Human platelet aggregation and shape change are coupled to separate thromboxane A2-prostaglandin H2 receptors

Dorn, Gerald W.; DeJesus, Albert

doi:10.1152/ajpheart.1991.260.2.h327

Cited by 12 publications

(11 citation statements)

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“…Low concentrations of I-BOP (Յ0.3 nM) that induced only shape change in the absence of platelet aggregation (32,34) failed to phosphorylate pleckstrin or induce aggregation (Fig. 2B).…”

Section: Resultsmentioning

confidence: 96%

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

Habib¹,

FitzGerald²,

Maclouf³

1999

Journal of Biological Chemistry

125

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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␣.

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“…Low concentrations of I-BOP (Յ0.3 nM) that induced only shape change in the absence of platelet aggregation (32,34) failed to phosphorylate pleckstrin or induce aggregation (Fig. 2B).…”

Section: Resultsmentioning

confidence: 96%

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

Habib¹,

FitzGerald²,

Maclouf³

1999

Journal of Biological Chemistry

125

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“…Some studies using the radiolabeled TXA 2 analogs as ligands demonstrated two classes of binding sites in platelets, and indicated that they might represent the receptor subtypes (5)(6)(7)(8). They further suggested that the two putative subtypes of the receptor may independently mediate shape change and aggregation (8,9). This suggestion has been supported by the reports that platelet shape change and aggregation can be differentiated by several TXA 2 analogs.…”

Section: Introductionmentioning

confidence: 88%

Two thromboxane A2 receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation.

Hirata¹,

Ushikubi²,

Kakizuka³

et al. 1996

J. Clin. Invest.

280

196

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Thromboxane A 2 (TXA 2 ) receptor is a key molecule in hemostasis as its abnormality leads to bleeding disorders. Two isoforms of the human TXA 2 receptor have been cloned; one from placenta and the other from endothelium, here referred to as TXR ␣ and TXR ␤ , respectively. These isoforms differ only in their carboxyl-terminal tails. We report that both isoforms are present in human platelets. The two isoforms expressed in cultured cells show similar ligand binding characteristics and phospholipase C (PLC) activation but oppositely regulate adenylyl cyclase activity; TXR ␣ activates adenylyl cyclase, while TXR ␤ inhibits it. The Arg 60 to Leu mutant of TXR ␣ , which has been shown to impair PLC activation (Hirata, T., A. Kakizuka, F. Ushikubi, I. Fuse, M. Okuma, and S. Narumiya. 1994. J. Clin. Invest. 94: 1662-1667), also impairs adenylyl cyclase stimulation, whereas that of TXR ␤ retains its activity to inhibit adenylyl cyclase. These findings suggest that the pathway linked to adenylyl cyclase inhibition might be involved in some of the TXA 2 -induced platelet responses such as shape change and phospholipase A 2 activation which remain unaffected in the patients with this mutation. ( J. Clin. Invest. 1996. 97:949-956.)

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“…The K d values correspond to the EC 50 values for I-BOP to induce shape change and aggregation. Thus, the binding of I-BOP to low affinity receptors induces secretion and aggregation, whereas its binding to high affinity receptors induces shape change (8). Similarly, a TP antagonist such as GR 32191 shows noncompetitive inhibition of platelet aggregation but competitive inhibition of platelet shape change, consistent with the existence of two subtypes of receptors (9).…”

mentioning

confidence: 90%

“…Evidence suggests that two different TPs within the platelets or two different affinity states of the same receptor may exist. Analysis of the binding of 125 I-labeled I-BOP by platelet membranes indicates two platelet binding sites of high affinity (K d ϭ 270 Ϯ 60 pM) and low affinity (K d ϭ 3.9 Ϯ 1-2 nM) (8). The K d values correspond to the EC 50 values for I-BOP to induce shape change and aggregation.…”

mentioning

confidence: 99%

Tyrosine Phosphorylation of Cortactin Associated with Syk Accompanies Thromboxane Analogue-induced Platelet Shape Change

Gallet¹,

Rosa

Habib

et al. 1999

Journal of Biological Chemistry

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Thromboxane A 2 (TxA 2 ) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7-3-(3-hydroxy-4-(4-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2.1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F 2 ␣. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal.Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA 2 -induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA 2 high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.

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Human platelet aggregation and shape change are coupled to separate thromboxane A2-prostaglandin H2 receptors

Cited by 12 publications

References 0 publications

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

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

Two thromboxane A2 receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation.

Tyrosine Phosphorylation of Cortactin Associated with Syk Accompanies Thromboxane Analogue-induced Platelet Shape Change

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