Phosphorylation of the Thromboxane Receptor α, the Predominant Isoform Expressed in Human Platelets

Abstract

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, ␣ and ␤. 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. Isoformspecific antibodies permitted detection of TP␣, but not TP␤, 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 (ϳ30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TP␣ 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␣ phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF 2␣ -III induced platelet shape change but not TP␣ 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␣ was blocked by protein kinase C inhibitors. TP␣ 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␣ -III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TP␣ in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TP␣.

Thromboxane A2 Signalling in Humans: a ‘Tail’ of Two Receptors

“…Additional immunofluorescence studies, employing TP isoform specific antibodies directed to peptide sequences within the unique C-tail domains of TPα and TPβ, also confirmed the expression and cellular localisation of the TP receptors [38,39]. Taken together, these studies investigating the expression and tissue distribution of the TPα and TPβ receptors indicate that they are subject to differential expression and regulation [34].…”

“…Consistent with this hypothesis, based on observations that TP isoform specific antibodies permitted detection of TPα, but not TPβ in human platelets, Habib et al, [39] have proposed that…”

Characterization of the effects of isoprostanes on platelet aggregation in human whole blood

“…Indeed, there is a growing body of evidence that illustrates interactions between 8-iso PGF 2a and TP receptors. In human platelets 8-iso PGF 2a binds to TP receptors, although its interaction at these sites appears complex and has not been fully de®ned Minuz et al, 1998;Habib et al, 1999). Using platelet-rich plasma, Pratico et al (1996) have shown that 8-iso PGF 2a can activate human platelets to induce shape change and facilitate the proaggregatory actions of other full agonists but cannot itself induce full aggregation.…”

“…Initially, this phenomenon could not be attributed to TP receptor activation. However, 8-iso PGF 2a was subsequently (Habib et al, 1999) shown to bind a speci®c site on TPa receptors and to trigger signal transduction distinct from thromboxane (Takahara et al, 1990). Using whole blood, we were not able to reliably determine e ects on shape change.…”

“…8-iso PGF 2a also has actions on isolated platelets where, in contrast to smooth muscle preparations, it may not apparently fully activate platelet TP receptors to cause aggregation Habib et al, 1999) and instead inhibits the proaggregatory e ects of the TP ligand U46619 (Morrow et al, 1992c;Yin et al, 1994). In addition, although the E 2 isoprostane, 8-iso PGE 2 , causes aggregation of platelets in a sub-population of human volunteers, it also inhibits U46619-induced aggregation (Longmire et al, 1994).…”