Human Platelet Thromboxane A2 Receptor Research Articles

Corrigendum

CORRIGENDACorrigendumPublished Online:01 Dec 2005https://doi.org/10.1152/ajpheart.01029.2005Original articleMoreSectionsPDF (27 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 289, August 2005 Volume 58, August 2005 Pages H578–H585: R. J. Gonzales, A. A. Ghaffari, S. P. Duckles, and D. N. Krause. “Testosterone treatment increases thromboxane function in rat cerebral arteries.” Reference 1 in this paper was incorrectly cited. It should appear as the following:Ajayi AA, Mathur R, and Halushka PV. Testosterone increases human platelet thromboxane A2 receptor density and aggregation responses. Circulation 91: 2742–2747, 1995.This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesTestosterone treatment increases thromboxane function in rat cerebral arteries 01 Aug 2005American Journal of Physiology-Heart and Circulatory Physiology More from this issue > Volume 289Issue 6December 2005Pages H2754-H2754 Copyright & PermissionsCopyright © 2005 by the American Physiological Societyhttps://doi.org/10.1152/ajpheart.01029.2005History Published online 1 December 2005 Published in print 1 December 2005 Metrics

Mutagenic Analysis of Platelet Thromboxane Receptor Cysteines: ROLES IN LIGAND BINDING AND RECEPTOR-EFFECTOR COUPLING

The human platelet thromboxane A2 receptor is a member of the G-protein-coupled superfamily of receptors. Previous pharmacologic studies examining the effects of biochemical reduction, oxidation, or sulfhydryl alkylation on thromboxane receptors have suggested a role for cysteines in determining receptor binding characteristics. To characterize the roles of individual cysteines, we employed site-directed mutagenesis to substitute serines for cysteines at seven positions throughout the human K562 thromboxane receptor and analyzed mutant receptor radioligand ([1S-(1alpha,2beta(5Z),3alpha- (1E,3S),4alpha]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-l-butenyl)-7-oxabicyclo-[2. 2.1]heptane-2-yl]-5-heptenoic acid) binding and calcium signaling. Replacing cysteines in the amino terminus (amino acid position 11), and transmembrane domains two and six (positions 68 and 257) had little effect on thromboxane receptor binding or signaling. Introduction of serines for cysteines in the first (position 105) or the second (position 183) extracellular loop eliminated thromboxane receptor binding, consistent with the existence of a critical disulfide bond between these positions. Mutation of a second cysteine in extracellular loop one (position 102) resulted in a receptor with decreased binding affinity and low binding capacity that transduced only a low amplitude calcium signal, suggesting the involvement of a free sulfhydryl group at this location in receptor-ligand interactions. Finally, mutation of the cysteine at position 223, located in intracellular loop three, resulted in a receptor with normal ligand binding characteristics, but which did not transduce a calcium signal. Some additional amino acid substitutions in this region of the receptor (Cys-223 --> Ala, Thr-221 --> Met) resulted in receptors that had normal binding but transduced low amplitude calcium signals, while other mutations in the same region (His-224 --> Arg and His-227 --> Arg) exhibited normal binding and calcium signaling characteristics. These findings demonstrate that cysteines in extracellular loops one and two contribute to proper ligand binding to thromboxane receptors and show the importance of discrete amino acid sequences in the third intracellular loop, especially cysteine 223, in thromboxane receptor-effector coupling.

Cloning and expression of cDNA for a human thromboxane A2 receptor

Thromboxane A2 is a very unstable arachidonate metabolite, yet a potent stimulator of platelet aggregation and a constrictor of vascular and respiratory smooth muscles. It has been implicated as a mediator in diseases such as myocardial infarction, stroke and bronchial asthma. Using a stable analogue of this compound we recently purified the human platelet thromboxane A2 receptor to apparent homogeneity. Using an oligonucleotide probe corresponding to its partial amino-acid sequence, we have obtained a complementary DNA clone encoding this receptor from human placenta and a partial clone from cultured human megakaryocytic leukaemia cells. The placenta cDNA encodes a protein of 343 amino acids with seven putative transmembrane domains. The protein expressed in COS-7 cells binds drugs with affinities identical to those of the platelet receptor, and that in Xenopus oocytes opens Ca2(+)-activated Cl- channel on agonist stimulation. Northern blot analysis and nucleotide sequences of the two clones suggest that an identical species of the thromboxane A2 receptor is present in platelets and vascular tissues. This first report on the molecular structure of an eicosanoid receptor will promote the molecular pharmacology and pathophysiology of these bioactive compounds.

Platelet thromboxane A2 receptors in type I diabetes

1. Human platelet thromboxane A2 receptor expression on the membrane surface is possibly dynamically regulated by changes either in the ligand concentration or in membrane fluidity. An increased thromboxane A2 production and a decreased membrane fluidity has been reported in diabetic patients. 2. In the present study the binding characteristics of platelet thromboxane A2 receptors have been investigated in nine diabetic patients (type I) and in 15 healthy control subjects by a radioligand-binding method using 9,11-dimethylmethane-11,12-methane-16-[3-125I-4-hydroxyphenyl]-13, 14- dihydro-13-aza-15-tetranor-thromboxane A2 as the radiolabelled ligand. 3. The maximum concentration of binding sites was 163 (SD 35) fmol/10(8) platelets (n = 15) with 987 (SD 209) receptors/platelet in controls, whereas in diabetic patients the maximum concentration of binding sites was 74.2 (SD 28) fmol/10(8) (n = 9) with 447 (SD 172) receptors/platelet (P less than 0.001). The dissociation constants were 18 (SD 4) nmol/l and 21 (SD 6) nmol/l (not significant) in control subjects and in diabetic patients, respectively. Glycated haemoglobin, which is reported to reduce membrane fluidity, was found to be negatively correlated (r = 0.60, P less than 0.05) with thromboxane A2 receptor number in the diabetic patient group. On the contrary, a positive linear correlation between the equilibrium dissociation constant and glycated haemoglobin was found in diabetic patients (r = 0.75, P less than 0.01).

Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium.

The inter-relationships between receptor occupancy, inositol phospholipid metabolism and elevation of cytosolic free Ca2+ in thromboxane A2-induced human platelet activation were investigated by using the stable thromboxane A2 mimetic, 9,11-epoxymethanoprostaglandin H2, and the thromboxane A2 receptor antagonist, EPO45. 9,11-Epoxymethanoprostaglandin H2 stimulated platelet phosphatidylinositol metabolism as indicated by the rapid accumulation of [32P]phosphatidate and later accumulation of [32P]phosphatidylinositol in platelets pre-labelled with [32P]Pi. These effects of 9,11-epoxymethanoprostaglandin H2 were concentration-dependent and half-maximal [32P]phosphatidate formation occurred at an agonist concentration of 54 +/- 8 nM. With platelets labelled with the fluorescent Ca2+ indicator quin 2, resting cytosolic free Ca2+ was 86 +/- 12 nM. 9,11-Epoxymethanoprostaglandin H2 induced a rapid, concentration-dependent elevation of cytosolic free Ca2+ to a maximum of 300-700 nM. Half-maximal stimulation was observed at an agonist concentration of 80 +/- 23 nM. The thromboxane A2 receptor antagonist EPO45 selectively inhibited 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and elevation of cytosolic free Ca2+, indicating that both events are sequelae of receptor occupancy. Human platelets contain a single class of stereospecific, saturable, high affinity (KD = 70 +/- 13 nM) binding sites for 9,11-epoxymethano[3H]prostaglandin H2. The concentration-response curve for receptor occupancy (9,11-epoxymethano-[3H]prostaglandin H2 binding) is similar to that for 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and for elevation of cytosolic free Ca2+. These observations indicate that human platelet thromboxane A2 receptor occupation is closely linked to inositol phospholipid metabolism and to elevation of cytosolic free Ca2+. Both such events may be necessary for thromboxane A2-induced human platelet activation.