Inhibition of Thrombin-induced Neuronal Cell Death by Recombinant Thrombomodulin and E5510, a Synthetic Thrombin Receptor Signaling Inhibitor

Summary

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a pivotal role in thrombosis and haemostasis. Thrombin acts as a mitogen and apoptosis inducer in a dose-dependent fashion. We have previously shown that thrombin caused proliferation of vascular smooth muscle cells (VSMCs). Here, we show that a low concentration of thrombin caused proliferation of mouse neuroblastoma (Neuro-2a) and human neuroblastoma (NB-1) cells, while higher concentrations affected cell viability in a time-dependent manner. Similar effects were observed when thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) was applied. The dying cells showed nuclear condensation and fragmentation, suggesting that cell death occurred by apoptosis. The extent to which thrombin induced cell death was significantly attenuated by recombinant thrombomodulin (rTM), or by a minimum functional domain of TM, termed E456. Furthermore, a synthetic compound that inhibits signaling from the thrombin receptor, 4-cyano-5,5-bis (4-methoxyphenyl)-4-pentanoic acid (E5510), and the antioxidant N-acetyl L-cysteine (NAC), efficiently prevented thrombin-induced Neuro-2a cell death. Thus, thrombin inhibitors and antioxidant appear to neutralize thrombin toxicity.

• References

• 1 Berg DT, Wiley MR, Grinnell BW. Enhanced protein C activation and inhibition of fibrinogen cleavage by a thrombin modulator. Science 1996; 273: 1389-91.
• 2 Fenton II JW. Thrombin. Ann NY Acad Sci 1986; 485: 5-15.
• 3 Bouton MC, Jandrot PM, Moog S, Cazenave JP, Guillin MC, Lanza F. Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system. Biochem J 1995; 305: 635-41.
• 4 Nakajima T, Kitajima I, Shin H, Takasaki I, Shigeta K, Abeyama K, Yamashita Y, Tokioka T, Soejima Y, Maruyama I. Involvement of NF-kB activation in thrombin-induced human vascular smooth muscle cell proliferation. Biochem Biophy Res Commun 1994; 204: 950-5.
• 5 Shin H, Nakajima T, Kitajima I, Shigeta K, Abeyama K, Imamura T, Okano T, Kawahara K, Nakamura T, Maruyama I. Thrombin receptor-mediated synovial proliferation in patients with rheumatoid arthritis. Clinical Immunol Immunopathol 1995; 76: 225-33.
• 6 Gurwitz D, Cunningham DD. Thrombin modulates and reverses neuroblastoma neurite outgrowth. Proc Natl Acad Sci USA 1988; 85: 3440-4.
• 7 Vu TK, 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.
• 8 Low DA, Scott RW, Baker JB, Cunningham DD. Cells regulate their mitogenic response to thrombin through release of protease nexin. Nature 1982; 298: 476-8.
• 9 Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tam T, Coughlin SR. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 1997; 386: 502-6.
• 10 Akiyama H, Ikeda K, Kondo H, McGeer PL. Thrombin accumulation in brains of patients with Alzheimer’s disease. Neurosci Lett 1992; 146: 152-4.
• 11 Hoffmann MC, Nitsch C, Scotti AL, Reinhard E, Monard D. The prolonged presence of glia-derived nexin an endogenous protease inhibitor in the hippocampus after ischemia-induced delayed neuronal death. Neuroscience 1992; 49: 397-408.
• 12 Choi BH, Suzuki M, Kim T, Wanger SL, Cunningham DD. Protease nexin-1 localization in the human brain suggest a protective role against extravasated serine proteases. Am J Pathol 1990; 137: 741-7.
• 13 Kappelmayer J, Berecki D, Misz M, Olah L, Fekete I, Csiba L, Blasko G. Monocytes express tissue factor in young patients with cerebral ischemia. Cerebrovasc Dis 1998; 8: 235-9.
• 14 Hirashima Y, Nakamura S, Suzuki M, Kurimoto M, Endo S, Ogawa A, Takaku A. Cerebrospinal fluid tissue factor and thrombin-antithrombin III complex as indicators of tissue injury after subarchanoid hemorrhage. Stroke 1997; 28: 1666-70.
• 15 McKinney M, Snider RM, Richelson E. Thrombin binding to human brain and spinal cord. Mayo Clin Proc 1983; 58: 829-31.
• 16 Snider RM, Richelson E. Thrombin stimulation of guanosine 3’,5’-monophosphate formation in murine neuroblastoma cells (clone N1E-115). Science 1983; 221: 566-8.
• 17 Nishino A, Suzuki M, Ohtani H, Motohashi O, Umezawa K, Nagura H, Yoshimoto T. Thrombin may contribute to the pathophysiology of central nervous system injury. J Neurotrauma 1993; 10: 167-79.
• 18 Vaughan PJ, Pike CJ, Cotman CW, Cunningham DD. Thrombin receptor activation protects neurons and astrocytes from cell death produced by environmental insults. J Neurosci 1995; 15: 5389-401.
• 19 Donovan FM, Pike CJ, Cotman CW, Cunningham DD. Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. J Neurosci 1997; 17: 5316-26.
• 20 Turgeon VL, Lloyd ED, Wang S, Festoff BW, Houenou LJ. Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation. J Neurosci 1998; 18: 6882-91.
• 21 Smirnova IV, Zhang SX, Citron BA, Arnold PM, Festoff BW. Thrombin is an extracellular signal that activates intracellular death protease pathways inducing apoptosis in model motor neurons. J Neurobiol 1998; 36: 64-80.
• 22 Suidan HS, Stone SR, Hemmings BA, Monard D. Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors. Neuron 1992; 8: 363-75.
• 23 Beecher KL, Anderson TT, Fenton II JW, Festoff BW. Thrombin receptor peptides induce shape change in neonatal murine astrocytes in culture. J Neurosci Res 1994; 37: 108-15.
• 24 Weiler-Guettler H, Aird WC, Rayburn H, Husain M, Rosenberg RD. Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos. Development 1996; 122: 2271-81.
• 25 Suzuki K, Kusumoto H, Deyashiki Y, Nishioka J, Maruyama I, Zushi M, Kawahara S, Honda G, Yamamoto S, Horiguchi S. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J 1987; 6: 1891-7.
• 26 Zushi M, Gomi K, Yamamoto S, Maruyama I, Hayashi T, Suzuki K. The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity. J Biol Chem 1989; 264: 10351-3.
• 27 Fujimori T, Harada K, Saeki T, Kogushi M, Yoshimura T, Katayama K. A new antiplatelet drug, E5510, has multiple suppressive sites during receptor-mediated signal transduction in human platelets. Jpn J Pharmacol 1991; 55: 81-91.
• 28 Kunimoto M. Possible involvement of the 440 kDa isoform of ankykrin B in neuritogenesis in human neuroblastoma NB-1 cells. FEBS Lett 1995; 357: 217-20.
• 29 Mangeney M, Schmitt JR, Leverrier Y, Thomas J, Marvel J, Brun G, Gillet G. The product of the v-src-inducible gene nr-13 is a potent anti-apoptotic factor. Oncogene 1996; 13: 1441-6.
• 30 Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 1989; 119: 203-10.
• 31 Maruyama Y, Maruyama I, Soejima Y. Thrombin receptor agonist peptide decreases thrombomodulin activity in cultured human umbilical vein endothelial cells. Biochem Biophy Res Commun 1994; 199: 1262-9.
• 32 Troyer DA, Bouton A, Bedolla R, Padilla R. Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells. J Am Soc Nephrol 1996; 7: 415-23.
• 33 Droogmans L, Cludts I, Cleuter Y, Kettmann R, Burny A. Nucleotide sequence of the bovine interleukin-6 gene promoter. DNA-Seq 1992; 3: 115-7.
• 34 Shin H, Kitajima I, Nakajima T, Shao Q, Tokioka T, Takasaki I, Hanyu N, Kubo T, Maruyam I. Thrombin receptor mediated signals induce expressions of interleukin-6 and granulocyte colony stimulating factor via NF-kB activation in synovial fibroblasts. Ann Rheum Dis 1999; 58: 55-60.
• 35 Blackwell TS, Holden EP, Blackwell TR, Delarco JE, Cristman JW. Cytokine-induced neutrophil chemoattractant mediates neutrophillic alveolitis in rats: association with nuclear factor kappa B activation. Am J Respir Cell Mol Biol 1994; 11: 464-72.
• 36 Himes SR, Coles LS, Katsikeros R, Lang RK, Shannon MF. HTLV-1 tax activation of the GM-CSF and G-CSF promoters requires the interaction of NF-kB with other transcription factor families. Oncogene 1993; 8: 3189-97.
• 37 Voraberger G, Schafer R, Stratowa C. Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5’-regulatory region. Induction by cytokines and phorbol ester. J Immunol 1991; 147: 2777-86.
• 38 Sugama Y, Tiruppathi C, Janakidevi K, Anderson TT, Fenton II JW, Malik AB. Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion. J Cell Biol 1992; 119: 935-44.
• 39 Zimmermann T, Bierhaus A, Nagel M, Albrecht S, Saeger HD. NF-kB activation-a central mediator of sepsis. Langenbechs Arch-Chir-Suppl-Kongressbd 1997; 114: 363-7.
• 40 Maruyama I, Majerus PW. The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin. J Biol Chem 1985; 260: 15432-8.
• 41 Tsiang M, Lentz SR, Dittman WA, Wen D, Scarpati EM, Sadler JE. Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues. Biochemistry 1990; 29: 10602-12.
• 42 Nakajima T, Kitajima I, Shin H, Matsumoto W, Soejima Y, Maruyama I. E5510 antagonizes thrombin receptor signals by inhibiting NF-kB activation. Biochem Biophys Res Commun 1994; 203: 1184-7.
• 43 Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng D, Moff S, Farese Jr RV, Tam C, Coughlin SR. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-4.
• 44 Valle A, Kinet JP. N-acetyl cysteine inhibits antigen-mediated Syk, but not Lyn tyrosine kinase activation in mast cells. FEBS Lett 1995; 357: 41-4.
• 45 Rezaul K, Sada K, Yamamura H. Involvement of reactive oxygen intermediates in lectin-induced protein-tyrosine phosphorylation of Syk in THP-1 cells. Biochem Biophys Res Commun 1998; 246: 863-7.
• 46 Chen YC, Lin-Shiau SY, Lin JK. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 1998; 177: 324-33.
• 47 Simizu S, Takada M, Omezawa K, Imoto M. Requirement of caspase-3 (-like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs. J Biol Chem 1998; 273: 26900-7.
• 48 Simizu S, Omezawa K, Takada M, Arber N, Imoto M. Induction of hydrogen peroxide production and Bax expression by caspase-3 (-like) proteases in tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma cells. Exp Cell Res 1998; 238: 197-203.
• 49 Behl C, Skutella T, Lezoualach F, Post A, Widmann M, Newton CJ, Holsboer F. Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol Pharmacol 1997; 51: 535-41.
• 50 Leoncini G, Signorello MG, Piana A, Carrubba M, Armani U. Hyperactivity and increased hydrogen peroxide formation in platelets of NIDDM patients. Thromb Res 1997; 86: 153-60.
• 51 Heye N, Cervos NJ. Microthromboemboli in acute infarcts: analysis of 40 autopsy cases. Stroke 1996; 27: 431-4.
• 52 Walz DA, Anderson GF, Ciaglowski RE, Aiken M, Fenton II JW. Thrombin-elicited contractile responses of aortic smooth muscle. Proc Soc Exp Biol Med 1985; 180: 518-26.