Abstract
Habutobin, thrombin-like enzyme that converts rabbit fibrinogen to fibrin, was purified from T. flavoviridis . Habutobin bound to β3 subunit of integrin αIIbβ3, and then habutobin inhibited the collagen-induced aggregation, in previously study. We investigated the effect of habutobin for intracellular signaling through tyrosine(Tyr)-phosphorylation of protein occurring in the collagen-induced aggregation. Rabbit platelet was washed with ACD-A and HEPES-Tyrode's sol (Ca2+, Mg2+ free) and the washed platelet was suspended in the Tyrode's sol containing Mg2+ (1 mM). From results of Western blot with anti-phosphotyrosine antibody(4G10), Tyr-phosphorylation of several intracellular proteins increased dramatically during collagen-induced aggregation. Tyr-phosphorylated proteins were observed in 55-60, 72, 85 and 116 kD at 90 sec after the addition of collagen. In the presence of habutobin, although Tyr-phosphorylated proteins was not observed at 90 sec after the addition of collagen, they were observed in 60, 72 and 116 kD at 3 min after the addition of collagen. In addition, in the presence of habutobin, FAK Tyr-phosphorylation was inhibited at an early stage of collagen-induced aggregation, but it was not inhibited at a late stage of collagen-induced aggregation. From the present study, it was suggested that the binding of habutobin to β3 subunit of integrin αIIbβ3 resulted to inhibit FAK Tyr-phosphorylation, and habutobin inhibits the β3 signaling, that is, outside-in signaling. [J Physiol Sci. 2006;56 Suppl:S139]
