Amendment history: Correction (July 1972) Cyclic 3′,5′-adenosine monophosphate in human blood platelets: II. Effect of N6-2′-0-dibutyryl cyclic 3′,5′-adenosine monophosphate on platelet function Edwin W. Salzman, Linda Levine J Clin Invest. 1971;50(1):131-141. https://doi.org/10.1172/JCI106467. The relation of cyclic 3′,5′-adenosine monophosphate to platelet function has been studied by investigating the influence of this compound and of its N6-2′-0-dibutyryl derivative on platelet aggregation and other aspects of platelet behavior after demonstration of adenyl cyclase activity in disrupted platelets. Dibutyryl cyclic AMP inhibited platelet aggregation induced by ADP, epinephrine, collagen, and thrombin. Cyclic AMP was also inhibitory but was less effective. The platelet “release reaction” was also inhibited; specifically, there was inhibition of the induction of platelet factor 3 activity and of the release of labeled 5-hydroxytryptamine. Platelet swelling produced by ADP was not inhibited. The action of dibutyryl cyclic AMP did not result from contamination with 5′-AMP, nor was it attributable to production of 5′-AMP by plasma enzymes. Dibutyryl cyclic AMP was degraded to 2′-O-monobutyryl cyclic AMP and to cyclic AMP in plasma, but plasma exhibited no cyclic nucleotide phosphodiesterase activity, and the production of 5′-AMP did not occur. The in vitro effects of dibutyryl cyclic AMP were associated with uptake of the compound by platelets. Adenyl cyclase activity of platelet homogenates was demonstrated with production of 9.27 × 10-11 (±2.62 × 10-11) mole cyclic AMP […] Find the latest version: https://jci.me/106467/pdf Cyclic 3',5'-Adenosine Monophosphate in Human Blood Platelets II. EFFECT OF N6-2'-O-DIBUTYRYL CYCLIC 3',5'- ADENOSINE MONOPHOSPHATE ON PLATELET FUNCTION EDwIN W. SALzMAN and LINDA LEVINE From the Department of Surgery, Harvard Medical School; Surgical Service, Beth Israel Hospital, Boston, Massachusetts 02215 A B S T R A C T The relation of cyclic 3',5'-adenosine propanolol. Adenyl cyclase activity was also inhibited monophosphate to platelet function has been studied by by collagen, 5-hydroxytryptamine, and thrombin. ADP, investigating the influence of this compound and of its dibutyryl cyclic AMP, and cyclic AMP did not alter N6-2'-O-dibutyryl derivative on platelet aggregation and adenyl cyclase activity. other aspects of platelet behavior after demonstration These observations are consistent with the hypothesis of adenyl cyclase activity in disrupted platelets. that platelet aggregation is favored by a decrease in Dibutyryl cyclic AMP inhibited platelet aggregation platelet cyclic AMP and inhibited by an increase in induced by ADP, epinephrine, collagen, and thrombin. cyclic AMP. Cyclic AMP was also inhibitory but was less effective. The platelet "release reaction" was also inhibited; spe- cifically, there was inhibition of the induction of platelet INTRODUCTION factor 3 activity and of the release of labeled 5-hydroxy- Inhibition of platelet aggregation by 3',5'-adenosine tryptamine. Platelet swelling produced by ADP was monophosphate (cyclic AMP, cAMP) was first reported not inhibited. by Marcus and Zucker in 1965 (1), but this observation The action of dibutyryl cyclic AMP did not result from attracted little attention for several years. Recently contamination with 5'-AMP, nor was it attributable to several laboratories have suggested a relation of cyclic production of 5'-AMP by plasma enzymes. Dibutyryl AMP to platelet function and have studied the metabo- cyclic AMP was degraded to 2'-O-monobutyryl cyclic lism of this nucleotide in platelets. Adenyl cyclase activ- AMP and to cyclic AMP in plasma, but plasma ex- ity in platelets was first described by Scott (2), who hibited no cyclic nucleotide phosphodiesterase activity, found the enzyme unresponsive to epinephrine. Abdullah and the production of 5'-AMP did not occur. The in (3) noted reversal of platelet aggregation by 10' M vitro effects of dibutyryl cyclic AMP were associated dibutyryl cyclic AMP but not by 10' M cyclic AMP, and with uptake of the compound by platelets. he described stimulation of platelet cyclic nucleotide Adenyl cyclase activity of platelet homogenates was phosphodiesterase by imidazole and nicotinic acid. demonstrated with production of 9.27 x 10-n (+2.62 x Butcher, Scott, and Sutherland (4) and Wolfe and 10-u) mole cyclic AMP per min per 1010 platelets. The Shulman (5) reported that adenyl cyclase activity in activity was increased by NaF and by prostaglandin platelet homogenates was stimulated by prostaglandin PGE1 and was decreased by epinephrine. The effect of PGE1, and the latter authors also observed stimulation epinephrine was blocked by phentolamine but not by by NaF. These findings were confirmed by Zieve and This work was presented in part to the American Physio- Greenough (6), who also described stimulation of plate- logical Society, April 1970, and was published as an abstract let adenyl cyclase by glucagon and inhibition by throm- (1970 Fed. Proc. 29: 316). Received for publication 15 July 1970 and in revised form bin, norepinephrine, epinephrine, and serotonin. Mar- 22 September 1970. quis, Vigdahl, and Tavormina (7) reported confirmatory The Journal of Clinical Investigation Volume 50 1971 131 results and observed inhibition of platelet phosphodi- at 4°C. Platelet counts were performed by the method of esterase by caffeine. Brecher and Cronkite (19). 2. Platelet aggregation was studied by the method of Born A reasonable hypothesis based on these observations (20). The optical density of PRP was measured at 600 m~u (8) would be that agents that produce or augment in a Coleman Jr. spectrophotometer (Coleman Instruments, platelet aggregation (e.g., epinephrine [9], nicotinic Maywood, Ill.) modified to permit magnetic stirring, control acid [3], and imidazole [3]) reduce the level of platelet of temperature at 37°C, and continuous recording. 3. Platelet factor 3 activity was measured by determina- cyclic AMP (by inhibition of adenyl cyclase or stimula- tion of the recalcified clotting time of PRP after activation tion of phosphodiesterase), and that agents with the with 10 mg/ml of kaolin according to the technique of opposite effect on platelet clumping (e.g., caffeine [10], Hardisty and Hutton (21). PGE1 [11]) elevate platelet cyclic AMP. Direct mea- 4. Release of serotonin was studied by a modification of surement of the cyclic AMP content of platelets has the method of Spaet and Zucker (22). To the citrate to be employed as anticoagulant (No. 1, above), was added supported this formulation; Salzman and Neri (12) 5-hydroxytryptamine-2-'4C (SA 5.2 ,Ci/mg; New England found platelet cAMP increased by caffeine and reduced Nuclear Corp., Boston, Mass.) to a final concentration of by epinephrine and by ADP. Robison, Arnold, and 1.0 ,uCi/40 ml of blood, and after preparation of PRP the Hartmann (13) reported an increase in platelet cyclic sample was separated into aliquots and incubated at 370C. AMP produced by PGE1 and observed that the effect 30-60 min after collection of the blood, agents to be tested were added to aliquots of the PRP and further incubated of epinephrine on the platelet content of cyclic AMP at 37°C for 5 min. The PRP was then centrifuged at 20°C was inhibited by phentolamine, an a-adrenergic inhibitor, for 15 min at 2,000 g, and aliquots of the supernatant were but not by the P-adrenergic inhibitor, propanolol. An added to scintillation fluid1 and counted for radioactivity in analogous relation of a-adrenergic and 8-adrenergic a Nuclear-Chicago Mark I liquid scintillation counter (Nuclear-Chicago, Des Plaines, Ill.). Release of labeled blocking agents has been described for platelet aggrega- serotonin by platelets was expressed as per cent of total tion induced by epinephrine (9, 14). uptake. In studies to be described, the relationship of cyclic 5. Platelet volume was measured at 37°C with the Coulter AMP to platelet aggregation was further examined by Counter Model B (Coulter Electronics Inc., Hialeah, Fla.) and histogram plotter according to our previously published use of the derivative compound, N6-2'-0-dibutyryl cyclic technique (23). 3',5'-AMP (dibutyryl cyclic AMP, DB-cAMP) as em- 6. The activated partial thromboplastin time (24) (Throm- ployed by Posternak, Sutherland, Falbriard, and Henion bofax; Ortho Pharmaceutical Corp., Raritan, N. J.), throm- (15, 16). In some circumstances acylated derivatives of bin clotting time (25) (Thrombin Topical, Parke, Davis & cyclic AMP are known to have pharmacologic effects Co., Detroit, Mich.), and one stage prothrombin time (25) (Thromboplastin; Dade Div., American Hospital Supply in vivo and in tissue slices greater than the parent Corp., Miami, Fla.) were performed according to previously compound (15, 17). The enhanced activity of dibutyryl published techniques. cyclic AMP has been attributed to its resistance to cyclic 7. Platelet uptake of NV-2'-0-dibutyryl cyclic 3',5'-AMP-3H nucleotide phosphodiesterase or, alternatively, by virtue (SA 2.8 Ci/mmoles; New England Nuclear) and of cyclic of its to a greater ability to penetrate 3',5'-AMP-3H (SA 16.3 Ci/mmoles; Schwarz Bio Research nonpolarity, Inc., Orangeburg, N. Y. was studied by a previously de- lipophilic cell membranes (15, 17). Data on this latter scribed technique (26). After incubation of 1.0 ml aliquots point have not, to our knowledge, been reported. of PRP with the tracer and carrier nucleotide at specified The effect of agents influencing platelet aggregation concentrations, the samples were filtered through cellulose acetate Millipore filters (EAwp 02500, 1.2-,g pores) and the was also explored by -assessment of adenyl cyclase fiiters were immersed in scintillation fluid 1 and counted. activity in platelet homogenates. Human albumin-"1'I (Abbott Laboratories, N. Chicago, Ill.) The data to be reported support the above mentioned (0.3 ,uCi added to each sample) was added to the tracer mix- ture as a plasma marker.