarche I, Dol F. Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. FEBS Lett 1992; 301: 155-158
• 31 Hedin U, Frebelius S, Snachez J, Dryjski M, Swedenborg J. Antithrombin III inhibits thrombin-induced proliferation in human arterial smooth muscle cells Arterioscler. Thromb 1994; 14: 254-260
• 32 Lombardi A, Nastri F, Galdiero S, Della Morte R, Stiano N, Pedone C, Pavone V. Rational design of enzymatically resistant, peptide based, multi-site directed, a-thrombin inhibitors. American Peptide Symposium, Columbus (Ohio, USA), June 18-23 1995 abstract n° P930
• 33 Chamley-Champbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev 1979; 59: 1-61
• 34 Jaffe EA. Culture of human endothelial cells. Transplant Proc 1980; 12: 49-53
• 35 Owens GK, Loeb A, Gordon D, Thompson M. Expression of smooth muscle cell-specific a isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation. J Cell Biol 1986; 102: 343-352
• 36 Crutchley DJ, Conanan LB, Toledo AW, Solomon DE, Que BG. Effects of prostacyclin analogues on human endothelial cell tissue factor expression. Arterioscler Thrombosis 1993; 13: 1082-1089
• 37 Mulder AB, Blom NR, Smit JW, Ruiters MH, van der Meer J, Halie MR, Bom VJJ. Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymo-trypsin instead of collagenase. Thromb Res 1995; 80: 399-411
• 38 Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues. Implication for disorders of hemostasis and thrombosis. Am J Pathol 1989; 134: 1087-1097
• 39 Brinkman HJM, Mertens K, Holthuis J, Zwart-Huinink LA, Grijm K, van Mourik JA. The activation of human blood coagulation factor X on the surface of endothelial cells: a comparison with various vascular cells, platelets and monocytes. Brit J haemat 1994; 87: 332-342
• 40 Kanthou C, Dennehy U, Benzakour O, Kakkar V. Conversion of prothrombin to an enzymatically and mitogenically active form at the surface of human vascular smooth muscle cells. Thromb Haemost 1995; 73: 911
• 41 Sekiya F, Usui H, Inoue K, Fukudome K, Morita T. Activation of prothrombin by a novel membrane-associated protease. J Biol Chem 1994; 269: 32441-32445
• 42 Nystedt S, Emilsson K, Wahlestedt C, Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci 1994; 91: 9208-9212
• 43 Nystedt S, Emilsson K, Larsson AK, Strombeck B, Sundelin J. Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem 1995; 232: 84-89
• 44 Santulli RJ, Derian CK, Darrow AL, Tomko KA, Eckardt AJ, Seiberg M, Scarborough RM, Andrade Gordon P. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc Natl Acad Sci 1995; 92: 9151-9155
• 45 Vouret-Craviari V, Van Obberghen-Schilling E, Rasmussen UB, Pavirani A, Lecocq JP, Poyssegur J. Synthetic a-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. Mol Biol of the Cell 1992; 3: 95-102
• 46 Vouret-Craviari V, Van Obberghen-Schilling E, Scimeca JC, Van Obberghen E, Pouyssegur J. Differential activation of p44maPk (ERK1) by a-thrombin and thrombin receptor peptide agonist. Biochem J 1993; 289: 14
• 47 Lau LF, Pumiglia K, Cote YP, Feinstein MB. Thrombin receptor agonist peptides, in contrast to thrombin itself are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators. Biochem J 1994; 303: 391-401
• 48 Connely TM, Condra C, Feng DM, Cook JJ, Stranieri MT, Reilly CF, Nutt RF, Gould RJ. Species variability in platelet and other cellular responsiveness to thrombin receptor-derived peptides. Thromb Haemost 1994; 72: 627-633
• 49 Kanthou C, Benzakour O, Patel G, Deasman J, Kakkar VV, Lupu F. Thrombin receptor activating peptide (TRAP) stimulates mitogenesis, c-fos and PDGF-A gene expression in human vascular smooth muscle cells. Thromb Haemost 1995; 74: 1340-1347
• 50 Kanthou C, Parry G, Eijelath E, Kakkar VV, Demoliou-Mason C. Thrombin-induced proliferation and expression of platelet-derived growth factor-A chain gene in human vascular smooth muscle cells. FEBS Lett 1992; 314: 143-148
• 51 Witting JI, Bourdon P, Brezniak DV, Maraganore JM, Fenton JW. Thrombin-specific inhibition by and slow cleavage of hirulog-1. Biochem J 1992; 283: 737-743
• 52 Newby AC, George SJ. Proposed roles for growth factors in mediating smooth muscle proliferation in vascular pathologies. Cardiovasc Res 1993; 27: 1173-1183
• 53 Benzakour O, Formstone C, Rahman S, Kanthou C, Dennehy U, Scully MF, Cooper DN, Kakkar VV. Evidence for protein S receptor(s) on human vascular smooth muscle cells. Analysis of the binding characteristics and mitogenic properties of protein S on human vascular smooth muscle cells. Thromb Haemost 1995; 73: 939
• 54 Harker LA, Hanson SR, Runge MS. Thrombin hypothesis of thrombus generation and vascular lesion formation. Am J Physiol 1995; 75: 12B-15B
• 55 Schwartz SM, Reidy MA, O’Brien ERM. Assessment of factors important in atherosclerotic occlusion and restenosis. Thromb Haemost 1995; 74: 541-551
• 56 Osterud B, Bajaj MS, Bajaj SP. Sites of tissue factor pathway inhibitor (TFPI) and tissue factor expression under physiologic and pathologic conditions. Thromb Haemost 1995; 73: 873-875
• 57 Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel and in the atherosclerotic plaque. Proc Natl Acad Sci 1989; 86: 2839-2843
• 58 Drake TA, Morrissey JH, Edgington TS. Immunohistochemical detection of tissue factor in human atherosclerotic plaques. Circ 1989; 80: 11-182
• 59 Weiss HJ, Turitto VT, Baumgartner HR, Nemerson Y, Hoffman T. Evidence for the presence of tissue factor activity on the subendothelium. Blood 1989; 73: 968-975
• 60 Speidel CM, Edgington TS, Eisenberg PR, Ab endshein DR. Tissue factor induces prolonged procoagulant activity on the luminal surface of rabbit aortas after balloon-induced injury. Circ 1994; 90: 1-344
• 61 Nelken NA, Soifer SJ, O’Keefe J, Vu TKH, Charo IF, Coughlin SR. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest 1992; 90: 1614-1621
• 62 Fischer EG, Wolfram R, Mueller BM. Tissue factor-initiated thrombin generation activates the signaling thrombin receptor on malignant melanoma cells. Cancer Res 1995; 55: 1629-1632