Direct Evidence for the Inhibition of Platelet Aggregation and Release by Intracellular Cyclic AMP Produced with a New Photoactivatable Derivative

Summary

An increase in platelet cyclic AMP (cAMP) via stimulation of adenylate cyclase is thought to be the underlying mechanism by which potent prostaglandins i.e. PGD₂ PGI₂ inhibit platelet functions. We report here new and direct evidence for the inhibitory effects of cAMP on platelet aggregation and serotonin release. Washed platelets from rat were incubated with a new photoactivatable cAMP analogue (4,5-dimethoxy-2-nitrobenzyl ester); this compound is almost physiologically inert before irradiation and liberates free cAMP (“cAMP jumps”) following light flashes. A single flash, delivered after 2 min incubation in 100-200 μM of the analogue, dramatically inhibited thrombin-induced aggregation, as compared with controls. Endogenous serotonin release, measured in the same samples by means of an electrochemically treated carbon electrode was undetectable after the cAMP jump. Pre-irradiated solutions added to platelets had no effect. The kinetics of the flash-induced effects were also studied. From these results we can conclude that: i) the photoactivatable cAMP derivative has to permeate through the platelet membrane; ii) the analogue remains photolabile; and, iii) intracellular cAMP, resulting from photolysis dramatically inhibits platelet aggregation and serotonin release. It is possible that cAMP exerts its effects by regulating cytoplasmic free calcium concentration and/or other actions affecting platelet activation.

Key words

Rat platelet aggregation - Endogenous serotonin release - Cyclic AMP - Photoactivatable cyclic AMP

• References

• 1 Gerrard JM, Peterson DA, White JG. Calcium mobilization. In: Platelets in Biology and Pathology. 2 Gordon JL. (ed.) Elsevier/ North-Holland; Amsterdam: 1981: 407-436
• 2 Feinstein MB, Rodan GA, Cutler LS. Cyclic AMP and calcium in platelet function. In: Platelets in Biology and Pathology. 2 Gordon JL. (ed.) Elsevier/ North-Holland; Amsterdam: 1981: 437-472
• 3 Fox JE B, Phillips DR. Role of phosphorylation in mediating the association of myosin with the cytoskeletal structures of human platelets. J Biol Chem 1982; 257: 4120-4126
• 4 Haslam RJ, Davidson MM L, Davies T, Lynham JA, McClenagham MD. Regulation of blood platelet function by cyclic nucleotides. Adv Cyclic Nucleotides Res 1978; 9: 533-552
• 5 Feinstein MB, Fraser C. Human platelet secretion and aggregation induced by calcium ionophores. J Gen Physiol 1975; 66: 561-581
• 6 Nerbonne JM, Richard S, Nargeot J, Lester JA. New photoactivatable cyclic nucleotides produce intracellular jumps in cyclic AMP and cyclic GMP concentrations. Nature 1984; 310: 74-76
• 7 Ciavatti M, Michel G, Renaud S. Biosynthesis of cholesterol and cholesterol precursors in platelets of female rats treated by oral contraceptives. Biochim Biophys Acta 1980; 620: 297-307
• 8 Marcenac F, Blache D. Endogenous platelet serotonin release monitored during aggregation by means of a new electrochemical technique. Biochim Biophys Acta 1985; 840: 377-382
• 9 Gonon FG, Buda M, Pujol JF. Treated carbon fiber electrodes for measuring catechols and ascorbic acid. In: Measurement of neurotransmitter release Mardsen CA. (ed.) 153-171 J. Willy & Sons; Chichester: 1984
• 10 Massini P, Lüscher EF. On the significance of the influx of calcium ions into stimulated human blood platelets. Biochim Biophys Acta 1976; 436: 652-663
• 11 Feinstein MB. Release of intracellular membrane-bound calcium precedes the onset of stimulus-induced exocytosis in platelets. Biochem Biophys Res Commun 1980; 93: 593-600
• 12 Rink TJ, Smith SW, Tsien RY. Cytoplasmic free Ca²⁺ in human platelets: Ca²⁺ thresholds and Ca-independent activation for shape-change and secretion. FEBS Lett 1982; 21-26
• 13 Bills TK, Smith JB, Silver MJ. Selective release of arachidonic acid from the phospholipids of human platelets in response to thrombin. J Clin Invest 1977; 60: 1-6
• 14 Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by fumor-promoting phosphol esters. J Biol chem 1982; 257: 7847-7851
• 15 Hathaway BR, Adelstein RS. Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for reactivity. Proc Natl Acad Sci USA 1979; 76: 1653-1657
• 16 Imai A, Hattori H, Takahashi M, Nozawa Y. Evidence that cyclic AMP may regulate Ca²⁺-mobilization and phospholipases in thrombin-stimulated human platelets. Biochem Biophys Res Commun 1983; 112: 693-700
• 17 Feinstein MB, Ega JJ, Sha’afi RI, White J. The cytoplasmic concentration of free calcium in platelets is controlled by stimulators of cyclic AMP production (PGD₂ PGE₁ Forskolin). Biochem Biophys Res Commun 1983; 113: 598-604
• 18 Yamanishi J, Kawahara Y, Fukuzaki H. Effect of cyclic AMP on cytoplasmic free calcium in human platelets stimulated by thrombin: direct measurements with Quin 2. Thromb Res 1983; 32: 183-188
• 19 Zavoico GB, Feinstein MB. Cytoplasmic Ca²⁺ in platelets is controlled by cyclic AMP: antagonism between stimulators and inhibitors of adenylate cyclase. Biochem Biophys Res Commun 1984; 120: 579-585
• 20 Seamon KB, Padgett W, Daly JW. Forskolin: Unique diterpene activator of adenylate cyclase in membranes and in fat cells. Proc Natl Acad Sci USA 1981; 78: 3363-3367
• 21 Feinstein MB, Egan JJ, Opas EE. Reversal of thrombin-induced myosin phosphorylation and the assembly of cytoskeletal structures in platelets by the adenylate cyclase stimulants PGD₂ and forskolin. J Biol Chem 1983; 258: 1260-1267