Activation of Phospholipase C as a Primary Target of the Thromboxane A(2)-mediated Amplification Mechanism in Thrombin-induced Rabbit Platelet Activation.
The inhibitory effects of a cyclooxygenase inhibitor, indomethacin, and a thromboxane (TX) A(2) receptor antagonist, S-145, on thrombin-stimulated rabbit platelet responses were examined for their contribution to the TXA-mediated amplification mechanism. Although thrombin (0.01-0.3 U/ml) induced the dosedependent aggregation and release of TXB(2) from washed rabbit platelets, indomethacin (30 μM) inhibited only the aggregation induced by a threshold dose of thrombin, even though it completely inhibited the formation of TXB(2). Indomethacin inhibited both the secretion of ATP and the elevation of the intracellular concentration of Ca(2+) ([Ca(2+)](i)) induced by all doses of thrombin used and induced a considerable rightward shift of the dose-response curves. S-145 also significantly inhibited the elevation of [Ca(2+)](i). Thrombin caused rapid accumulation of [(3)H]-InsP(3) or of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]. This accumulation was also inhibited by indomethacin to about 70% of the control level. STA(2), a stable analogue of TXA(2), and arachidonic acid caused accumulation of InsP, and that induced by the latter was completely inhibited by indomethacin (1 μM). Thrombin-induced aggregation peaked at a lower level of [Ca(2+)](i) than that required for the secretion of ATP. The apparent contribution of TXA(2) to aggregation therefore appears to be restricted to that induced by lower doses of thrombin. These results suggest that in thrombin-stimulated rabbit platelets, activation of phospholipase C, which is regulated by TXA(2) receptors, is a primary target of the TXA(2)-mediated amplification mechanism. Through its effect on the accumulation of Ins(1,4,5)P(3), this amplification mechanism may contribute to about 25-30% of the elevation of (Ca(2+)](i), in addition to the thrombin receptor-mediated mechanism.
Department of Pharmacology, Kitasato University School of Medicine, Kitasato 1-51 1, Sagamihara, Kanagawa, 228, Japan.
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Kawamura
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Huang
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Harada
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Katori