DIFFERENTIAL INHIBITION BY LOW-DOSE ASPIRIN OF HUMAN VENOUS PROSTACYCLIN SYNTHESIS AND PLATELET THROMBOXANE SYNTHESIS

969

DIFFERENTIAL INHIBITION BY LOW-DOSE ASPIRIN OF HUMAN VENOUS PROSTACYCLIN

preoperatively, and the other 47 were asked to take a single dose of aspirin (40 mg, 81 mg, or 300 mg) either 14, 24, or 48 h

SYNTHESIS AND PLATELET THROMBOXANE

preoperatively. Immediately after operation the adventitious tissue was removed

SYNTHESIS

JANE BEVAN

S. P. HANLEY S. R. COCKBILL

S. HEPTINSTALL

Department of Medicine, University Hospital, Nottingham NG7 2UH The capacity of venous tissue for prostacyclin synthesis was determined in 68 patients undergoing surgery for removal of varicose veins. A single dose of aspirin (81 mg or 300 mg) taken 14 h preoperatively strongly inhibited its synthesis, and the effect of 300 mg was still evident 48 h after ingestion. A single dose of 40 mg aspirin taken 14 h preoperatively had no effect on prostacyclin synthesis. The capacity of blood platelets to synthesise thromboxane (measured as malondialdehyde) was

Summary

determined in volunteers before and

at

various times after

ingestion of 300 mg or 40 mg aspirin. Both doses had an inhibitory effect that lasted for at least 96 h. The length of time for which the amount of thromboxane synthesised was insufficient to support platelet aggregation and the platelet release reaction depended on both the donor and the dose of aspirin. If prostacyclin and thromboxane are important in the pathogenesis of thrombosis, then doses of aspirin much lower than those used previously should be tested. The long-lasting effect of 300 mg aspirin on both venous tissue and platelets indicates that this dose is unlikely to produce a favourable

prostacyclin/thromboxane balance. Introduction ASPIRIN inhibits the production in platelets of thromboxane A2 (TXA2), a material that can induce platelets to aggregate and release other pro-aggregatory materials from the cells. 1.4 By this action aspirin may have antithrombotic activity. Trials to test this hypothesis in stroke’-’ and myocardial infarction 7,8 have given conflicting or disappointing results, but we now know that aspirin not only prevents the formation of TXA2 in platelets but also inhibits the production of prostacyclin (PGI2) in the blood-vessel wall.9,10 The latter is a powerful inhibitor of platelet aggregation.9,11 If TXA2 and PGI2 production are both suppressed then the thrombotic process may not be modified. In-vivo studies in animals together with studies on platelets and cultured vascular cells from animals and man have indicated that the enzymes involved in TXA2 and PGI2 synthesis may not be suppressed equally by aspirin. The cyclo-oxygenase in vessel walls may be less sensitive to aspirin than is platelet cyclo-oxygenase,IO,12-15 and its effect on the vascular enzyme may be of shorter duration than its effect on the platelet enzyme.14,16-19 Such studies suggest that low doses of aspirin, or conventional doses of aspirin used infrequently, may have a beneficial effect on the prostacyclin/thromboxane balance in man. In the present study we have determined the effect of different doses of aspirin on venous PGI2 systhesis, on platelet thromboxane synthesis, and on platelet behaviour. The results suggest that a low dose of aspirin may be of value in thrombotic conditions, whereas the infrequent use of 300 :n2 aspirin may be of no value.

and the vein sectioned longitudinally. 6 mm discs were punched out and transferred to ice-cold 0 - 05 mol/1 Tris-HCl buffer (pH 7 · 5). For each determination two segments were then transferred to a tube containing platelet-free citrated plasma (160 µl) to which a solution of sodium arachidonate (22 µl) had been added (to give a final concentration of 1 mmol/1); the tube was placed in a 37°C water bath for 3 min, and duplicate volumes (10-60 µl) of the incubate were then analysed for PGlz by bioassay. This was achieved by determining their inhibitory effect on the adenosinediphosphate-induced aggregation of platelets in platelet-rich citrated plasma containing 0 -3mmol/1 aspirin to obviate the effect of any residual arachidonate transferred with the PGI2. The extent of aggregation was determined with the multichannel aggregometer described by Adams et al.20 and synthetic PGIwas used to construct standard curves immediately before the assay was carried out. Two sets of determinations were made on tissue from most patients and the mean result was obtained. The amount produced was expressed as ng PGIproduced by two vessel segments during the 3 min incubation. The capacity of the tissue for PGlz synthesis was determined within 3 h of operation. The material produced was judged to be PG12 because it inhibited platelet aggregation in the same way as synthetic PGlz and its formation was inhibited when the vessels were pre-incubated with aspirin or tranylcypromine. On five occasions the amount of PGlz determined with this bioassay was compared with the amount of 6-keto-PGFl a (the stable derivative of PGlz) produced. In these experiments 0 -3mmol/1 aspirin was added to the incubate at the end of the 3 min incubation, after removal of the samples for bioassay, to prevent any further PGlz production. The PGlz produced was converted to 6-ketoPGFI a by adjusting the pH of the sample to 6-0, and this was measured by radioimmunoassay. The respective amounts of PG12 and 6-keto-PGF1 a measured on five occasions were, 8 -5ng and 6 -88

ng, 3.4 ng and 3.4 ng, 4.3 ng and 5.7 ng; 0 ng and 1.38 ng, 2.4 ng and 3 - 4

Platelets were prepared from blood (18 ml) taken from volunteers before and then 2, 24, 48, 96, and 192 h after a single dose of aspirin (40 mg or 300 mg). On each occasion the blood was collected into two tubes that each contained 1 ml of 3 - 8% (w/v) trisodium citrate dihydrate and 6 µ1 of a solution of 14Cserotonin. The latter was obtained from the Radiochemical Centre, Amersham, and contained 50 µCi of 14C-serotonin/ml (specific activity 58 mCi/mmol). Platelet-rich plasma (PRP) was obtained by centrifuging the blood at 300 g for 10 min, and platelet-poor plasma (PPP) was obtained by recentrifuging the residual blood after the PRP had been removed. The PPP was used to dilute the PRP to give a final count of 3 ×1011 platelets/litre. The capacity of the platelets to synthesise TXAz was determined by measuring the amount of malondialdehyde (MDA) that was produced when the PRP was stirred with 1 mmol/1 sodium arachidonate. This was always expressed as nmol MDA produced by 109 platelets. The amount of 14C-serotonin released from the platelets during the procedure served as a measure of the extent of the platelet release reaction. This was always expressed as a percentage of the amount of 14C-serotonin that had been taken up by the platelets during their preparation. The experimental procedures and analytical techniques have been described in detail elsewhere. 21 =

Statistical Analysis The various treatments

68 patients admitted to hospital for removal of varicose veins were B;.!:d 21 of the

patients

were

asked

to

take

no

medicines

compared by

means

of Student’s

Results Vessel Studies

of PG12 generated by venous tissue from the who had been asked to take no medicines preoperatively ranged from 1-0 to 12 -0 ng. Differences in the amounts of PGIz produced did not relate to the age, weight, or sex of the patient. amounts

patients

Vessel Studies

were

t-test.

The

Patients and Methods

ng.

Platelet Studies

970 TABLE I-EFFECTS OF 300 MG ASPIRIN ON PLATELET MDA PRODUCTION (NMOL MDA/ 109 PLATELETS)

TABLE II-EFFECTS OF 40 MG ASPIRIN ON PLATELET MDA PRODUCTION (NMOL MDA/109 PLATELETS)

Fig. 1-Mean levels (±SEM) ofPGI2 produced by venous tissue from patients who had received no medication or had taken a single dose of aspirin 14 h preoperatively. n=no. of patients. The effects on PGI2 production of different single doses of aspirin taken 14 h preoperatively are shown in fig. 1. Whereas a dose of 300 mg or 81 mg aspirin taken 14 h earlier significantly reduced PGI2 production (p<0’001), 40 mg aspirin had no effect. The effects on PGI2 production of a single dose of 300 mg aspirin taken at various times before veins were removed are shown in fig. 2. PGI2 production was still significantly lower than in controls (p<0. 001) 14 h, 24 h, and 48 h after the had been ingested. aspirin

Platelet Studies The amounts of MDA generated by platelets before and after volunteers had ingested 300 mg or 40 mg aspirin are shown in tables I and II. Both doses reduced MDA production for at least 96 h. 300 mg aspirin reduced production to less than 2 ·6 nmol/109 platelets for either 48 h or 96 h. 40 mg aspirin reduced MDA production to less than 2 - 6 nmol/109 platelets for 96 h in donor I, 48 h in donor vi, and 24 h in donors II and vni. Platelets from donor Vn were relatively unaffected by 40 mg aspirin. The amounts of 14 C-serotonin released from the platelets are shown in tables III and IV. In general, either a large amount

of 14C-serotonin was released (>40%

of the 14C-serotonin that had been accumulated by the platelets) or none. Whenever 14C-serotonin was liberated the platelets aggregated in the sample tube. The release reaction occurred only when more than 2 6 nmol MDA was generated by the platelets. Discussion dose of aspirin that had no effect on PGI, this study was 40 mg. This dose was always sufficient to reduce platelet thromboxane synthesis, but the length of time for which the amount of thromboxane synthesised was insufficient to induce platelet aggregation and the platelet release reaction differed in different volunteers. Studies similar to our own but using small numbers of subjects and relatively high doses of aspirin 22,23 have shown that ingestion of 150-1500 mg aspirin inhibits PGI, synthesis for at least 8 h; thromboxane production was inhibited for at least 72 h. Our own study substantiates and extends these observations. It shows that venous PGI, production is still inhibited 48 h after ingestion of 300 mg aspirin whereas the effect of this dose on thromboxane production was still evident after 96 h. As a result the aggregation and release reaction that accompanies thromThe

only synthesis in

TABLE III-EFFECTS OF 300 MG ASPIRIN ON PLATELET RELEASE REACTION (% 14C-SEROTONIN RELEASED)

TABLE IV-EFFECTS OF 40 MG ASPIRIN ON PLATELET RELEASE REACTION (% 14C-SEROTONIN RELEASED)

Fig. 2-Mean levels (±SEM) of PGIproduced by venous tissue from patients who had received no medication or had taken a single dose of 300 mg aspirin at various times preoperatively. n=no. of patients.

971 boxane synthesis was completely inhibited for 48-96 h. The prolonged effect of 300 mg aspirin on both vessels and platelets indicates that this dose may have no value in thrombosis. Masotti et al. have studied the effect of ingested aspirin on PGI2 synthesis by measuring the level of6-keto-PGFQ’ (the stable derivative of PGI2) in whole blood after forearm ischaemia.24 They concluded that a dose of approximately 175 mg (2.55 mg/kg body weight) had no effect on PGI2 synthesis, whereas larger doses (3’5-10 mg/kg body weight) significantly inhibited PGI2 synthesis with full recovery by 48 h. The source of PGI2during forearm ischaemia is not known but perhaps it comes from arteries rather than veins. There is some evidence from studies in rats that the cyclooxygenase in arterial tissue may be less sensitive to aspirin than is the enzyme in venous tissue and that its effect on arterial tissue may be of shorter duration.z5 However, the opposite is seen in rabbits.26 On the other hand Baenziger et al. have shown that human vessel endothelial cells and human arterial smooth-muscle cells are similarly sensitive to aspirin in vitro." Viinikka and Ylikorkala were unable to detect an effect of ingested aspirin on the level of 6-keto-PGFra in

clotted blood.27 In the clinical studies that tested the ability of aspirin to prevent stroke and myocardial infarction the aspirin doses used ranged from 900 to 1300 mg daily.s8 Such doses will have inhibited both platelet and vascular cyclo-oxygenase activity, and this may explain why these trials failed to demonstrate a beneficial effect. Much lower doses need to be tested, but what dose and with what frequency? Hoogendijk and Ten Cate have shown that 40 mg aspirin taken daily can have a cumulative inhibitory effect on thromboxane synthesis.28 Perhaps long-term administration of 40 mg aspirin daily has a cumulative effect on PGIzsynthesis as well. This possibility is now being investigated in our laboratory. The person-to-person variation in response to 40 mg aspirin revealed in. the present study and also noted by orhers28,29 may also be important in determining the

appropriate treatment regimen. We thank Mr G. S. Makin and Mr B. R. Hopkinson for obtaining the surgical specimens; Dr J. Pike, of the Upjohn Co., for a gift ofPGI2; Dr M. J. Parry, of Pfizer Research Ltd., for carrying out the radioimmunoassays for 6-keto-PGF,a; and Prof. J. R. A. Mitchell for his encouragement and for reading the manuscript. The work was carried out with the aid ofa programme grant from the Medical Research Council.

Requests for reprints should be addressed to S.

P. H.

REFERENCES

1 Smith JB, Willis AL. Aspirin selectively inhibits prostaglandin production in human platelets. Nature 1971; 231: 235-37. 2 Roth GJ, Majerus PW. The mechanism of the effect of aspirin on human platelets. I. Acetylation ofa particulate fraction protein. J Clin Invest 1975; 56: 624-32. 3 Roth GJ, Stanford N, Majerus PW. Acetylation of prostaglandin synthase by aspirin. Proc Nat Acad Sci USA 1975, 72: 3073-76.

4 Hamberg M, SvenssonJ, Samuelsson B. A new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Nat Acad Sci USA 1975; 72: 2994-98.

5 Fields WS Lemak NA, Frankowski RF, Hardy RJ. Controlled trial of aspirin cerebral ischaemia. Stroke

1977; 8:

in

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5 Canadian Cooperative Study Group A randomised trial of aspirin and sulfinpyrazone in threatened stroke N Engl JMed 1978; 299: 53-59. Elwood PC, Sweetnam PM. Aspirin and secondary mortality after myocardial infarction Lancet 1979; ii: 1313-15. 8 Persantine-Aspirin Reinfarction Study Research Group. Persantine and Aspirin in coronary heart disease. Circulation 1980; 62: 449-61. 9 Moncada J, Higgs EA, Vane JR Human arterial and venous tissue generates prostacyclin, a potent inhibitor of platelet aggregation. Lancet 1977; i: 18-21. : Burch JW, Baenziger NL, Stanford N, Majerus PW Sensitivity of fatty acid cyclooxygenase from human aorta to acetylation of aspirin. Proc Nat Acad Sci USA 1978; 75: 5181-84 Moncada S, Gryglewski R, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation Nature 1976; 263: 663-65.

COLORECTAL CANCER AND

SCHISTOSOMIASIS CHEN MING-CHAI CHANG PEI-YU CHUANG CHI-YUAN CHEN YI-JEN WANG FU-PAN TANG YANG-CHUAN CHOU SHUN-CHUAN

Departments of Surgery and Pathology, Soochow Medical College Hospital, Soochow Ku 215006, People’s Republic of China The risk of colorectal cancer is known to be increased in patients with long-standing schistosomal colitis. A retrospective review of clinical data and surgical specimens from 60 patients with schistosomal granulomatous disease of the large intestine but without carcinoma demonstrated that 36 of them had mild to severe grades of colonic epithelial dysplasia. This was either focal or diffuse in distribution and occurred in flat mucosa, in pseudopolyps, or in regenerating epithelium at the edges of ulcers. These dysplastic changes are regarded as the pathological

Summary

basis for the malignant potential of schistosomal colitis, and they resemble the changes found in long-standing chronic ulcerative colitis. Introduction LONG-STANDING schistosomal colitis may predispose patients to the development of colorectal cancer.’ Colorectal cancer is one of the common malignancies seen in the People’s Republic of China.2 In some areas endemic for schistosomiasis, the prevalence rate for colorectal cancer is as

M, Dobranowski J, Gryglewski RJ. Prostacyclin and thromboxane generating in rabbits pretreated with aspirin Pharmacol Res Comm 1978; 10: 759-63. 13. Baenziger NL, Becherer PR, Majerus PW. Characterisation of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts. Cells 1979; 16: 967-74. 14. Ellis EF, Wright KF, Jones PS, Richardson DW, Ellis CK. Effect of oral aspirin dose on platelet aggregation and vascular prostacyclin (PGI2) synthesis in humans and rabbits. J Cardiovasc Pharmacol 1980; 2: 387-97. 15. Shaikh BS, Bott SJ, Demers LM. The differential inhibition of prostaglandin synthesis in platelets and vascular tissue in response to aspirin. Prostaglandins Med 1980; 4: 12. Basista

systems

439-47. 16. O’Brien JR. Effect of salicylates on human platelets. Lancet 1968; i: 779-83 17. Kocsis JJ, Hernandovich J, Silver MJ, Smith JB, Ingerman C. Duration of inhibition of platelet prostaglandin formation and aggregation by ingested aspirin or mdomethacin. Prostaglandins 1973; 3: 141-44. 18. Burch JW, Stanford N, Majerus PW. Inhibition of platelet prostaglandin synthetase by oral aspirin. J Clin Invest 1978; 61: 314-19. 19. Jaffe EA, Weksler BB. Recovery of endothelial cell prostacyclin production after inhibition by low doses of aspirin. J Clin Invest 1979; 63: 532-35. 20. Adams J, Heptinstall S, Mitchell JRA A six-channel automated platelet aggregometer. Thromb Diath Haemorrh 1975; 34: 821-24. 21. Heptinstall S, Bevan J, Coclbill SR, Hanley SP, Parry MJ. Effects of a selective inhibitor of thromboxane synthetase on human blood platelet behaviour. Thromb Res 1980; 20: 219-30. 22. Pareti FI, D’Angelo A, Mannucci PM, Smith JB. Platelets and the vessel wall: How much aspirin? Lancet 1980; i: 371-72. 23. Preston FE, Whipps S, Jackson CA, French AJ, Wyld PJ, Stoddard CJ. Inhibition of prostacyclin and platelet thromboxane A2 after low dose aspirin N EnglJ Med 1981; 304: 76-79 24. Masotti G, Galanti G, Poggesi L, Abbate R, Neri Serneri GG. Differential inhibition of prostacyclin production and platelet aggregation by aspirin. Lancet 1979; ii: 1213-16. 25. Villa S, Livio M, De Gaetano G. The inhibitory effect of aspirin on platelet and vascular prostaglandins in rats cannot be completely dissociated. Br J Haematol 1979; 42: 425-31. 26. Buchanan MR, Dejana E, Cazenave JP, Richardson M, Mustard JF, Hirsh J. Differences in inhibition of PGI2 production by aspirin in rabbit artery and vein segments. Thromb Res 1980; 20: 447-60. 27. Viinikka L, Ylikorkala O. Effect of various doses of acetylsalicylic acid in combination with dipyridamole on the balance between prostacyclin and thromboxane in human serum. Br J Pharmacol 1981; 72: 299-303 28. Hoogendijk EMG, Ten Cate JW Aspirin and platelets. Lancet 1980; i: 93-94. 29 O’Brien JR. Platelets and the vessel wall. How much aspirin? 1980; i: 372-73.