Inhibition of thrombin-induced neuronal cell death by recombinant thrombomodulin and E5510, a synthetic thrombin receptor signaling inhibitor

Thrombin, a serine protease generated by the activation of the blood coagul ation cascade following vessel injury, converts fibrinogen to fibrin, activ ates platelets and several coagulation factors, and plays a pivotal role in thrombosis and haemostasis. Thrombin acts as a mitogen and apoptosis induc er in a dose-dependent fashion. We have previously shown that thrombin caus ed proliferation of vascular smooth muscle cells (VSMCs). Here, we show tha t a low concentration of thrombin caused proliferation of mouse neuroblasto ma (Neuro-2a) and human neuroblastoma (NB-1) cells, while higher concentrat ions affected cell viability in a time-dependent manner. Similar effects we re observed when thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) w as applied. The dying cells showed nuclear condensation and fragmentation, suggesting that cell death occurred by apoptosis. The extent to which throm bin induced cell death was significantly attenuated by recombinant thrombom odulin (rTM), or by a minimum functional domain of TM, termed E456. Further more. a synthetic compound that inhibits signaling from the thrombin recept or, 4-cyano-5,5-bis (4-methoxyphenyl)-4-pentanoic acid (E5510), and the ant ioxidant N-acetyl L-cysteine (NAC), efficiently prevented thrombin-induced Neuro-2a cell death. Thus, thrombin inhibitors and antioxidant appear to ne utralize thrombin toxicity.