Effect of cAMP Phosphodiesterase Inhibitors on ADP-Induced Shape Change, cAMP and Nucleoside Diphosphokinase Activity of Rabbit Platelets

Summary

The effects of cAMP phosphodiesterase inhibitors on ADP-induced shape change and cAMP concentrations have been studied. Caffeine (10 mM), theophylline (8 mM), dipyridamole (0.2 mM), or papaverine (0.05 mM) prevented the shape change of washed rabbit platelets induced by 0.4 μM ADP. At these concentrations, none of these cAMP phosphodiesterase inhibitors increased ¹⁴C-cAMP in platelets in which the cytoplasmic adenine nucleotides had been labelled with ¹⁴C-adenine. By a protein binding assay, only papaverine by itself increased platelet cAMP above its basal level. These results indicate that two pools of cAMP may exist in platelets. Both methods showed that stimulation of platelet adenylate cyclase with PGE₁ (1 μM) resulted in an increase in platelet cAMP and all these cAMP phosphodiesterase inhibitors potentiated this increase caused by PGE₁. By themselves, some of these compounds may act through mechanisms that do not involve platelet cAMP. The effects of these cAMP phosphodiesterase inhibitors on platelet nucleoside diphosphokinase (NDK) activity were also investigated. At concentrations that prevented ADP-induced shape change, papaverine and dipyridamole had no effect on the formation of ¹⁴C-ATP from ¹⁴C-ADP by washed rabbit platelets. The methylxanthines partially inhibited NDK activity of washed rabbit platelets and of isolated platelet membranes, probably due to the structural similarity between the adenine ring of ADP and these substances. However, adenine (8 mM) inhibited ADP-induced shape change and platelet NDK activity but was a less effective inhibitor of ADP-induced platelet aggregation. Thus it seems unlikely that interference with platelet NDK or the ADP receptor is the major mechanism by which the methylxanthines inhibit platelet functions.

Key words

Platelets - cAMP phosphodiesterase inhibitors - Dipyridamole - Adenine - Nucleoside diphosphokinase

• References

• 1 Mills DCB, Smith JB. The influence on platelet aggregation of drugs that affect the accumulation of adenosine 3':5'-cyclic monophosphate in platelets. Biochem J 1971; 121: 185-196
• 2 Salzman EW. Cyclic AMP and platelet function. N Engl J Med 1972; 286: 358-363
• 3 Haslam RJ. Roles of cyclic nucleotides in platelet function. In: Biochemistry and Pharmacology of Platelets. Ciba Foundation Symposium 35. 121-151 (new series) Elsevier Excerpta Medica; North Holland: 1975
• 4 Holmsen H. Classification and possible mechanisms of action of some drugs that inhibit platelet aggregation. Ser Haematol 1976; 8: 50-80
• 5 Haslam RJ, Davidson MML, Davies T, Lynham JA, McClenaghan MD. Regulation of blood platelet function by cyclic nucleotides. Adv Cyclic Nucleotide Res 1978; 9: 533-552
• 6 Marquis NR, Vigdahl RL, Tavormina PA. Platelet aggregation. I. Regulation by cyclic AMP and prostaglandin E₁ . Biochem Biophys Res Comm 1969; 36: 965-972
• 7 Wolfe SM, Shulman NR. Adenyl cyclase activity in human platelets. Biochem Biophys Res Comm 1969; 35: 265-272
• 8 Haslam RJ. Interactions of the pharmacological receptors of blood platelets with adenylate cyclase. Ser Haematol 1973; 6: 333-350
• 9 McDonald JWD, Stuart RK. Regulation of cyclic-AMP levels and aggregation in human platelets by prostaglandin E₁ . J Lab Clin Med 1973; 81: 838-849
• 10 Salzman EW. Prostaglandins, cyclic AMP, and platelet function. Thrombos Diathes Haemorrh 1974; (Suppl. 60) 311-319
• 11 Michel H, Caen JP, Born GVR, Miller R, D’Auriac GA, Meyer P. Relation between the inhibition of aggregation and the concentration of cAMP in human and rat platelets. Br J Haematol 1976; 33: 27-38
• 12 Best LC, Martin TJ, Russel RGG, Preston FE. Prostacyclin increases cyclic AMP levels and adenylate cyclase activity in platelets. Nature 1977; 267: 850-852
• 13 Gorman RR, Bunting S, Miller OV. Modulation of human platelet adenylate cyclase by prostacyclin (PGX). Prostaglandins 1977; 13: 377-388
• 14 Tateson JE, Moncada S, Vane JR. Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets. Prostaglandins 1977; 13: 389-397
• 15 Ardlie NG, Glew G, Schultz BG, Schwartz CJ. Inhibition and reversal of platelet aggregation by methyl xanthines. Thrombos Diathes Haemorrh 1967; 18: 670-673
• 16 Ball G, Brereton GG, Fulwood M, Ireland DM, Yates P. Effect of prostaglandin E₁ alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3' 5'-cyclic monophosphate. Biochem J 1970; 120: 709-718
• 17 Cucuianu MP, Nishizawa EE, Mustard JF. Effect of pyrimido-pyrimidine compounds on platelet function. J Lab Clin Med 1971; 77: 958-974
• 18 Niewiarowski S, Lukasiewicz H, Nath N, Sha AT. Inhibition of human platelet aggregation by dipyridamole and two related compounds and its modification by add glycoproteins of human plasma. J Lab Clin Med 1975; 86: 64-76
• 19 Subbarao K, Rucinski B, Niewiarowski S. Effect of dipyridamole and its monoglucuronide derivative on adenosine uptake by human platelets and ADP-induced platelet aggregation. Biochem Pharmacol 1977; 26: 906-907
• 20 Best LC, McGuire MB, Jones PBB, Holland TK, Martin TJ, Preston FE, Segal DS, Russell RGG. Mode of action of dipyridamole on human platelets. Thromb Res 1979; 16: 367-379
• 21 Tang SS, Frojmovic MM. Inhibition of platelet function by antithrombotic agents which selectively inhibit low-Km cyclic 3', 5'-adenosine monophosphate phosphodiesterase. J Lab Clin Med 1980; 95: 241-257
• 22 Guccione MA, Packham MA, Kinlough-Rathbone RL, Mustard JF. Reactions of ¹⁴C-ADP and ¹⁴C-ATP with washed platelets from rabbits. Blood 1971; 37: 542-555
• 23 Feinberg H, Belamarich F, Born GVR. Transphosphorylation in human and rabbit platelet suspensions. Fed Proc 1973; 32: 220
• 24 Mustard JF, Packham MA, Perry DW, Guccione MA, Kinlough-Rathbone RL. Enzyme activities on the platelet surface in relation to the action of adenosine diphosphate. In: Biochemistry and Pharmacology of Platelets. Ciba Foundation Symposium 35. 47-75 (new series) Elsevier Excerpta Medica; North Holland: 1975
• 25 Packham MA, Guccione MA, Perry DW, Kinlough-Rathbone RL, Mustard JF. AMP inhibition of reactions of ADP with washed platelets from human and rabbits. Am J Physiol 1972; 223: 419-424
• 26 Lips JPM, Sixma JJ, Schiphorst ME. Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes. Biochim Biophys Acta 1980; 628: 451-467
• 27 Kinlough-Rathbone RL, Packham MA, Mustard JF. The effect of prostaglandin E₁ on platelet function in vitro and in vivo. Br J Haematol 1970; 19: 559-571
• 28 Aster RH, Jandl JH. Platelet sequestration in man. I. Methods. J Clin Invest 1964; 43: 843-855
• 29 Ardlie NG, Packham MA, Mustard JF. Adenosine diphosphate-induced platelet aggregation in suspensions of washed rabbit platelets. Br J Haematol 1970; 19: 7-17
• 30 Ardlie NG, Perry DW, Packham MA, Mustard JF. Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc Soc Exptl Biol Med 1971; 136: 1021-1023
• 31 Molnar J, Lorand L. Studies on apyrases. Arch Biochem Biophys 1961; 93: 353-363
• 32 Nurden AT, Caen JP. Specific roles for platelet surface glycoproteins in platelet function. Nature 1975; 255: 720-722
• 33 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275
• 34 Mustard JF, Packham MA. Platelet aggregation and the platelet release reaction in thromboembolism. Can Med Assoc J 1970; 103: 859-863
• 35 Lam SC-T, Harfenist EJ, Packham MA, Mustard JF. Investigation of possible mechanisms of pyridoxal 5'-phosphate inhibition of platelet reactions. Thromb Res 1980; 20: 633-645
• 36 Haslam RJ, Rosson GM. Effects of adenosine on levels of adenosine cyclic 3',5'-monophosphate in human blood platelets in relation to adenosine incorporation and platelet aggregation. Mol Pharmacol 1975; 11: 528-544
• 37 Brown BL, Albano JDM, Ekins RP, Sgherzi AM. A simple and sensitive saturation assay method for the measurement of adenosine 3':5'-cyclic monophosphate. Biochem J 1971; 121: 561-562
• 38 McElroy FA, Philp RB. Relative potendes of dipyridamole and related agents as inhibitors of cyclic nucleotide phosphodiesterases: Possible explanation of mechanism of inhibition of platelet function. Life Sci 1975; 17: 1479-1494
• 39 Radomski M, Swies J, Gryglewski RJ. Disaggregatory action of phosphodiesterase inhibitors. Pharmacol Res Comm 1981; 13: 41-47
• 40 Adler JR, Handin RI. Solubilization and characterization of a platelet membrane ADP-binding protein. J Biol Chem 1979; 254: 3866-3872
• 41 Cooper DMF, Rodbell M. ADP is a potent inhibitor of human platelet plasma membrane adenylate cyclase. Nature 1979; 282: 517-518
• 42 Markwardt F, Hoffmann A. Effects of papaverine derivatives on cyclic AMP phosphodiesterase of human platelets. Biochem Pharmacol 1970; 19: 2519-2520
• 43 Vigdahl RL, Mongin J Jr, Marquis NR. Platelet aggregation. IV. Platelet phosphodiesterase and its inhibition by vasodilators. Biochem Biophys Res Comm 1971; 42: 1088-1094
• 44 Rozenberg MC, Walker CM. The effect of pyrimidine compounds on the potentiation of adenosine inhibition of aggregation, on adenosine phosphorylation and phosphodiesterase activity of blood platelets. Br J Haematol 1973; 24: 409-418
• 45 Asano T, Ochiai Y, Hidaka A. Selective inhibition of separated forms of human platelet cyclic nucleotide phosphodiesterase by platelet aggregation inhibitors. Mol Pharmacol 1977; 13: 400-406
• 46 Tsien WH, Sheppard H. The lack of correlation between inhibition of platelet aggregation and cAMP levels. Fed Proc 1981; 40: 809
• 47 Zahavi M, Kakkar VV. Adenosine 3',5'-cyclic monophosphate (c-AMP) and inhibitors of platelet aggregation: A possibility of a double c-AMP pool in human platelets. Thromb Haemostas 1981; 46: 415 (Abstr)
• 48 Amer MS, Mayol RF. Studies with phosphodiesterase. III. Two forms of the enzyme from human blood platelets. Biochim Biophys Acta 1973; 309: 149-156
• 49 Pichard A-L, Hanoune J, Kaplan J-C. Multiple forms of cyclic adenosine 3',5'-monophosphate phosphodiesterase from human blood platelets. I. Kinetic and electrophoretic characterization of two molecular spedes. Biochim Biophys Acta 1973; 315: 370-377
• 50 Hidaka H, Asano T. Human blood platelet 3',5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase. Biochim Biophys Acta 1976; 429: 485-497
• 51 Lecomte M-C, Galand C, Boivin P. Inhibition of human erythrocyte casein kinase by methylxanthines. FEBS Lett 1980; 116: 45-47