Effects of U73122 and U73343 on human platelet calcium signalling and protein tyrosine phosphorylation

We have investigated the actions of the PLC inhibitor, U73122, and its close analogue, U73343, which does not inhibit PLC, in Fura-2-loaded human platelets. Rises in [Ca2+](i) evoked by thrombin and collagen, and the TxA(2)-dependent rise in [Ca2+](i) evoked by thapsigargin, were abolished by U73122, indicating that it inhibits the activity of both beta and gamma isoforms of PLC. The supposed control compound, U73343, was found to inhibit TxA(2) formation; it therefore partially inhibited the rise in [Ca2+](i) evoked by low concentrations of thrombin, by thapsigargin or by collagen. U73343 had a greater effect than aspirin on the action of collagen, indicating an action on the TxA(2)-independent component of the signal, via PLC gamma(2). U73343 lowered TxA(2) production by inhibiting the activation of cPLA(2), probably at a tyrosine phosphorylation step. U73343 seems to inhibit only the tyrosine kinases involved in the activation of PL(gamma)and the generation of TxA(2). In contrast, U73122 increased tyrosine phosphorylation of platelet proteins, perhaps by inhibiting receptor-independent tyrosine phosphatases, but inhibited all further tyrosine phosphorylation on addition of thrombin or other agonists.