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Title: Identification and characterization of a putative human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ receptor

Abstract

The thromboxane A/sub 2/ (TXA/sub 2/) analog, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15..cap alpha beta..-omega-tetranor TXA/sub 2/ (I-PTA-OH) was characterized as a competitive antagonist of TXA/sub 2/ mimetic-induced platelet aggregation, with a K/sub d/ of 190 nM in platelet rich plasma. This antagonism was specific for the putative thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor, since I-PTA-OH had no inhibitory effects on platelet aggregation stimulated by agonists which act independently of TXA/sub 2//PGH/sub 2/, and did not inhibit platelet TXA/sub 2/ synthesis. (/sup 125/I)-PTA-OH binding to a particulate fraction from human platelets was saturable, displaceable, and linear with protein concentration. Scatchard analysis of equilibrium binding revealed a single class of high affinity binding sites, with a K/sub d/ of 30 +/- 4 nM and a B/sub max/ of 1.8 +/- 0.3 pmol/mg protein. Kinetic analysis yielded a k/sub 1/ of 1.35 x 10/sup 6/ M/sup -1/ x min/sup -1/ and a k..sqrt../sub 1/ of 0.032 min/sup -1/, K/sub d/ = k..sqrt../sub 1//k/sub 1/ = 24 nM. The subcellular localization of the putative TXA/sub 2//PGH/sub 2/ receptor was determined using (/sup 125/I)-PTA-OH binding as a marker for the receptor. (/sup 125/I)-PTA-OH binding as a marker for the receptor. (/sup 125/I)-PTA-OH binding, was coenriched withmore » markers for plasma membranes and dense tubular system; but not with markers for cytoplasmic constituents, mitochondria, or granules.« less

Authors:
Publication Date:
Research Org.:
Medical Univ. of South Carolina, Charleston (USA)
OSTI Identifier:
6550635
Resource Type:
Thesis/Dissertation
Resource Relation:
Other Information: Thesis (Ph. D)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BLOOD PLATELETS; BLOOD COAGULATION; PROSTAGLANDINS; BIOCHEMICAL REACTION KINETICS; RECEPTORS; CHEMICAL COMPOSITION; BIOLOGICAL MARKERS; CELL MEMBRANES; IODINE 125; MAN; TRACER TECHNIQUES; ANIMALS; BETA DECAY RADIOISOTOPES; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CELL CONSTITUENTS; DAYS LIVING RADIOISOTOPES; ELECTRON CAPTURE RADIOISOTOPES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; MAMMALS; MATERIALS; MEMBRANE PROTEINS; MEMBRANES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PRIMATES; PROTEINS; RADIOISOTOPES; REACTION KINETICS; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Saussy, D.L. Jr.. Identification and characterization of a putative human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ receptor. United States: N. p., 1986. Web.
Saussy, D.L. Jr.. Identification and characterization of a putative human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ receptor. United States.
Saussy, D.L. Jr.. Wed . "Identification and characterization of a putative human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ receptor". United States. doi:.
@article{osti_6550635,
title = {Identification and characterization of a putative human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ receptor},
author = {Saussy, D.L. Jr.},
abstractNote = {The thromboxane A/sub 2/ (TXA/sub 2/) analog, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15..cap alpha beta..-omega-tetranor TXA/sub 2/ (I-PTA-OH) was characterized as a competitive antagonist of TXA/sub 2/ mimetic-induced platelet aggregation, with a K/sub d/ of 190 nM in platelet rich plasma. This antagonism was specific for the putative thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor, since I-PTA-OH had no inhibitory effects on platelet aggregation stimulated by agonists which act independently of TXA/sub 2//PGH/sub 2/, and did not inhibit platelet TXA/sub 2/ synthesis. (/sup 125/I)-PTA-OH binding to a particulate fraction from human platelets was saturable, displaceable, and linear with protein concentration. Scatchard analysis of equilibrium binding revealed a single class of high affinity binding sites, with a K/sub d/ of 30 +/- 4 nM and a B/sub max/ of 1.8 +/- 0.3 pmol/mg protein. Kinetic analysis yielded a k/sub 1/ of 1.35 x 10/sup 6/ M/sup -1/ x min/sup -1/ and a k..sqrt../sub 1/ of 0.032 min/sup -1/, K/sub d/ = k..sqrt../sub 1//k/sub 1/ = 24 nM. The subcellular localization of the putative TXA/sub 2//PGH/sub 2/ receptor was determined using (/sup 125/I)-PTA-OH binding as a marker for the receptor. (/sup 125/I)-PTA-OH binding as a marker for the receptor. (/sup 125/I)-PTA-OH binding, was coenriched with markers for plasma membranes and dense tubular system; but not with markers for cytoplasmic constituents, mitochondria, or granules.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed Jan 01 00:00:00 EST 1986},
month = {Wed Jan 01 00:00:00 EST 1986}
}

Thesis/Dissertation:
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  • This work investigated the interactions of several compounds with the TXA/sub 2//PGH/sub 2/ receptor in intact human platelets using direct ligand binding techniques. The compound studied were: (1) U46619, a chemically stable TXA/sub 2//PGH/sub 2/ mimetic; (2) four putative TXA/sub 2//PGH/sub 2/ receptor antagonists (namely SQ 29, 548, ONO 3708, BM 13.177 and 13-azaprostanoic acid); and (3) I-APA-PhN/sub 3/, a derivative of 13-azaprostanoic acid which incorporates a photoactive iodophenylazide group. Using (/sup 3/H)-U46619 as the radioligand, a binding assay was developed for use in intact, unactivated human platelets.
  • The TXA/sub 2//PGH/sub 2/ receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific /sup 3/H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg ofmore » protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA/sub 2//PGH/sub 2/ receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor.« less
  • Binding of [[sup 3]H]-SQ 29,548 was characterized to soluble thromboxane A[sub 2]/prostaglandin H[sub 2] (TP) receptors from human platelet membranes as a means of examining ligand-receptor interactions outside the lipophilic environment of the cell membrane. Kinetic determination revealed a rate of ligand-receptor association of 1.4 x 10[sup 7] [plus minus] 0.2M[sup [minus]1] [times] min[sup [minus]1] and a rate of dissociation of 0.5 [plus minus]0.07 min[sup [minus]1]. The resultant equilibrium affinity constant was 36.3 [plus minus] 5.8 nM. Saturation binding analysis revealed a single class of [[sup 3]H]-SQ 29,548 binding sites with an affinity constant of 39.7 [plus minus] 4.3 nMmore » and a B[sub max] of 1735.7 [plus minus] 69.1 fmol/mg protein. Specific [[sup 3]H]-SQ 29,548 binding was inhibited by specific TP receptor antagonists and agonists in a rank order of potency similar to that seen in platelet membranes: SQ 33,961 [much gt] SQ 29,548 > BM 13,505 [ge] U 46619 > BM 13,177. PGD[sub 2], PGE[sub 2] and PGl[sub 2] did not appreciably inhibit the specific binidng of [[sup 3]H]-SQ 29,548. These data indicate that [[sup 3]H]-SQ 29,548 binding of soluble human platelet TP receptors was specific, saturable, and reversible.« less
  • Transient elevations in murine secondary palatal adenosine 3',5'-monophosphate (cAMP) levels occur during palate ontogeny. Since palatal processes exposed to dibutyryl cAMP differentiate precociously, increases in palatal cAMP levels are of interest. Prostaglandin E/sub 2/ (PGE/sub 2/), which is synthesized by murine embryonic palate mesenchyme cells (MEPM), regulates cAMP levels in adult tissues via specific membrane bound receptors coupled to adenylate cyclase. Therefore, a PGE/sub 2/ receptor-adenylate cyclase systems was proposed in the developing murine secondary palate. Utilizing a radioligand binding assay, it was determined that murine palatal tissue on day 13 of gestation contained PGE/sub 2/ receptors that were saturable,more » of high affinity and low capacity. Specific (/sup 3/H)-PGE/sub 2/ binding was reversible by 30 min. The order of prostanoid binding affinity at specific PGE/sub 2/ binding sites was E/sub 2/ > F/sub 2//sub ..cap alpha../ > A/sub 2/ > E/sub 1/ = D/sub 2/ indicating specificity of the receptor for PGE/sub 2/. The ability of MEPM cells to respond to PGE/sub 2/ with dose-dependent accumulations of intracellular cAMP demonstrated the functional nature of these binding sites. Analysis of palatal PGE/sub 2/ receptor characteristics on days 12 and 14 of palate development indicated temporal alterations in receptor affinity and density during palate ontogeny.« less
  • Thrombocytopenia is often associated with immune complex disease and may in part be due to the interaction of circulating (IgG) immune complexes with an Fc{sub {gamma}} receptor on the platelet surface. Characterization of the immune complex-platelet interaction should provide for a better understanding of the pathophysiology of immune thrombocytpenia. To this end, a ligand binding assay, employing {sup 125}I-IgG trimer, was established. Receptor expression was determined by measuring the saturable binding of radiolabeled trimer to platelets at equilibrium. Normal human platelets were observed to express 8559 {plus minus} 852 binding sites for IgG trimer with a Kd of 12.5 {plusmore » minus} 1.7 {times} 10{sup {minus}8} M. Binding of IgG trimer to human platelets was blocked following preincubation of the cells with an anti-Fc{sub {gamma}}RII monoclonal antibody. Furthermore, this binding was ionic-strength dependent but was unaffected by the presence of Mg{sup ++} or cytochalasin B. Platelet Fc{sub {gamma}} receptor modulation was examined by assessing the effects of various physiologic and pharmacologic on the ability of platelets to bind IgG trimer. Platelet Fc{sub {gamma}} receptor expression was not affected by thrombin, ADP, or {gamma}-interferon. However, in 7/12 normal donors, treatment of platelets with dexamethasone resulted in a decrease in the number of Fc{sub {gamma}} receptors expressed.« less