Thrombomodulin Modulates the Mitogenic Response to Thrombin of Human Umbilical Vein Endothelial Cells

Monique Lafay; Reyes Laguna; Bernard F. Le Bonniec; Dominique Lasne; Martine Aiach; Francine Rendu

doi:10.1055/s-0037-1615076

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 1998; 79(04): 848-852
DOI: 10.1055/s-0037-1615076

Rapid Communication

Schattauer GmbH

Thrombomodulin Modulates the Mitogenic Response to Thrombin of Human Umbilical Vein Endothelial Cells

Monique Lafay

¹From the Unité INSERM 428, Faculté de Pharmacie, Paris, France

,

Reyes Laguna

²From the Laboratorio de Farmacologia, Faculdad de Farmacia, Santiago de Compostela, Spain

,

Bernard F. Le Bonniec

¹From the Unité INSERM 428, Faculté de Pharmacie, Paris, France

,

Dominique Lasne

¹From the Unité INSERM 428, Faculté de Pharmacie, Paris, France

,

Martine Aiach

¹From the Unité INSERM 428, Faculté de Pharmacie, Paris, France

,

Francine Rendu

¹From the Unité INSERM 428, Faculté de Pharmacie, Paris, France

› Institutsangaben

Abstract

Summary

Thrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 μM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

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References
1 Gospodarowicz D, Brown KD, Birdwell CR, Zetter BR. Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth, and thrombin. J Cell Biol 1978; 77: 774-88.
2 Dupuy E, Bikfalvi A, Rendu F, Lévy Toledano S, Tobelem G. Thrombin mitogenic responses and protein phosphorylation are different in cultured human endothelial cells derived from large and microvessels. Exp Cell Res 1989; 185: 363-72.
3 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: 1057-68.
4 Gerszten RE, Chen J, Ishii M, Ishii K, Wang L, Nanevicz T, Turck CW, Vu TKH, Coughlin SR. Specifity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 1994; 368: 648-51.
5 Vouret-Craviari. Van Obberghen-Shilling E, Rasmussen UB, Pavirani A, Lecocq JP, Pouysségur J. Synthetic α-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell 1992; 3: 95-102.
6 Garcia JGN. Molecular mechanisms of thrombin-induced human and bovine endothelial cell activation. J Lab Clin Med 1992; 120: 513-9.
7 Brass LF. Homologous desensitization of HEL cell thrombin receptors. J Biol Chem 1992; 267: 6044-50.
8 Zacharias U, He CJ, Nguyen G, Sraer JD, Rondeau E. Long-term effects of thrombin require sustained activation of the functional thrombin receptor. FEBS Lett. 1993; 334: 225-8.
9 Lasne D, Donato J, Falet H, Rendu F. Different abilities of thrombin receptor activating peptide and thrombin to induce platelet calcium rise and full release reaction. Thromb Haemost 1995; 74: 1323-8.
10 McNamara CA, Sarembock IJ, Gimple LW, Fenton JW, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993; 91: 94-8.
11 Albrightson CR, Zabko-Potapovich B, Dytko G, Bryan WM, Hoyle K, Moore ML, Stadel JM. Analogues of the thrombin receptor tethered-ligand enhance mesangial cell proliferation. Cellular Signalling 1994; 6: 743-50.
12 Van Obberghen-Schilling E, Rasmussen UB, Vouret-Craviari V, Lentes KU, Pavirani A, Pouysségur J. Structure-activity analysis of synthetic α-thrombin-receptor-activating peptides. Biochem J 1993; 292: 667-71.
13 Schaeffer P, Riera E, Dupuy E, Herbert JM. Nonproteolytic activation of the thrombin receptor promotes human umbilical endothelial cell growth but not intracellular Ca²⁺, prostacyclin, or permeability. Biochem Pharmacol 1997; 53: 487-91.
14 Esmon CT. Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB J 1995; 9: 946-55.
15 Grinnell BW, Berg DT. Surface thrombomodulin modulates thrombin receptor responses on vascular smooth muscle cells. Am J Physiol 1996; 270: H603-H9.
16 Jaffé EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest 1973; 52: 2745-56.
17 Le Bonniec BF, MacGillivray RTA, Esmon CT. Thrombin Glu³⁹ restricts the P’₃ specificity to nonacidic residues. J Biol Chem 1991; 266: 13796-803.
18 Le Bonniec BF, Guinto ER, Esmon CT. Interaction of thrombin-des-ETW with antithrombin III, the Kunitz inhibitors and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Tyr insertion of thrombin. J Biol Chem 1992; 267: 19341-8.
19 Stone SR, Le Bonniec BF. Inhibitory mechanism of serpins. Identification of steps involving the active-site serine residue of the protease. J Mol Biol 1997; 265: 344-62.
20 Arnaud E, Lafay M, Gaussem P, Picard V, Jandrot-Perrus M Aiach, Rendu F. An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation. Blood 1994; 84: 1843-50.
21 Coller BS, Ward P, Ceruso M, Scudder LE, Springer K, Kutok J, Prestwich GD. Thrombin receptor activating peptides: importance of the N-terminal serine and its ionization state as judged by pH dependence, nuclear magnetic resonance spectroscopy, and cleavage by aminopeptidase M. Biochemistry 1992; 31: 11713-20.
22 Nystedt S, Emilsson K, Wahlestedt C, Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 1994; 91: 9208-12.
23 Garcia JGN, Patterson C, Bahler C, Aschner J, Hart CM, English D. Thrombin receptor activating peptides induce Ca²⁺ mobilization, barrier dysfunction, prostaglandin synthesis, and platelet-derived growth factor mRNA expression in cultured endothelium. J Cell Physiol 1993; 156: 541-9.
24 Amiral J, Adam A, Mimilla F, Larrivaz I, Chambrette B, Boffa MC. Design and validation of a new immunoassay for soluble forms of thrombomodulin and studies on plasma. Hybridoma 1994; 13: 205-13.
25 DiMuzio PJ, Pratt KJ, Park PK, Carabasi RA. Role of thrombin in endothelial cell monolayer repair in vitro. J Vasc Surg 1994; 20: 621-8.
26 Nystedt S, Ramakrishnan V, Sundelin J. The proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells. Comparison with the thrombin receptor. J Biol Chem 1996; 271: 14910-5.
27 Mirza H, Yatsula V, Bahou WF. The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells. Molecular characterization and evidence for functional coupling to the thrombin receptor. J Clin Invest 1996; 97: 1705-14.
28 Herbert JM, Dupuy E, Laplace MC, Zini JM, Bar-Shavit R, Tobelem G. Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway. Biochem J 1994; 303: 227-31.
29 Molino M, Woolkalis MJ, Reavy-Cantwell J, Pratico D, Andrade-Gordon P, Barnathan ES, Brass LF. Endothelial cell thrombin receptors and PAR-2. Two protease-activated receptors located in a single cellular environment. J Biol Chem 1997; 272: 11133-41.
30 Lerner DJ, Chen M, Tram T, Coughlin SR. Agonist recognition by proteinase-activated receptor 2 and thrombin receptor. Importance of extracellular loop interactions for receptor function. J Biol Chem 1996; 271: 13943-7.
31 Ngaiza JR, Jaffé EA. A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells. Biochem Biophys Res Commun 1991; 179: 1656-61.
32 Maruyama Y, Maruyama I, Soejima Y. Thrombin receptor agonist peptide decreases thrombomodulin activity in cultured human umbilical vein endothelial cells. Biochem Biophys Res Commun 1994; 199: 1262-9.
33 Hollenberg MD, Mokashi S, Leblond L, DiMaio J. Synergistic actions of a thrombin-derived synthetic peptide and a thrombin receptor-activating peptide in stimulating fibroblast mitogenesis. J Cell Physiol 1996; 169: 491-6.
34 Maruyama I, Majerus PW. The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. J Biol Chem 1985; 260: 15432-8.
35 Ishii K, Chen J, Ishii M, Koch WJ, Freedman NJ, Lefkowitz RJ, Coughlin SR. Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. Functional specificity among G-protein coupled receptor kinases. J Biol Chem 1994; 269: 1125-30.
36 Hoxie JA, Menaka A, Belmonte E, Pizarro S, Parton R, Brass LF. Internalization and recycling of activated thrombin receptors. J Biol Chem 1993; 268: 13756-63.
37 Woolkalis MJ, DeMelfi TM, Blanchard N, Hoxie JA, Brass LF. Regulation of thrombin receptors on human umbilical vein endothelial cells. J Biol Chem 1995; 270: 9868-75.
38 Blackhart BD, Emilsson K, Nguyen D, Teng W, Martelli AJ, Nystedt S, Sundelin J, Scarborough RM. Ligand cross-reactivity within the protease-activated receptor family. J Biol Chem 1996; 271: 16466-71.
39 Conway EM, Pollefeyt S, Collen D, Steiner-Mosonyi M. The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. Blood 1997; 89: 652-61.