Phosphorylation of the thromboxane receptor alpha, the predominant isoform expressed in human platelets.

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, alpha 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 TPalpha, but not TPbeta, 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 ( approximately 30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TPalpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TPalpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF2alpha-III induced platelet shape change but not TPalpha 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 TPalpha was blocked by protein kinase C inhibitors. TPalpha 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, iPF2alpha-III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TPalpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TPalpha.