Thromboxane-insensitive dog platelets have impaired activation of phospholipase C due to receptor-linked G protein dysfunction

Human platelet thromboxane A₂/prostaglandin H₂ (TXA₂/PGH₂) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the G(q) class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G proteins. Platelets from most dogs are TXA₂ insensitive (TXA₂-); i.e., they do not aggregate irreversibly or secrete although they bind TXA₂, but they respond normally to thrombin. In contrast, a minority of dogs have TXA₂-sensitive (TXA₂+) platelets that are responsive to TXA₂. To determine the mechanism responsible for TXA₂- platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA₂/PGH₂ receptor agonists, [¹²⁵I]BOP and [³H]U46619, and antagonist, [³H]SQ29,548, revealed comparable high-affinity binding to TXA₂-, TXA₂+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA₂- platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP₃) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA₂- platelets. Antisera to G(q) class α subunits abolished U46619-induced GTPase activity in TXA₂-, TXA₂+, and human platelets. Direct G protein stimulation by GTPγS yielded significantly less PA and IP₃ in TXA₂- platelets. Immunotransfer blotting revealed comparable quantities of G(q) class α- subunits in all three platelet types. Thus, TXA₂- dog platelets have impaired PI-PLC activation in response to TXA₂/PGH₂ receptor agonists secondary to G protein dysfunction, presumably involving a member of the G(q) class.