A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, cu and beta. Both isoforms are rapidly phosphorylated in response to thromboxane agonists when overexpressed in human embryonic kidney 293 cells; this phenomenon is only slightly altered by inhibitors of protein kinase C. Pharmacological studies have defined two classes of TP in human platelets; sites that bind the agonist I-BOP with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoform-specific antibodies permitted detection of TP alpha, but not TP beta, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50-60 kDa, reflecting the glycosylated receptor, was phosphorylated upon activation of platelets for 2 min with I-BOP, This was a rapid (similar to 30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TP alpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism, Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TP alpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change, Also, activation of these sites with the isoprostame iPF(2 alpha)-III induced platelet shape change but not TP alpha phosphorylation. Heterologous TP phosphorylation was observed in aspirin-treated platelets exposed to thrombin, high concentrations of collagen, and the calcium ionophore A 23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TP alpha was blocked by protein kinase C inhibitors. TP alpha was the only isoform detectably translated in human platelets, This appeared to correspond to the activation of the low affinity site defined by the antagonist GR 32191 and not activated by the high affinity agonist, iPF(2 alpha)-III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TP alpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TP alpha.
