Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1983 Mar;71(3):420–428. doi: 10.1172/JCI110786

2-Methylthioadenosine[beta-32P]diphosphate. An agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets.

D E Macfarlane, P C Srivastava, D C Mills
PMCID: PMC436889  PMID: 6298277

Abstract

2-Methylthio-ADP and its radioactive analogue [beta-32P]2-methylthio-ADP were synthesized and used to investigate platelet receptors for ADP. 2-Methylthio-ADP induced platelet aggregation and shape change, and inhibited cyclic AMP accumulation in platelets exposed to prostaglandin E1. Compared with ADP, 2-methylthio-ADP was 3-5 times as active as an aggregating agent and 150-200 times as active as an inhibitor of cyclic AMP accumulation. Binding of [beta-32P]2-methylthio-ADP to platelets was measured after centrifuging them through silicone oil to separate platelets from their suspension medium. Binding was reversible, saturable, and specific, with between 400 and 1,200 sites/cell in different platelet preparations. There was no evidence for a second class of binding sites with different affinity. The second order association rate constant was approximately 3.5 X 10(6) M-1 S-1, and the first order dissociation rate was 0.024 s-1, both measured at 23 degrees C. The dissociation equilibrium constant (approximately 15 nM) was about three times higher than the concentration giving half-maximal inhibition of prostaglandin E1-stimulated cyclic AMP accumulation in platelet-rich plasma. Binding was inhibited by ADP (Ki = 3.5 microM), ATP (7 microM), 2-azido-ADP (0.12 microM), inosine diphosphate (IDP, 150 microM), guanosine diphosphate (GDP, 350 microM), and AMP (800 microM). Binding of 2-methylthio-ADP was also blocked by the non-cell-penetrating thiol reagent, p-mercuribenzene sulphonate, a reagent that blocks the inhibition of adenylate cyclase by ADP, but which does not block the ability of ADP to induce aggregation or platelet shape change. The amount of 2-methylthio-ADP bound at saturation was independent of pH in the range 6-8, but the affinity was reduced at pH 6 compared with pH 6.5-8.0. The dissociation constant was not temperature dependent in the range 32 degrees -40 degrees C, whereas the rate of dissociation of 2-methylthio-ADP from platelets after the addition of an excess of ADP approximately doubled over this range. The activation energy for dissociation was approximately 15 kcal/mol. Our results support the conclusion that platelets have a receptor for ADP, which inhibits cyclic AMP accumulation, and which has a sulphydryl group in the binding pocket.

Full text

PDF
420

Selected References

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

  1. Born G. V., Feinberg H. Binding of adenosine diphosphate to intact human platelets. J Physiol. 1975 Oct;251(3):803–816. doi: 10.1113/jphysiol.1975.sp011123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cooper D. M., Rodbell M. ADP is a potent inhibitor of human platelet plasma membrane adenylate cyclase. Nature. 1979 Nov 29;282(5738):517–518. doi: 10.1038/282517a0. [DOI] [PubMed] [Google Scholar]
  3. Gough G., Maguire M. H., Penglis F. Analogues of adenosine 5'-diphosphate-new platelet aggregators. Influence of purine ring and and phosphate chain substitutions on the platelet-aggregating potency of adenosine 5'-diphosphate. Mol Pharmacol. 1972 Mar;8(2):170–177. [PubMed] [Google Scholar]
  4. Macfarlane D. E. Bidirectional collision coupling in the regulation of the adenylate cyclase. The allozyme hypothesis for receptor function. Mol Pharmacol. 1982 Nov;22(3):580–588. [PubMed] [Google Scholar]
  5. Macfarlane D. E., Mills D. C. Inhibition by ADP of prostaglandin induced accumulation of cyclic AMP in intact human platelets. J Cyclic Nucleotide Res. 1981;7(1):1–11. [PubMed] [Google Scholar]
  6. Macfarlane D. E., Mills D. C., Srivastava P. C. Binding of 2-azidoadenosine [beta-32P]diphosphate to the receptor on intact human blood platelets which inhibits adenylate cyclase. Biochemistry. 1982 Feb 2;21(3):544–549. doi: 10.1021/bi00532a020. [DOI] [PubMed] [Google Scholar]
  7. Macfarlane D. E., Mills D. C. The effects of ATP on platelets: evidence against the central role of released ADP in primary aggregation. Blood. 1975 Sep;46(3):309–320. [PubMed] [Google Scholar]
  8. Macfarlane D. E., Stump D. C. Parallel observation of the occupancy of the alpha 2-adrenergic receptor in intact platelets and its ability to inhibit the adenylate cyclase. Mol Pharmacol. 1982 Nov;22(3):574–579. [PubMed] [Google Scholar]
  9. Macfarlane D. E., Wright B. L., Stump D. C. Use of [methyl-3H]Yohimbine as a radioligand for alpha-2 adrenoreceptors on intact platelets. Comparison with dihydroergocryptine. Thromb Res. 1981 Oct 1;24(1-2):31–43. doi: 10.1016/0049-3848(81)90029-3. [DOI] [PubMed] [Google Scholar]
  10. Mustard J. F., Packham M. A., Perry D. W., Guccione M. A., Kinlough-Rathbone R. L. Enzyme activities on the platelet surface in relation to the action of adenosine diphosphate. Ciba Found Symp. 1975;35:47–75. doi: 10.1002/9780470720172.ch4. [DOI] [PubMed] [Google Scholar]
  11. Walsh P. N., Mills D. C., White J. G. Metabolism and function of human platelets washed by albumin density gradient separation. Br J Haematol. 1977 Jun;36(2):287–296. doi: 10.1111/j.1365-2141.1977.tb00649.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES