Human Platelet Aggregation and camp System camp Level, Adenyl Cyclase, Phosphodiesterase *

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 8, No. 5 Copyright 1978, Institute for Clinical Science Human Platelet Aggregation and camp System camp Level, Adenyl Cyclase, Phosphodiesterase * TEH Y. WANG, Ph.D., CLARA V. HUSSEY, M.D., EDWARD A. SASSE, Ph.D. and JANE E. FOBIAN, B.S. Department of Pathology, The Medical College of Wisconsin, Milwaukee County Medical Complex, Milwaukee, Wl ABSTRACT T he possibility of a direct and casual relationship betw een various param eters of the adenosine 3', 5'-cyclic monophosphate (camp) system, e.g., the level of camp, adenyl cyclase activity and phosphodiesterase activity, and platelet aggregation was studied by m easuring the effects of various environmental conditions, as well as metabolic inhibitors, aggregating agents and aggregation inhibitors upon these parameters. A competitive binding technique using 3H labeled camp was used to determ ine the level of camp in intact platelets, and a high performance liquid chromatographic m ethod was developed to m easure the adenyl cyclase and phosphodiesterase activities in the platelet m em brane fraction. Although the availability of substrate adenosine triphosphate (ATP) correlated well with the amount of camp produced, and in turn the availability of camp seem ed to have a direct effect upon the reversibility of shape changes induced by the various stimuli, the only effect upon aggregation concerned the extent to which it occurred. No direct correlation of the level of camp with either the actual inhibition or activation of the aggregation m echanism was observed. Introduction bin, ep in ep h rin e, collagen, serotonin and adenosine diphosphate (ADP) have all been reported to be accompanied by a decrease in camp, either in the presence or absence of prostaglandin E t (PGE j).» ,23 However, Robison et al4,19 w ere unable to dem onstrate consistently a fall in camp from the normal baseline level in response to either ADP or epinephrine, and Droller and Wolfe5 reported an increase in platelet camp in response to throm bin. Similarly, an in /78/ $01.50 Institute for Clinical Science, Inc. T he p late le t camp level has been shown to parallel platelet aggregability. H ow ever, the im m ediate relationship b e tw e en p la te le t aggregability and cam P levels has not b e e n d efin ed. Platelet aggregation induced by throm * Address for reprint request: Clara V. Hussey, M.D., Departm ent of Pathology, The Medical College of Wisconsin, 8700 W. Wisconsin Avenue, Milwaukee, WI

2 404 W ANG, HUSSEY, SASSE AND FOBIAN crease in response to epinephrine was observed by Haslam and Taylor.8 Additional controversy is encountered regarding inhibitors of p latelet aggregation. Although the effects of inhibitors have been thought to be m ediated through the camp system, ,20 23,25 other factors have not b een ruled out.15,21 Thus, it remains uncertain as to w hether changes in the level of platelet camp directly effect aggregation or reflect secondarily the effects of certain aggregating agents and aggregation inhibitors. The purpose, therefore, of this study is to test the validity of the existence of a direct and causal relationship betw een various parameters of the camp system, e.g., the level of camp, adenyl cyclase activity, phosphodiesterase activity and platelet aggregation. To accomplish this, the effects of various environm ental conditions, metabolic inhibitors, aggregating agents and aggregation inhibitors upon the level of camp in intact platelets and adenyl cyclase and phosphodiesterase activities in the platelet membrane fraction are evaluated. In addition, the effects of several aggregation inhibitors on different aggregating agents are studied to establish w hether or not factors outside the camp system also contribute to platelet aggregation. Reagents and M aterials PGE! Prostaglandin E x (U-10136, Lot VDV-54) was kindly supplied.* It was prepared in 95 percent ethanol and used in the same volume as other aggregating inhibitors. The final concentration in all experiments was 1 X 10_5M unless otherwise specified. A g g r e g a t in g A g e n t s ADP (adenosine 5'-d ip h o sp h ate, sodium salt),f R F (bovine fibrinogen),! * Supplied by Dr. J. E. Pike of the Upjohn Co. f Sigma. throm bin, NaF (sodium fluoride) and ep in e p h rin e (adrenalin chloride, injection) I were prepared or diluted in 0.05M tris HC1 buffer in normal saline, ph 7.4 (tris buffer-saline) for aggregation studies. A g g r e g a t in g I n h ib it o r s ATP (adenosine 5'-triphosphate, disodium salt),f AMP (adenosine 5'-monophosphate, sodium salt),f theophylline,! NEM (N -ethyl m aleim ide),! salyrgan (m ersalyl sodium ),! KCN (potassium cy an id e), iodoacetate (sodium iodoacetate),! sodium azide! and ASA (acetyl salicylic acid)! w ere prepared in tris buffer-saline for aggregation studies. U n w a s h e d P l a t e l e t s Pooled platelet rich plasma for camp determ inations was prepared by centrifuging freshly drawn EDTA whole blood from a minimum of 10 individuals at 75 x g for 10 minutes. The final platelet counts were 3 to 5 x 108 per ml. W a s h e d P l a t e l e t s Platelets from 10 to 20 individual samples of freshly drawn ethylenediam ine tetraacetic acid (EDTA) w hole blood were pooled and then washed three times with citrated saline (0.38 percent sodium citrate in normal saline, ph adjusted to 6.5 with HC1). The washed packed platelets were either sonicated and used for measurem ent of adenyl cyclase and phosphodiesterase activities or resuspended in tris buffer-saline for use in the camp determination. The final platelet counts were adjusted to 3 to 5 x 108 per ml. Methods P l a t e l e t A g g r e g a t io n T e s t The preparation of unwashed platelets as well as the methodology of platelet aggregation have b e e n previously d e scribed.11,27 { Parke-Davis. Merck. II K&K Laboratories, Inc.

3 A d e n y l C y c l a s e A c t iv it y HUM AN PLA T E LE T AGGREGATION AND CAMP SYSTEM 405 Platelet membrane fraction. W ashed packed platelets from 100 ml of freshly drawn EDTA whole blood w ere resuspended in 0.5 ml of 0.05M tris-hcl buffer, ph 7.4, containing 0.015M EDTA. Precisely 4.0 ml of distilled water were added, and the cells w ere disrupted by sonification.* The resultant pellet was treated according to Wolfe et al29 and then resuspended in 3.0 ml of 0.05M tris-hcl buffer, ph 7.4. The protein content measured 7 to 9 mg per ml with a m icrobiuret m ethod.12 Incubation. The incubation mixture was prepared by mixing 0.1 ml of the previously described platelet preparation w ith 0.01 ml PGE t and the following compounds, all of which were prepared in the 0.05M tris-h Cl buffer: 0.02 ml of M gc l2-atp m ixture, 0.01 ml of theophylline and 0.01 ml of test reagent. The control contained 0.01 ml of the 0.05M tris-hcl buffer in place of the test reagent. The final concentrations were 2.5 mm ATP, 5.0 mm Mg++, 5.0 mm theophylline and 1 x 10_5M PG E!. The blank contained 0.02 ml of the 0.05M tris-hcl buffer in place of the M gcl2-atp mixture. After incubation at 37 C for 30 minutes, 0.05 ml each of 0.25M solutions of Z n S 0 4 and Ba(OH)2 was added to precipitate nucleotides other than cam P.14 High pressure liquid chrom atography was performed on ml of supernatant to determ ine the level of camp. Separation and quantitation of camp were performed with a liquid chrom atograph f using a 254 nm ultraviolet flow cell, SAX column, temperature of 23 C, inlet pressure of 700 psig, flow rate of 0.3 ml per min and mobile phase consisting of dilute H 2S 0 4, ph Cyclic AMP in concentrations as low as 100 p moles can be reliably detected under these conditions. The specificity of this m easurem ent of adenyl cyclase activ * Sonifier Cell Disruptor, Mode W185D, Heat System-Ultrasonics, Inc., Plainview, NY. t DuPont 820 Liquid Chromatograph. ity was dem onstrated by first precipitating the non-cyclic nucleotides with ZnSO*- Ba(OH)2 treatm ent and then separating camp from the nucleosides by chromatography. Adenyl cyclase activity was calculated from the camp produced following the enzym atic reaction. P h o s p h o d i e s t e r a s e A c t iv it y Enzym e preparation. W ashed packed platelets w ere sonicated and dialyzed according to Mills et al.17 The final preparation in 0.02M tris-hcl buffer, ph 7.4, contained about 5 mg per ml of protein. Adenyl cyclase activity could not be dem onstrated in this preparation. Incubation. The incubation mixture contained 0.4 ml of 0.02M tris-hcl buffer, ph 7.4, 0.1 ml of the protein preparation and 0.05 ml of test reagent prepared in the 0.02M tris-h Cl buffer. The control contained 0.05 ml of the 0.02M tris-hcl buffer in place of the test reagent. Exactly 0.5 ml of the M gcl2-camp mixture prepared in the 0.02M tris-hcl buffer was added to initiate the reaction. The final concentrations were l.omm camp and 5.0mM Mg++. The blank contained 0.5 ml of the 0.02M tris-h C l buffer in place of the M gcl2-camp mixture. After incubation at 37 C for 15 m inutes, 0.5 ml each of 0.25M solutions of Z n S 0 4 and B a(o H )2 was added and ml of the resu ltin g supernatant was used for camp determ i nation by liquid chromatography. The same chrom atographic operating conditions as for adenyl cyclase activity were observed. Phosphodiesterase activity was calculated from the decrease in camp following the enzym atic reaction. D e t e r m in a t io n o f camp L e v e l s in I n t a c t P l a t e l e t s P la telet extra cts. W ashed or u n w ashed p latelets from freshly draw n EDTA w hole blood were pooled and tested in 5.0 ml aliquots either with or without preincubation. The effect of various test reagents on these platelets was

4 406 W ANG, HUSSEY, SASSE AND FOBIAN studied by adding 2.0 ml of each prepared in tris buffer-saline to the platelets, followed by incubation at 37 C for 15 minutes, unless otherw ise specified. The samples were then centrifuged at 600 X g for 10 m inutes and washed once with citrated saline. After addition of 1.0 ml of 0.1N HC1, the platelet pellet was sonicated and then treated with 0.25 ml of trichloroacetic acid (final concentration 4 percent). The supernatant was extracted five times with two volumes of ether. The extract was then dried at 60 C and redissolved in 0.5 ml of sodium acetate/acetic acid, ph 4.0 (NaAc).6 camp assay. Reagent preparation and methodology followed the procedure of Gilman.6 The reaction mixture was prepared by adding 0.05 ml each of platelet extract, protein-kinase inhibitor preparation and 3HcAMP to 0.1 ml of NaAc. The reaction was initiated by the addition of 0.1 ml of binding protein. Optimal assay conditions w ere achieved with concentrations of 5 to 8 p m oles3 HcAMP, binding protein sufficient to bind approxim ately 30 percent of the nucleotide and a maximally effective concentration of proteinkinase inhibitor preparation. A liquid scin tillatio n spectrophotom eter* was used, and efficiency was approximately 37 percent. For standardization, several concentrations of camp ranging from 1 to 40 p moles per 0.05 ml in NaAc were treated as platelet extract and run with each experiment. A linear curve was obtained w hen bound radioactivity was p lo tted ag ain st th e co n cen tratio n of camp on a log-log scale. Results P l a t e l e t camp L e v e l s U n d e r V a r io u s C o n d it io n s * Packard Tri-Carb. The basal level of camp obtained from several e x p erim en ts was 12 to 15 p m oles/109 platelets. Platelets preincubated at 4 C for one hour demonstrated slightly elevated levels of camp, while those preincubated at 37 C for one hour e x h ib ite d slig h tly d e c reased levels. W ashed p late le ts dem o n strated high levels of camp. Incubation with P G E 1resulted in a marked elevation of the camp level, while incubation with theophylline caused only a slight elevation. C onversely, incubation with NaF resulted in a slight decrease in camp levels (figure 1). I n h ib i t o r s o f G l u c o s e M e t a b o l is m a n d t h e camp Sy s t e m In the presence of KCN, the camp level of intact washed platelets was reduced to 89 percent of that of control platelets, while iodoacetate reduced camp to 59 percent of the control level. In the presence of both KCN and iodoacetate, the level was markedly reduced to 18 percent of the control (table I). Similar results were obtained with unwashed platelets (figure 2). In order to determ ine w hether the decrease in camp resulted from decreased formation of its precursor, ATP, or from suppression of adenyl cyclase activity by the inhibitors of glucose metabolism, the effect of these inhibitors on adenyl cyclase activity was studied using the platelet m em brane fraction in the presence of abundant ATP. It was found that KCN exerted no effect w hile iodoacetate reduced adenyl cyclase activity to 69 percent of the control (table I). KCN and iodoacetate in combination did not inhibit activity m ore than iodoacetate alone. Study of p h o sp h o d ie stera se activity show ed that iodoacetate had no effect w hile KCN slightly stim ulated the en zyme activity (table I). A g g r e g a t i n g A g e n t s a n d T h e i r E f f e c t U p o n t h e P l a t e l e t cam P Sy s t e m in t h e P r e s e n c e o f P G E! T he effects of several aggregating agents, e.g., ADP, throm bin, N af and

5 HUMAN PLA TELET AGGREGATION AND CAMP SYSTEM 407 F i g u r e 1. A representative exam ple of p latelet camp levels under various conditions. U nwashed platelets were preincubated alone at 37 C or 4 C for one hour, or with test reagents at 37 C for 10 minutes; no pretreatm ent was conducted with control unw ashed or freshly washed platelets. Each value represents the average result of duplicate determinations performed on each of two separate experim ents using one batch of platelet preparation. The range of all determ inations (x 4) is indicated by vertical brackets. (Authors note: D ifferent batches of platelets reacted Control 4 C 37 C Theophylline PGE, Ix I0-5 M similarly, although their camp content varied.) NaF Washed Ixl0-J-M platelets bovine fibrinogen, on the camp level of intact platelets and the adenyl cyclase and p h o sp h o d ie stera se activ ities o f the platelet membrane fraction were studied (table II). ADP effected no change in the lev el of cam P in w ashed p late le ts. Thrombin, bovine fibrinogen and NaF decreased the level of camp to 91 percent, 79 percent and 60 percent, respectively, of the level in untreated washed platelets. Similar effects were observed in unw ashed platelets. H ow ever, w hile ADP and bovine fibrinogen exerted little or no effect upon adenyl cyclase activity, throm bin d e pressed it to 72 percent of the control value and NaF m arkedly increased it. Thus, no direct correlation betw een the effects of these agents on the camp level and adenyl cyclase activity was observed. In addition, no m ediation involving phosphodiesterase could be im plicated, as only NaF produced a change in its activity, decreasing it to 86 percent of the control level. In further studies with thrombin and heparin, adenyl cyclase activity was increasingly inhibited as the concentration of thrombin or heparin was increased. Inhibition was even more pronounced when heparin and throm bin w ere added together (figure 3). However, at the same tim e, heparin counteracted the aggregating effect of throm bin, which suggests that TABLE I Effects of Inhibitors of Glucose Metabolism on the camp Level of Intact Washed Platelets and the Adenyl Cyclase and Phosphodiesterase Activities of the Platelet Membrane Fraction camp L evel (P ercent) A denyl C yc la se A c t i v i t y (P ercent) Phosphod ie s t e r a s e A c t i v i t y (P ercent) Control KCN (1 x 10_ 3M) 89 ± ± ± 3 Iodoacetate 59 ± 7 69 ± ± 4 (4 x 10_3M) KCN (1 x 10_ 3M) + Iodoacetate (4 x 10 3M) 18 ± 5 68 ± ± 2 PGE^ (1 x 10 ^M) and theophylline (5 x 10~^M) were present in addition to the indicated inhibitor(s) in the assays of camp and adenyl cyclase activity. Each figure represents the average result and range of duplicate determinations performed on each of two separate experiments using one batch of platelet preparation.

6 408 WANG, HUSSEY, SASSE AND FO BIA N Control KCN lodoacetate Ix I0-3M 4 x I0-3M lodoacetate 4 x I O ' 3 M Control KCN lodoacete IxlO-^M 4 x I0 3M lo d o a ceta te 4x10'% F i g u r e 2. Comparison of the effects of inhibitors of glucose metabolism on camp levels in washed (A) and unw ashed (B) platelets. P G E t (1 x 10-5M) and theophylline (5 x 10~3M) were present in addition to the indicated inhibitor(s). Each value represents the average result of duplicate determ inations perform ed on each of two separate experiments using one batch of platelet preparation. The range of all determinations (x 4) is indicated by vertical brackets. the induction of platelet aggregation is not prim arily m ediated by suppression of adenyl cyclase activity. E f f e c t s o f A g g r e g a t io n I n h ib it o r s o n t h e P l a t e l e t c A M P Sy s t e m The effects of several aggregation inhibitors on the camp system were also studied. As shown in table III, AMP (3 x 10_3M), sodium azide (1 x 10-3M) and acetyl salicylic acid (1 X 10_3M) had no significant effect upon the level of camp, TABLE I I Effects of Aggregating Agents on the camp Level of Intact Washed Platelets and the Adenyl Cyclase and Phosphodiesterase Activities of the Platelet Membrane Fraction camp Level (Percent) Adenyl C yclase A c tiv ity (Percent) Phosphod ie stera se A c tiv ity (Percent) C o n tr o l ADP (1 X 1CT5M) 101 ± ± ± 2 Throm bin ( 0.5 U /ml) 91 ± 7 72 ± ± 2 B o v in e f i b r i n o g e n 79 ± 6 96 ± ± 3 (20 m g /d l) NaF (1 x 1CT2M) 6 0 ± 6 * 86 ± 3 PGE^ (1 x 1 0 " % ) an d t h e o p h y l l i n e (5 x 10 M) w e re p r e s e n t i n t h e a s s a y s o f camp a n d a d e n y l c y c l a s e, e x c e p t when NaF w as u s e d, s i n c e NaF a lo n e m a rk e d ly s ti m u l a t e d a d e n y l c y c l a s e a c t i v i t y ( * ). E a c h f i g u r e r e p r e s e n t s t h e a v e r a g e r e s u l t a n d r a n g e o f d u p l i c a t e d e t e r m i n a t i o n s p e rfo rm e d o n e a c h o f tw o s e p a r a t e e x p e r im e n ts u s in g o n e b a tc h o f p l a t e l e t p r e p a r a t i o n. adenyl cyclase activity and phosphodiesterase activity. Salyrgan, however, appeared to be a strong inhibitor of adenyl cyclse. It was also a m uch stronger inh ib ito r of p h o sp h o d ie stera se than theophylline which inhibited the enzyme activity by 44 percent at a concentration of 5 x 10_3M. In one experiment, salyrgan, at a concentration as low as 3 X 10-5M, inhibited phosphodiesterase by 36 percent. While NEM appeared to be as potent an inhibitor of adenyl cyclase as salyrgan, in contrast it did not exhibit the same marked inhibition of phosphodiesterase activity. These results could explain why, in addition to inhibiting adenyl cyclase activity, salyrgan nevertheless increased the camp level in intact platelets even though NEM drastically decreased it. However, it is also possible that the elevation of camp reflects an indirect rather than direct net effect of salyrgan on adenyl cyclase and phosphodiesterase, since salyrgan has been shown to penetrate effectively the platelet m em brane only gradually.1 E f f e c t s o f A g g r e g a t io n I n h ib it o r s o n D i f f e r e n t A g g r e g a t in g A g e n t s The effects of the various inhibitors on platelet aggregation induced by ADP,

7 HUM AN PLA TELET AGGREGATION AND CAMP SYSTEM 409 F i g u r e 3. Effects of thrombin and heparin in the presence of P G E j (1 x 10-5M) and theophylline (5 x 10-3M) on the adenyl cyclase activity of the platelet membrane fraction. The values are the average of duplicate determ inations perform ed on a single experiment. e p in e p h rin e, b ovine fib rin o g en and throm bin were also studied (table IV). W ithout exception, all of the inhibitors effectively suppressed the secondary phase of aggregation induced by these agents. However, their effect upon the primary p h ase of aggregation ran g ed from in hibitory to stimulatory depending upon the agent used for induction. No direct correlation of these results with any involvement of the camp system could be made (tables I, II, III and IV). D iscussion The level of camp in platelets varied considerably w hen platelets w ere exposed to different environm ental conditions. Washing and incubation at 4 C resulted in higher levels than incubation at room tem perature. In a previous study, it was observed by us that with washing, p latelets becam e irregularly shaped, pseudopods were extended, microtubules disappeared and the canalicular system enlarged.26,27 These same changes were also observed, although to a lesser degree, in platelets incubated in the cold. However, in each instance, increasing the level of camp restored the original disc shape of th e p latelets,27 although pro longed stimulation of the camp system resulted in depletion of the p latelet s energy resources.28 Although the m echanism of camp elevation in platelets subjected to cold tem peratures or w ashing TABLE I I I Effects of Inhibitors of Platelet Aggregation on the camp Level of Intact Washed Platelets and the Adenyl Cyclase and Phosphodiesterase Activities of the Platelet Membrane Fraction camp L e v e l (P e r c e n t) A d en yl C y c la s e A c t i v i t y (P e r c e n t) P h o sp h o - d i e s t e r a s e A c t i v i t y (P e r c e n t) Control AMP (3 x 10"3M) 106 ± 5 96 ± 3 70 ± 4 Sodium azide 91 ± ± ± 2 (1 x 10_ 3M) NEM (1 x 10-3M) 30 ± 4 28 ± 2 77 ± 3 Salyrgan 121 ± 7 35 ± 4 4 ± 2 (1 X 10"3M) ASA (1 x 1CT3M) 91 t 7 92 ± ± 3 PGE-l (1 x 10-5M) and theophylline (5 x 10 3M) were present in addition to the indicated inhibitor(s) in the assays of camp and adenyl cyclase activity. Each figure represents the average result and range of duplicate determinations performed on each of two separate experiments using one batch of platelet preparation.

8 410 W ANG, HUSSEY, SASSE AND FOBIAN has not been studied, the previous findings may indicate the existence of a type of defense mechanism which would supply the energy n e e d ed to restore norm al platelet shape. The hypothesis that the level of camp is dependent upon the amount of ATP is substantiated by the observation that, in combination, the inhibitors of both the aerobic and anaero b ic pathw ays of glycolysis m arkedly reduce the camp level, w hile separately they are only weakly effective. T hat N af m arkedly stimulates the adenyl cyclase activity of a broken cell preparation but decreases the camp level of intact cells m ight therefore be explained by inhibition of substrate ATP formation in intact platelets. This hypothesis is consistent with the results of an earlier study in which it was found that N af only d e p le ted p late le t glycogen stores to 9.6 ig from 10.0 /xg per 108 platelets w hile PG E j d ep leted the stores to 5.4 ju.g.27 However, our results with NaF are inconsistent w ith the finding of Robison et al19 that it elevated the camp level of intact platelets. No correlation betw een the level of camp and the individual activities of the aggregating agents could be drawn. Low concentrations of ADP demonstrate little effect upon the camp system. Although thrombin, bovine fibrinogen and NaF decrease the camp level of intact cells, this effect is probably not directly associated w ith any effect of these agents upon adenyl cyclase and phosphodiesterase. Upon closer evaluation, the fact that thrombin inhibits adenyl cyclase activity appears wholly unrelated to the role of thrombin in initiating aggregation, since heparin, w hich is also an inhibitor of adenyl cyclase, effectively suppresses aggregation induced by thrombin, even though it potentiates the inhibitory effect of throm bin upon adenyl cyclase. The finding that throm bin inhibits the adenyl cyclase activity of the platelet membrane, however, was not supported by Brodie et al.3 T he increase or decrease of camp levels in response to throm bin, epinephrine, collagen, ADP and serotonin has b een the subject of repeated controversy. TABLE IV Effects of Aggregation Inhibitors on the Primary and Secondary Phases of Platelet Aggregation Induced by Various Aggregating Agents F in a l ADP E p in e p h r in e BF T h rom bin C o n c e n tr a tio n x 1 0 ' 6M 1 x 1 0~ 4M 20 mg p e r d l 0.3 U/m l I n h i b i t o r s (M) AMP 1 X 10" ' KCN 1 X 10" X 10~2-44- Iodoacetate 4 X Theophylline 1 X io-3 4 4,4, Sodium azide 1 X 1er ' ASA 0.5 X 10-3 si Salyrgan 1 X io-4 si X NEM o 1 ) u 1 X 44-4,4,4. 1 X 10" H O1 t-t- 4' - 4,4,4, si 4 si i 4,4.4, si -t- - si + si 4 4,4,4, 4 4,4, sl 4-4,4,4. si 4-4 si ,4,4, ,4, si ,4,4, -t- 4- si 4-4~4 si 4 + si + 4, Inhibitors were added to unwashed platelets immediately before addition of the aggregating agents. Data represent the average of three or more separate experiments. The inhibitory (or stimulatory) effect was defined arbitrarily as no effect (-), slight (si! or sl+) if less than 10 percent, weak (4- or +) if between 10 and 20 percent, moderate (4-+ or ti) if between 20 and 50 percent, and strong (44-4 or +++) if between 50 and 100 percent of inhibition or enhancement.

9 HUMAN PLA TELET AGGREGATION AND C A M P SYSTEM These differences in opinion may merely reflect differences in experim ental conditions. Although a prim ary causal relationship betw een decreased levels of camp and aggregation cannot be established, it is possible that aggregating agents indirectly influence camp levels through the availability of ATP, as it has been proposed that aggregating agents increase ATP utilization.13,24,30 Compartmentalization of m etabolically active ATP9,10 appears to be an important factor in effecting the level of camp, and it is conceivable that the ATP pool in closest proximity to the adenyl cyclase should prim arily reflect these changes. C onsistent w ith the inability to correlate camp levels with the activity of various aggregating agents is the apparent lack of correlation betw een the camp level and the effects of various aggregation inhibitors. Although theophylline increases the level of camp while inhibiting platelet aggregation, its effect is an immediate one, and in this respect it resembles the majority of inhibitors. Salyrgan also increases the camp level, for it is a more potent phosphodiesterase inhibitor than theophylline. W hile salyrgan in h ib its A D P-induced prim ary aggregation, it nevertheless stim ulates primary aggregation induced by bovine fibrinogen. On the other hand, NEM decreases the camp level and strongly inhibits the aggregation of unwashed platelets with ADP, epinephrine and bovine fibrinogen, although at 1 x 10_3M it enhances throm bin-induced aggregation in u n w ashed platelets (table IV). Since this effect of NEM cannot be reproduced if w ashed p la te le ts su sp en d e d in tris buffer-saline are used, it appears that the enhancem ent may involve some constituent of plasma, i.e., one or more antithrombins. Indeed, it was demonstrated by the present authors that NEM (5 x 10-4M) inhibits antithrom bin II (unpublished data). It appears, therefore, that factors other than camp are also responsible for aggregation inhibition. In conclusion, the level of p latelet camp may become elevated, but only in response to experim entally induced alterations of normal platelet physiology. In addition, the camp level, especially in intact platelets, may also depend upon the availability of substrate ATP. No ev i dence, however, was found to support changes induced in the camp level by any of the aggregating agents as a primary cause of aggregation. Similarly, none of the aggregation inhibitors appeared to exert th e ir im m ediate effect through camp. Acknowledgment This investigation was supported by the Medical College of Wisconsin, Department of Pathology, Jon V. Straumfjord, M.D., Ph.D., Chairman. References 1. BEHNKE, O.: Effects of some chemicals on blood platelet microtubules, platelet shape and some platelet function in vitro. Scand. J. Haemat. 7: , BRINSON, K.: Effect of aminophylline on blood platelet reactions. Atherosclerosis 16: , Br o d ie, G. N., Ba e n zig e r, N. L., Chase, L. R., and MAJERUS, P. W.: The effects of thrombin on adenyl cyclase activity and a membrane protein from human platelets. J. Clin. Invest. 52:81-88, Co l e, B., Ro biso n, G. A., and H artman, R. C.: Studies on the role of cyclic AMP in platelet function, cyclic amp and cell function. Ann. N. Y. Acad. Sci. 185: , D r o l l e r, M. J. and Wo l f e, S. M.: Thrombininduced increase in intracellular cyclic 3', 5'- adenosine monophosphate in human platelets. J. Clin. Invest. 51: , Gil m a n, A. G.: A protein binding assay for adenosine 3', 5'-cyclic monophosphate. Proc. Nat. Acad. Sci. 67: , HASLAM, R. J.: Interaction of the pharmacological receptors of blood platelets with adenyl cyclase. Microvasc. Res. 6:255, H a slam, R. J. and Ta y l o r, A.: Effects of catecholamines on the formation of adenosine 3':5 -cyclic monophosphate in human blood platelets. Biochemistry 125: , HOLMSEN, H.: Changes in radioactivity of 32Plabeled acid-soluble organophosphates in blood platelets during collagen and adenosine diphosphate induced platelet aggregation. Scand. J. Clin. Lab. Invest. 17: , 1965.

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