Thromboxane A(2)-mediated shape change: Independent of Gq-phospholipase C-Ca2+ pathway in rabbit platelets

1 Thromboxane A(2) (TXA(2)) receptor-mediated signal transduction was investigated in washed rabbit platelets to clarify the mechanisms of induction of shape change and aggregation. 2 The TXA(2) agonist, U46619 (1 nM to 10 mu M) caused shape change and aggregation in a concentration-dependent manner. A forty-times higher concentration of U46619 was needed for aggregation (EC(50) of 0.58 mu M) than shape change (EC(50) of 0.013 mu M). The aggregation occurred only when external 1 mM Ca2+ was present, but the shape change could occur in the absence of Ca2+. 3 SQ29548 at 30 nM and GR32191B at 0.3 mu M (TXA(2) receptor antagonists) competitively inhibited U46619-induced shape change and aggregation with similar potency, showing that both aggregation and shape change induced by U46619 were TXA(2) receptor-mediated events. However, ONO NT-126 at 1 nM, another TXA(2) receptor antagonist, inhibited U46619-induced aggregation much more potently than the shape change, suggesting the possible existence of TXA(2) receptor subtypes. 4 ONO NT-126 (2 nM to 3 mu M) by itself caused a shape change without aggregation in a concentration-dependent manner, independent of external Ca2+. Therefore, ONO NT-126 is a partial agonist at the TXA(2) receptor in rabbit platelets. 5 U46619 (10 nM to 10 mu M) increased internal Ca2+ concentration ([Ca2+](i)) and activated phosphoinositide (PI) hydrolysis in a concentration-dependent manner with a similar concentration-dependency. 6 U46619 (3 nM to 10 mu M) also activated GTPase concentration-dependently in the membranes derived from platelets. U46619-induced activation of GTPase was partly inhibited by treatment of membranes with QL, an antibody against G(q/11). 7 The EC(50) values of U46619 in Ca2+ mobilization (0.15 mu M), PI hydrolysis (0.20 mu M) and increase in GTPase activity (0.12 mu M) were similar, but different from the EC(50) value in shape change (0.013 mu M), suggesting that activation of TXA(2) receptors might cause shape change via an unknown mechanism. 8 U46619-induced shape change was unaffected by W-7 (30 mu M), a calmodulin antagonist or ML-7 (30 mu M), a myosin light-chain kinase inhibitor, indicating that an increase in [Ca2+](i) might not be involved in the shape change. In fact, U46619 (10 nM) could cause shape change without affecting [Ca2+](i) level, determined by simultaneous recordings. 9 [H-3]-SQ29548 and [H-3]-U46619 bound to platelets at a single site with a K-d value of 14.88 nM and B-max of 106.1 fmol/10(8) platelets and a K-d value of 129.8 nM and B-max of 170.4 fmol/10(8) platelets, respectively. The inhibitory constant K-i value for U46619 as an inhibitor of H-3-ligand binding was similar to the EC(50) value of U46619 in GTPase activity, phosphoinositide hydrolysis and Ca2+ mobilization, but significantly different (P < 0.001 by Student's t test) from the effect on shape change. 10 Neither U46619 nor ONO NT-126 affected the adenosine 3',5'-cyclic monophosphate (cyclic AMP) level in the presence or absence of external Ca2+ and/or isobutyl methylxanthine. 11 The results indicate that TXA(2) receptor stimulation causes phospholipase C activation and increase in [Ca2+](i) via a G protein of the G(q/11) family leading to aggregation in the presence of external Ca2+, and that shape change induced by TXA(2) receptor stimulation might occur without involvement of the G(q)-phospholipase, C-Ca2+ pathway.