THROMBOSIS RESEARCH 13; 1089-1101 @pergamon Press Ltd.1978. printed

in Great Britain 00I+9-3848/:8,‘1201-1089

$02.00/o

EFFECT ON PLATELETACTIVITY OF INHIBITION OF ADENYLATECYCLASE E.W. Salzman, D.E. MacIntyre, M.L. Steer, and J. L. Gordon Department of Surgery, Harvard Medical School and Beth Israel Hospital, and A.R.C. Institute of Animal Physiology, Babraham, Boston, Ma., U.S.A., and University Department of Pathology, Cambridge, England

(Received 18.7.1978; in Accepted by Editor

revised form 22.9.1978. S. Niewiarowski)

ABSTRACT 5422536 (9-[tetrahydro-2-furyl]-adenine), an inhibitor of adenylate cyclase, blocks the inhibition of platelet function by substances that stimulate this enzyme, including PGEl, PGI2, PGD2, and adenosine. 5422536 enhances platelet aggregation and serotonin release induced by prostaglandin endoperoxides and their analogs but does not consistently affect the platelet response to ADP or other agonists. 5422536 reduces platelet cyclic AMP and inhibits the effect on this The elevation of cyclic GMP innucleotide of PGE and adenosine. duced by ADP is pievented by 5422536. INTRODUCTION Many inhibitors of platelet function increase the platelet level of cyclic AMP by stimulation of adenylate cyclase (e.g., prostaglandin El and D,, (l-3) or adenosine (4)) or by inhibition of platelet phosphodiesterase (e.g., papaverine (5)) dipyridamole (S), and methyl xanthines (5)). In contrast, a reduction in cyclic AMP accompanies platelet stimulation by many agents, including ADP, epinephrine, thrombin, collagen (6, 7), arachidonic acid (8), prostaglandin endoperoxide PGG2 (y), and physical stimuli such as stirring with kaolin powder or centrifugation (10). The decrement in cyclic AMP levels induced by these stimuli is greater in magnitude if the basal cyclic AMP concentration is first elevated by agents that activate adenylate cyclase or inhibit phosphodiesterase (11). These observations have suggested that cyclic AMP acts as a “second messenger” in platelets. The mechanism by which platelet cyclic AMP is reduced during platelet activation is not certain, although some agents (e.g., epinephrine, collagen, and thrombin) have been shown to inhibit platelet adenylate cyclase directly in broken cell preparations (2). Because the fall in basal levels of cyclic AMP accompanying platelet activation is inhibited by nonsteroidal antiinflammatory agents (12), an association with synthesis of prostaglandin endoperoxides is assumed. On the other hand, the reduction in platelet cyclic AMP from levels elevated by PGEl or adenosine is not blocked by aspirin or other 1089

1090

PLATELET

ADE?;kZ.ATE

CYCLXSE

~01.13,No.6

antiinflammatory drngs. It may therefore reflect a direct effect of platelet aggregating agents on adenylate syclase. The significance of these reduc,\gyregation and release can in platelet cyclic XI? is not certain. t ions occur while cyclic .MP is elevated above basal levels (13), so a low cyclic FurtherA?dP level does not seem to be essential for activation of platelets. in cyclic .&VPis not a sufficient stimulus to initiate platemore, reduction PGE, in low concentrations reduces cvclic A&IF let secretion or aggregation. activity, it does not initiate it. (14); although it enhances platelei Harris and associates (15) described a family of purine derivatives that inhibit lung adenylatc cyclase and observed that these substances prevent the rise in platelet cyclic .&VP induced by prostaglandin E (PGE ) and reverse WA havelstudied one of the inhibitory effect of PCEl on platelet function. SQ22536 (9- [tetrahydro-2-fury11 -adenine), and have found these substances, it to inhibit platelet adenylate cyclasc and to reverse the inhibition of platelet function by agents that stimulate adenylate cyclnse. We employed SQ22536 in experiments designed to investigate the effect of inhibition of adenylate cyclase on platelet function. METHOD&YD ?.t-\TERIALS Blood was collected by venipuncture from human volunteer donors or by vena caval puncture from anesthetized rats (16) and was anticoagulated with trisodium citrate 0.013 IV or acid-citrate (17) 6 vol to 3.J parts blood or Platelet rich plasma (PRP) and platelet porcine mucosal heparin 10 u/ml. poor plasma (PPF) were prepared by differential centrifugation as previously Incubation with [JH]-S-hydroxytryptamine and the release of described (2). labclled serotonin were assessed by methods previously reported (18). PlateIct aggregation was measured photometrically (19). Separation of platelets from PRP was performed by filtration through a column of Sepharose ZB gel according to Tangen et al. (20) as modified by Linden and associates (21). Cyclic ANP in platelet rich plasma ~3s measured by a modification of the method of Gilman (22), described in detail elsewhere (10). The rationale for expressing cyclic .&VP levels in tcrlns of the concentration of the nucleotide in platelet rich plasma has been discussed (10). Platelet adenylate cyclase activity was assayed by a modification of the method of Salomon et al. (23). Washed human platelets were frozen in liqgid For assay, the tubes were thawed by incubation at 30 C nitrogen until use. for 1.5 minutes. Test agents suspended in 0.05 ml doubly-distilled water were added and, after an additional minute at 30°C, the reaction was initiated by addition of 0.1 ml containing substrate, buffer, phosphodiesterase inhibiFinal concentrations of agents in the tor and an ATP-regenerating solution. assay mixture were Tris-HCl (pH 7.6), 30 mkl; b!gC12, S&l; a-[j2-PI-ATP (4050 CPM/pmole) , 0.25 m:!; pyruvate kinase, 0.3 units/ml; phosphoenolp,yruvate, After either 5 or 10 2.8 mN; caffeine, 33 mM; and protein, 0.25-0.30 mg/ml. was terminated by addiminutes at 30 C in a shaking water bath, the reaction tion of 0.1 ml containing 4 m&lATP and 1.4 mM CAMP in 2% sodium dodecyl sulfate. Recovery was monitored by addition of 0.05 nl containing S-10,000 CPF1of [3H]-C.&VP. Samples were placed in boiling water for 3 minutes and [32P]-ctilP counted after passage over Dowex and neutral alumina columns (24). Recovery was 70-80”; and duplicate samples gave results which varied no more than +5%. Under the conditions described, adenylate cyclase activity was linear for more than 20 minutes. Protein concentrations were measured according to the method of Lowry et al. (25) using bovine serum albumin as the standard,

Vol.

?3.XO.c;

PLATELET

XDEXYLXTE

TO?1

CYCLXSE

Change in platelet shape was studied by a modification of the method of Prostaglandins E and F were measured by a modification (10) of Born (26). Mafondialdehyde was assayed by a modificathe method of Jaffe et al. (27). tion (9) of the method of sfacfarlane and associates (28). Special reagents included stable prostaglandins, prostaglandin endoper11, [epoxymethano] prostadienoic oxide analog U-44069 (15 [S] hydroxy-9,, acid), and prostacyclin (PGI2) (Dr. John Pike, Upjohn Corp., Kalamazoo, l4ich.) , bovine Achilles tendon collagen (Sigma, St. Louis, Fio.) , bovine thrombin (thrombin topical, Parke-Davis, Detroit, :+lich.) , arachidonic acid (Nuchek, Elysian, Minn.), prostaglandin endoperoxide PGG2 (Prof. Bengt Samuelsson, Karolinska Institutet, Stockholm) and PGH2 (Dr. Robert Corman, Upjohn Corp., Kalamazoo, Mich.), endoperoxide analog Azo PGH2 (15 [S] hydroxy9 u, 11, [azo] prostadienoic acid) (Prof. E.J. Corey, Harvard Univ., Cambridge, Indianapolis, Ind.), and 2-n-amylthio AMP (Dr. K. Sias s . ) A23187 (Eli Lilly SQ22536 was obtained from Dr. D.N. Harris, Kikugaia, Kohjin Co., Japan). Squibb, Princeton, N.J. Other chemicals were reagent grade or better and were obtained from commercial sources. RESULTS Addition of SQ22536 10-6!M to 10W4Mto platelet rich plasma anticoagulated with citrate or heparin did not result in a change in platelet shape, platelet aggregation or release of radioactive serotonin, or generation of prostaglandins E or F or of malondialdehyde. Effect on inhibitors of platelet aggregation. Addition of SQ2253G 10 3, L to platelet rich plasma together with PGI2 markedly reduced the inhibition of platelet aggregation and secretion by the latter compound (Fig. 1).

ADP

5x!@M

0

FGI,

0.1

(0)

10-e M

L0.D. SQ 22-6 +PG12

(10)

0.2

(28)

0.3

lo-4M 4

COnild 1 min.

04

FIG. 1 Platelet aggre ation and secretion of serotonin induced in titrated PRP by ADP 5 x lo- %M. Numbers in parentheses in this and subsequent figures refer to percent release of [3H]-5-hydroxytryptamine. Addition of PGI2 10-&t one minute before ADP inhibited the response to ADP. In the presence of SQ22536 lo-4M, the inhibitory effect of PG12 was reduced. Time of addition of ADP is indicated by the injection artifact. SQ22536 also antagonized the inhibition of platelet activity by adenosine (9), In contrast, SQ22536 had no effect on the PGEl , and PGD2 (data not shown). inhibition of platelet aggregation and serotonin release by 2-n-amylthio-5*AMP (Fig. 2)) a more specific inhibitor of ADP-induced platelet activation

'092

PLAT'ELEX

ADETWLATE

Vol.

CYCLASE

13,Bo.F;

AMP is a weak stimulant to platelet adenylate (29) * Although 2-n-amylthio cyclase in broken cell preparations, its inhibitory action on platelets is probably due to competition for an ADP receptor rather than to stimulation of adenylate cyclase (29).

A00

R-At+=

2-n-amylthio-J’-mp

FIG. 2 Addition of 2-n-amylthio-S’-AMP (“R-AMP”) one minute before ADP inhibited The effect of 2-n-amylthio-S’-AMP was not affected the platelet response. by SQ22536 added at the same time or one minute before (data not shown).

Effect on agents that stimulate aggregation. In 1975 an extensive series and preliminary results of these of experiments with SQ22536 was undertaken, Over a narrow range of ADP concenstudies have been presented (8, 9, 30). trations at which there occurred a transition from “primary” aggregation to “second phase” aggregation and serotonin release (on the average, 1 x 10W6M to 3 x 10~%I), the platelet response was enhanced by the presence of SQ22536. SQ22536 also appeared to enhance platelet activation induced by collagen, arachidonic acid, thrombin, and epinephrine (8). Unfortunately, a second batch of the material furnished by the manufacturer in September, 1977, which was identical to the first batch in its effect on platelet adenylate cyclase (see below) and on reversal of the inhibitory action on platelet function of adenylate cyclase activators, failed to demonstrate the same degree of enhancement of platelet secretion or aggregation with these stimuli. Subsequently a third batch of SQ22536 was also inactive in this respect. he conclude that our original reports concerning ADP, epinephrine, collagen, thrombin, and arachidonic acid were incorrect, most likely because of a contaminant in the SQ22536 preparation (see Discussion). In contrast to the variability in the influence of different batches of SQ22536 on the platelet response induced by ADP, epinephrine, collagen, or arachidonic acid, enhancement of aggregation and release induced by addition of PGH2 to PRP. was a consistent finding with all batches of SQ22536 (Fig. 3).

Effect of SQ22536 on FIG. 3. platelet aggregation and serotonin release induced in citrate:! PRP by PCH,.

32

AOD. 33

34

0.5

Similar results were obtained with PGG2 (9). SQ22536 also enhanced PGH2-induced aggregation of gel filtered platelets suspended in Tyrode’s buffer (Fig. 4).

Gel-flltered

Effect of SQ22536 on FIG. 4. PGH2-induced aggregation of gel filtered platelets suspended in modified Tyrode’s buffer.

platelets

PGHz

031,~M

-01 SQ 22536

AOD -02

Similarly, platelet aggregation and serotonin secretion induced by the endoperoxide analog U-44069 n‘ere enhanced by SQZ2536 in human PRP (Fig. 5). Enhancement of the response to the endoperoxide analog Azo PGH2 was observed The platelet response to U-44069 was also tested in heparinized (Fig. 6). rat PRP, and augmentation of the effect by 3422536 was readily demonstrated (Fig. 7). Effect on platelet cyclic nucleotides. Addition of 5422536 to human platelet rich plasma led to a modest fall in cyclic A&IFlevels (Table I). SQ22536 reduced the elevation of cyclic AMP induced by prostaglandin PGEl or cyclase activity in adenos ine . SQ22536 reduced the basal level of adenylate

‘0?4

PLATELET

u-

ADENYLATE

CYCLME

0.3pM

Am

-0

con*o(

Vol.l3,No.6

PGHz

2 # lo-’ M

(0) - 0.1 A0.D. - 0.2

m3w -a3 so 22536 u9t.A - 0.4 :I I min.

FIG. 5

FIG. 6

Effect of SQ22536 on platelet aggregationand serotonin release induced in titrated PRP by U-44069 (5) or Azo PGH2 (6).

FIG. 7. Effect of SQ22536 on platelet aggregationand serotonin release induced in heparinizedrat PRP by U-44069.

platelets disrupted by freezing and thawing (Fig. 8). Adenylate cyclase activity reduced by epinephrinewas further decreased by SQ22536. Stimulation of platelet adenylate cyclase activity by PGEl or sodium fluoride was diminished in the presence of SQ22536. We measured cyclic G?+iP levels in titrated PRP before and after induction of platelet aggregationby ADP. In the presence of SQ22536, aggregationof platelets by ADP was not inhibited,but the usual rise in cyclic Gt4Pinduced

TXBLE I Cyclic

.-LIP, paoles/mi

;\denosine Control

PR.P TGZ, 5 s 1D-6 \,

5 x 10-o >I

._- _ 153+3.!

lS.O:D.9S

15.lO.W

SQ2’;36 10-l !.I 15.O+O.S6 Citrated platelet with SQ22536 101’ 8 enosine 5 s 10-6 bt, utes 0.1 vol of cold nixing, and assay of carried out.

69-2.1 _-cl for one ai:lutc rich plasm3 ~3s insub3ted at s or an equal volume of c’.lJS !I !;aCl and then xi:tfter thrc.e minor PGEl 3 x 10e6 !4 was added. 100”; trichloracetic acid xas added with l:igorous cyclic .UP by Gilman’s mztlio,i. (22) i;ns ti1en 15.5+0.7

Results shown are the me3n and S.D. of dGplicate assayed in triplicate. Platelets 63S,700/mn3.

OL, 0

001

samples,

i‘ach

0.1 M

22536

mM

FIG. S Effect of SQ27.536 on adenylate let preparation. For details,

cyclase activity see Xethods.

of

3 broken

plate-

a reduction in cyclic 041’ \