Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Nov;82(21):7434–7438. doi: 10.1073/pnas.82.21.7434

Solubilization of a thromboxane A2/prostaglandin H2 antagonist binding site from human platelets.

R M Burch, D E Mais, D L Saussy Jr, P V Halushka
PMCID: PMC391359  PMID: 2997791

Abstract

A binding site for 9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-hydroxyph eny l)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranorthromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 antagonist, was solubilized into the 200,000 X g supernatant from human platelet membranes by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Binding to the solubilized site was saturable, displaceable, and reversible. Displaceable binding was not affected by sodium, potassium, or phosphate concentrations up to 50 mM or by magnesium to 5 mM but was increased 14% (P less than 0.05) by 5 mM calcium. A pH optimum for displaceable binding occurred between pH 7.0 and 7.5. Scatchard analysis of [125I]-PTA-OH binding to the solubilized binding site revealed a single class of sites, having a dissociation constant (Kd) of 66 +/- 16 nM (n = 3) and a Bmax of 750 +/- 80 fmol/mg of protein. The Kd for the membranes prior to solubilization was 47 +/- 11 nM (n = 3) and the Bmax was 700 +/- 90 fmol sites per mg of protein. The association rate constant, k1, was 1.57 X 10(7) M-1 X min-1 and the dissociation rate constant, k-1, was 0.61 +/- 0.04 min-1 (n = 4), yielding a Kd (k-1/k1) of 39 nM. Several thromboxane A2/prostaglandin H2 agonists and antagonists displaced bound [125I]-PTA-OH at concentrations similar to those at which they affect platelet aggregation. Collectively, these observations suggest that the solubilized protein is the thromboxane A2/prostaglandin H2 binding site that mediates platelet aggregation.

Full text

PDF
7434

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bennett J. S., Vilaire G., Burch J. W. A role for prostaglandins and thromboxanes in the exposure of platelet fibrinogen receptors. J Clin Invest. 1981 Oct;68(4):981–987. doi: 10.1172/JCI110352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bruns R. F., Lawson-Wendling K., Pugsley T. A. A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Anal Biochem. 1983 Jul 1;132(1):74–81. doi: 10.1016/0003-2697(83)90427-x. [DOI] [PubMed] [Google Scholar]
  3. Cuatrecasas P. Isolation of the insulin receptor of liver and fat-cell membranes (detergent-solubilized-( 125 I)insulin-polyethylene glycol precipitation-sephadex). Proc Natl Acad Sci U S A. 1972 Feb;69(2):318–322. doi: 10.1073/pnas.69.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Halushka P. V., MacDermot J., Knapp D. R., Eller T., Saussy D. L., Jr, Mais D., Blair I. A., Dollery C. T. A novel approach for the study of thromboxane A2 and prostaglandin H2 receptors using an 125I-labeled ligand. Biochem Pharmacol. 1985 Apr 15;34(8):1165–1170. doi: 10.1016/0006-2952(85)90490-3. [DOI] [PubMed] [Google Scholar]
  5. Hjelmeland L. M. A nondenaturing zwitterionic detergent for membrane biochemistry: design and synthesis. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6368–6370. doi: 10.1073/pnas.77.11.6368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Horn P. T., Kohli J. D., LeBreton G. C., Venton D. L. Antagonism of prostanoid-induced vascular contraction by 13-azaprostanoic acid (13-APA). J Cardiovasc Pharmacol. 1984 Jul-Aug;6(4):609–613. doi: 10.1097/00005344-198407000-00009. [DOI] [PubMed] [Google Scholar]
  7. Hung S. C., Ghali N. I., Venton D. L., Le Breton G. C. Prostaglandin F2 alpha antagonizes thromboxane A2-induced human platelet aggregation. Prostaglandins. 1982 Aug;24(2):195–206. doi: 10.1016/0090-6980(82)90145-9. [DOI] [PubMed] [Google Scholar]
  8. Kidric M., Petrović J., Soskić V., Trajković D. Solubilization of dopamine-D2 receptors from synaptosomal membranes of the bovine caudate nucleus. Br J Pharmacol. 1984 Nov;83(3):687–695. doi: 10.1111/j.1476-5381.1984.tb16222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Le Breton G. C., Venton D. L., Enke S. E., Halushka P. V. 13-Azaprostanoic acid: a specific antagonist of the human blood platelet thromboxane/endoperoxide receptor. Proc Natl Acad Sci U S A. 1979 Aug;76(8):4097–4101. doi: 10.1073/pnas.76.8.4097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lefkowitz R. J., Haber E., O'Hara D. Identification of the cardiac beta-adrenergic receptor protein: solubilization and purification by affinity chromatography. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2828–2832. doi: 10.1073/pnas.69.10.2828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Leigh P. J., Cramp W. A., MacDermot J. Identification of the prostacyclin receptor by radiation inactivation. J Biol Chem. 1984 Oct 25;259(20):12431–12436. [PubMed] [Google Scholar]
  13. Mais D. E., Kochel P. J., Saussy D. L., Jr, Halushka P. V. Binding of an 125I-labeled thromboxane A2/prostaglandin H2 receptor antagonist to washed canine platelets. Mol Pharmacol. 1985 Aug;28(2):163–169. [PubMed] [Google Scholar]
  14. Mais D. E., Saussy D. L., Jr, Chaikhouni A., Kochel P. J., Knapp D. R., Hamanaka N., Halushka P. V. Pharmacologic characterization of human and canine thromboxane A2/prostaglandin H2 receptors in platelets and blood vessels: evidence for different receptors. J Pharmacol Exp Ther. 1985 May;233(2):418–424. [PubMed] [Google Scholar]
  15. Matsui H., Imafuku J., Asakura M., Tsukamoto T., Ino M., Saitoh N., Miyamura S., Hasegawa K. Solubilization of active alpha-2 adrenergic receptor from rat brain: regulation by cations and GTP. Biochem Pharmacol. 1984 Oct 15;33(20):3311–3314. doi: 10.1016/0006-2952(84)90098-4. [DOI] [PubMed] [Google Scholar]
  16. Nicolaou K. C., Magolda R. L., Smith J. B., Aharony D., Smith E. F., Lefer A. M. Synthesis and biological properties of pinane-thromboxane A2, a selective inhibitor of coronary artery constriction, platelet aggregation, and thromboxane formation. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2566–2570. doi: 10.1073/pnas.76.6.2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schafer A. I., Cooper B., O'Hara D., Handin R. I. Identification of platelet receptors for prostaglandin I2 and D2. J Biol Chem. 1979 Apr 25;254(8):2914–2917. [PubMed] [Google Scholar]
  18. Schrör K., Darius H., Matzky R., Ohlendorf R. The antiplatelet and cardiovascular actions of a new carbacyclin derivative (ZK 36 374)--equipotent to PGI2 in vitro. Naunyn Schmiedebergs Arch Pharmacol. 1981 Jun;316(3):252–255. doi: 10.1007/BF00505658. [DOI] [PubMed] [Google Scholar]
  19. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  20. Siegl A. M., Smith J. B., Silver M. J. Specific binding sites for prostaglandin D2 on human platelets. Biochem Biophys Res Commun. 1979 Sep 12;90(1):291–296. doi: 10.1016/0006-291x(79)91623-1. [DOI] [PubMed] [Google Scholar]
  21. Weiland G. A., Molinoff P. B. Quantitative analysis of drug-receptor interactions: I. Determination of kinetic and equilibrium properties. Life Sci. 1981 Jul 27;29(4):313–330. doi: 10.1016/0024-3205(81)90324-6. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES