1213 DIFFERENTIAL INHIBITION OF PROSTACYCLIN PRODUCTION AND PLATELET AGGREGATION BY ASPIRIN* G. GALANTI G. MASOTTI R. ABBATE L POGGESI G. G. NERI SERNERI
Consiglio Nazionale delle Ricerche, Rome, and Istituto di Patologia Medica II dell’Università di Firenze, Florence, Italy
for at least 2 weeks, were investigated before and after a single dose of aspirin (2, 2-5, 3-5, 5, 8, and 10 mg/kg). Subjects were allotted to the various dosage groups of aspirin, by means of a list of random numbers. The subjects did not know which dose of aspirin they were taking. One to three tablets of 320 mg effervescent aspirin (Bayer) were dissolved in 1 dl of tapwater. Then a volume of solution, calculated according to the subjects’ weight, was made up to 1 dl with tap-water. PGI2 production, platelet aggregation, and malondialdehyde (MDA) formation were investigated before and 2, 12, 24, 48, and 72 h after aspirin administration.
PGI2Assay The effect of aspirin
ingestion on platelet aggregation, malondialdehyde (MDA) formation, and prostacyclin (PGI2) production by the blood-vessel wall was investigated in twenty-five healthy young volunteers. PGI2 production induced by ischæmia in the arm was assayed in venous blood. MDA formation and platelet aggregation induced by adenosine disphosphate (ADP), collagen, and adrenaline were inhibited by doses of aspirin smaller than those inhibiting prostacyclin production. The doses which inhibited 50% of platelet aggregation (ID 50) were 3·2 mg/kg for adrenaline, 3·4 for ADP, and 3·2 for collagen, whereas the ID 50 for prostacyclin production was 4·9 mg/kg. Inhibition of platelet aggregation increased only slightly with increasing doses, whereas inhibition of PGI2 production increased linearly up to 8 mg/kg. Inhibition of platelet aggregation and MDA formation was still present after 72 h, whereas prostacyclin inhibition reversed within 24 h in all subjects after 2, 3·5, and 5 mg/kg and in 6 out of 9 subjects after 8 and 10 mg/kg. These findings indicate that inhibition of platelet cyclo-oxygenase occurs with smaller doses of aspirin and lasts longer than inhibition of vessel-wall cyclo-oxygenase. 3·5 mg/kg is the dose of aspirin most likely to produce a consistent inhibition of platelet aggregation and only a slight inhibition of prostacyclin production. Summary
Introduction ASPIRIN strongly inhibits platelet aggregation by blocking cyclo-oxygenase.1-3 Endothelial cells in the vessel wall synthesise prostacyclin (PGI2),4-6 a substance which causes vasodilatation and potently inhibits platelet aggregation. Since synthesis of prostacyclin is also cyclo-oxygenase dependent, the use of aspirin as an antiaggregating agent for prevention of thrombosis might not be without risk, because aspirin inhibits not only thromboxane Az formation, but also prostacyclin production. Ischsemia of 3 min duration can induce PGIz release in circulating blood.7 Prostacyclin production by the vessel wall is measured in this way. We compared the effects of different doses of aspirin on platelet aggregation and PGIz production by vessel wall after
ischxmia.
Prostacyclin was assayed in venous blood by Vane’s "superfusion" technique3modified by Ferreira and De Souza Costa9 and by Bult et al.’° with bovine coronary artery (BCA), rat stomach strip (RSS), and chick rectum (CR) as test tissues. The tissues were connected to Harvard muscle transducers and contractions were recorded on a multichannel Rikadenki pen recorder. A solution containing indomethacin (1µg/ml), which blocked endogenous generation of endoperoxides and prostaglandins by the tissues, and a mixture of antagonists which rendered the tissues insensitive to acetylcholine, hydroxytryptamine, catecholamines, and histamine was used. The ratio of antagonist solution to test blood was 1/10. The tissues were first perfused with Krebs’ solution at a rate of 0-7ml/min until they were stabilised. Then the tissues were perfused with venous blood from the subject under study. As soon as the tissues had again reached a baseline, prostacyclin produced by the vessel wall after 3 minutes’ ischmmia of the forearm was assayed. Ischoemia was produced by inflating the cuff of the sphygmomanometer to 20 mm Hg above the systolic pressure of the subject. Prostacyclin production was expressed as PGI2 output (concentration in ng/ml multiplied by blood-flow in ml min-’ 100 g’ tissue). Blood-flow was measured by straingauge plethysmography. Measurements were made every 15 s and the total flow of the first minute was calculated by measuring the area under the curve. Blood supply to the test tissues was maintained at a rate of 0-7 ml/min by an infusion pump. This rate enables the draining of venous blood for 60-90 min without discomfort to the subject. The interval between the withdrawal of the blood from the vein and its arrival on the first tissue of the cascade was 60 s, this permitted the detection of highly unstable substances such as prostacyclin. Synthetic prostacyclin (kindly supplied by Dr J. R. Vane, Wellcome Research Laboratories, Beckenham, Kent) was used as a standard reference and infused in Krebs’ solution, in a concentration which produced a response in the test tissues to that observed in experimental procedures. The infusion of standard prostacyclin was maintained for one minute since prostacyclin is released almost exclusively during the first minute of post-ischoeniic hyperæmia.7
equivalent
Platelet Aggregation Platelet-rich plasma (PRP) was obtained from venous blood anticoagulated with trisodium citrate 3-8% (1/10) and centrifuged at 180 g for 10 min. Platelet aggretation was investigated in a Born apparatus. PRP was aggregated by ADP (2 µmol/l), by adrenaline (10 µmol/l), and by collagen (Stago) (5 Variations of optical density and percentage aggregacalculated. The occurrence of the second wave of aggregation was recorded.
pLg/ml).
tion
Materials and Methods
,
were
Subjects
Malondialdehyde Formation
Twenty-five young healthy volunteers (fourteen men and eleven women) aged 18-30 years, who had not taken aspirin
MDA production by platelet buttons from 1 ml of PRP stimulated by thrombin (5 U/ml) was assayed according to the method of Okuma et al." Prostacyclin, platelet aggregation and malondialdehyde were assayed by three different observers who did not compare results until the study was completed.
*Preliminary results were presented at the 5th International Congress on Thromboembolism, Bologna, May 29-June 2, 1978.
1214 Results
Effects of Aspirin on Platelet Aggregation and MDA Production MDA production was completely inhibited 2 h after the administration of 2 mg/kg of aspirin. Adrenaline and collagen-induced platelet aggregation were partially inhibited (23 and 30%) and ADP-induced aggregation was only slightly affected (table I). A dose of 2.5 mg/kg reduced ADP, adrenaline, and collagen induced platelet aggregation, by 25%, 35.6% and 35% respectively. The second wave of aggregation by ADP was inhibited in two out of four patients, and the second wave of aggregation by adrenaline was inhibited in all patients. The inhibition of platelet aggregation was almost maximum for all three agents 2 h after the administration of 3.5 mg/kg of aspirin. The doses necessary to inhibit platelet aggregation by 50% (ID 50) were 3.2 mg/kg for adrenalin, 3.4 mg/kg for ADP, and 3.5mg/kg for collagen (fig. 1). Further increase in the dose (5, 8, and 10 mg/kg) only provoked a slight increase in inhibition, which was not proportional to the increase in dose (fig. 1). Whatever dosage was used, aspirin never completely inhibited platelet aggregation induced by any of the aggregating agents. At a dosage of 3.5mg/kg the second wave of aggregation induced by adrenaline or ADP was inhibited in all patients. The duration of aspirin-induced inhibition of platelet aggregation varied with dose and
1—Effect of increasing doses of aspirin on prostacyclin production and platelet aggregation 2 h after administration.
Fig.
Mean values in different patient groups treated with doses of aspirin are given (SD shown in table I and II).
increasing
aggregating agent. Adrenaline-induced aggregation was longer than ADP and collagen induced aggregation (table I). 72 h after a 3.5mg/kg dose of aspirin, adrenaline-induced aggregationwas still inhibited by 35%, ADP-induced aggregation by 22.5%, and collageninduced aggregation by 18%. 72 h after a 5 mg/kg dose of aspirin, platelet aggregation was similarly inhibited (table i and fig. 2). 3.5mg/kg and 5 mg/kg of aspirin inhibited
TABLE I-EFFECTS OF INCREASING DOSES OF ASPIRIN ON PLATELET AGGREGATION AND MDA FORMATION
(PERCENTAGE ASPIRIN)
INHIBITION COMPARED WITH VALUES BEFORE
inhibited the second wave of adrenaline-induced aggreand 72 h (two out of four patients) and the second wave of aggregation induced by ADP for 24 and 48 h (two out of four subjects). MDA production was still markedly inhibited 72 h after 3.5-10 mg/kg (table
gation for 48
I).
Hours Fig.
2-Duration of effect of one dose of aspirin (5 mg/kg) on platelet aggregation induced by collagen, adrenaline, and ADP, and on production of prostacyclin and malondialdehyde
(MDA). Mean values of 4 cases
(SD in table i).
1215 TABLE II-EFFECTS OF INCREASING DOSES OF ASPIRIN ON OUTPUT IN
PROSTACYCLIN PRODUCTION INDUCED BY
ng/min)
(PROSTACYCLIN
3 MINUTES’
ISCHEMIA IN ARM
Effects of Aspirin on Prostacyclin Production by Ischæmia
Prostacyclin production induced by ischoemia was affected by aspirin only at doses higher than 2.5mg/kg. A dose of 2 mg/kg completely inhibited MDA production, but did not affect prostacyclin production (table n). 2 h after a dose of 3.55 mg/kg production of prostacyclin was inhibited by 29-61%, a dose of 5 mg/kg produced an average inhibition of 50-96%. Almost complete inhibition (90-07%) of PGIz production occurred after 8 mg/kg. The inhibition of prostacyclin is short-lasting. 24 h after 2, 3 - 5, and 5 mg/kg aspirin, PGIz had reverted to normal in all subjects (table i). Nevertheless, in three out of nine subjects treated with 8 and 10 mg/kg of aspirin, prostacyclin was still inhibited by 81-7%, 70%, and 65 - 8% (data not shown). However, 48 h after aspirin, prostacyclin production had returned to pretreatment values. Discussion
Aspirin inhibition of platelet aggregation
was
propor-
tional to doses in the range 2-5 mg/kg, but not for larger doses (8 and 10 mg/kg). These results differ from the observations in the rat12 but they accord with other studies in man. 13 Production of prostacyclin by the vessel wall, MDA formation by platelets, and platelet aggregation were differently affected by increasing doses of aspirin. Platelet cyclo-oxygenase proved particularly sensitive to the action of aspirin; MDA formation was inhibited by 100% and 95%, 2 and 24 h after the administration of 2 mg/kg of aspirin. At this dose platelet aggregation was poorly inhibited and PGI2 production by the vessel wall was unaffected. The different dosage of aspirin needed to produce a 50% inhibition of platelet aggregation and
prostacyclin production after ischsemia reflects the different effects of aspirin on platelet and vessel-wall cyclooxygenase-i.e., 3.2 mg/kg for adrenalin-induced aggregation, 3-4 mg/kg for ADP-induced aggregation, 335 mg/kg for collagen-induced aggregation, and 5 mg/kg for prostacyclin production. The degree of platelet cyclooxygenase inhibition obtained in the present study, as judged by MDA production, resembled that reported by Burch et al.14 who measured cyclo-oxygenase acetylation in platelets and found an inhibition of 82% 24 hours after a 160 mg dose of aspirin. However, the explanation for differences in inhibition of platelet and endothelial cyclo-oxygenase by aspirin may lie in drug distribution. Nevertheless, there is evidence that the aspirin sensitivity of cyclo-oxygenase varies with the tissues of origin. In cell cultures, platelet cyclo-oxygenase proved more sensitive to aspirin than vessel-wall cyclo-oxygenase.15 Prostaglandin synthesis in intact platelets in vitro was sixfold more sensitive than human synovium to aspirin. 16 Halushka et al.17 demonstrated that large doses (30 mg/kg intravenously) of aspirin in dogs completely block platelet aggregation and platelet arachidonic-acid metabolism but have no effect on myocardial synthesis of immunoreactive prostaglandin-E-like material. O’Grady and Moncada 18 demonstrated that low doses of aspirin prolong the bleeding-time in man; this accords with the view that low doses affect platelets without inhibiting prostacyclin formation. Burch et al.14 demonstrated that platelet cyclo-oxygenase is nearly 30 times more sensitive to aspirin than the cyclo-oxygenase from sheep seminal vesicles. The durations of inhibition of vessel-wall cyclo-oxygenase (assayed by the production ofPGI2), and of platelet cyclo-oxygenase (assessed by MDA production) were also remarkably different. Vessel-wall cyclo-oxygenase activity usually returned to normal 24 h after aspirin administration, whereas platelet cyclo-oxygenase was still inhibited 72 h after aspirin administration. Our results accord with the slow recovery of platelet cyclo-oxygenase observed by Roth et al. after aspirin, and with the findings of Moncada and Korbut’9 in rabbits, that vessel-wall cyclo-oxygenase quickly returns to normal. As the acetylation of cyclo-oxygenase by aspirin is irreversiblethe longer duration of inhibition of platelet cyclo-oxygenase compared with that of endothelial cyclo-oxygenase is likely to be due both to the inability of platelets to synthesise cyclo-oxygenase3and probably to the action of aspirin on megakaryocyte cyclo-oxygenase.’" Our results confirm that the metabolism of arachidonic acid via cyclo-oxygenase is not essential to the which can occur during complete inhibition of MDA. The arachidonic-acid pathway may modulate the reactions involved in platelet aggregation by modifying the threshold of aggregating stimuli or by potentiating the effect of aggregating agents via the release reaction caused by thromboxane formation. 14.20 Doses of 3-5 mg/kg of aspirin, given at 3-day intervals, seem to be able to induce maximum in-vivo inhibition of platelet aggregation without significantly affecting prostacyclin production by the vessel wall. Higher doses significantly inhibit PGIz prodution but only slightly increase inhibition of platelet aggregation. The identification of a dose of aspirin capable of giving the maximum of antiaggregating action and the minimum
platelet aggregation
1216
of prostacyclin inhibition could be useful in the prevenr tion of thrombosis. The administration of doses much greater than the optimum one could be the explanation for the poor results sometimes achieved in the prophylaxis of arterial thrombosis with aspirin. We thank the Wellcome Research Laboratories (Beckenham, Kent) for providing the standard prostacyclin. Requests for reprints should be addressed to G.M., Istituto di Patologia Medica II, Viale Morgagni 85, 50134 Firenze, Italy. REFERENCES 1. Smith JB, Willis AL. Aspirin selectively inhibits prostaglandin production in human platelets. Nature 1971; 231: 235-37. 2. Roth GJ, Stanford N, Majerus PW. Acetylation of prostaglandin synthetase by aspirin. Proc Natn Acad Sci U.S.A. 1975; 72: 3073-76. 3. Roth GJ, Majerus PW. The mechanism of the effect of aspirin in human platelets. J Clin Invest 1975; 56: 624-32. 4. Moncada S, Gryglewski RJ, 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. 5. Gryglewski RJ, Bunting S, Moncada S, Flower RJ, Vane JR. Arterial walls are protected against deposition of platelet thrombi by a substance (Prostaglandin X) which they make from prostaglandin endoperoxides. Prosta-
glandins 1976; 12: 685-711. 6. Moncada S, Higgs EA, Vane JR. Human arterial and venous tissues generate prostacyclin (prostaglandin X) a potent inhibitor of platelet aggregation. Lancet 1977; i: 18-20. 7. Neri Serneri GG, Masotti G, Poggesi L, Galanti G. Release of PGI2 in humans after local blood flow changes (post-ischemic hyperemia and venous stasis). Fifth International Congress on Thromboembolism,
JR. The use of isolated organs for detecting active substances in the circulating blood. Br J Pharm 1964; 23: 360-73. 9. Ferreira SH, De Souza Costa F. A laminar flow superfusion technique with much increased sensitivity for the detection of smooth muscle stimulatingsubstance. Eur J Pharmac 1976; 39: 379-81. 10. Bult H, Parnham MJ, Bonta IL. Bioassay by cascade superfusion using a highly sensitive laminar flow technique. J Pharm Pharmac 1977; 29: 8. Vane
369-70. 11. Okuma M, Steiner M, Baldini M. Studies on lipid peroxides in platelets. I. Method of assay and effect storage. J Lab Clin Med 1970; 75: 283-96. 12. Seuter F. Inhibition of platelet aggregation by acetylsalicylic acid on other inhibitors. Hæmostasis 1976; 5: 85-95. 13. Stuart RK. Platelet function studies in human beings receiving 300 mg of aspirin per day. J Lab Clin Med 1970; 75: 463-71. 14. Burch JW, Stanford N, Maherus PW. Inhibition of platelet prostaglandin synthetase by oral aspirin. Clin Invest 1978; 61: 314-19. 15. Baenziger NL. Dillender MJ, Majerus PW. Cultured human skin fibroblasts and arterial cells produce a labile platelet-inhibitory prostaglandin. Biochem Biophys Res commun 1977; 78: 301. 16. Patrono C, Ciabattoni G, Greco F, Grossi-Belloni D. Comparative evaluation of the inhibitory effects of aspirin like drugs on prostaglandin production by human platelets and synovial tissue. Adv Prostaglandin Throm-
boxaneRes 1976; 1: 125-31. 17. Halushka PV, Daniell HB, Miller WL, Thiboudeaux H. Increased coronary sinus prostaglandin E-like material (PGE) during myocardial infarction (MI): beneficial effects of aspirin (ASA). Clin Res 1977; 25: 225A. 18. O’Grady J, Moncada S. Aspirin: a paradoxical effect on bleeding time. Lancet 1978; ii: 780. 19. Moncada S, Korbut R. Dipyridamole and other phosphodiesterase inhibitors act as antithrombotic agents by potentiating endogenous prostacyclin. Lancet 1978; i: 1286-89. 20. Majerus PW. Why aspirin? Circulation 1976; 54: 357-59.
Bologna, May 29-June 2, 1978, p. 45 (Abstract).
Preliminary Communications EFFICACY OF IMMUNE PLASMA IN TREATMENT OF ARGENTINE HÆMORRHAGIC FEVER AND ASSOCIATION BETWEEN TREATMENT AND A LATE NEUROLOGICAL SYNDROME
JULIO I. MAIZTEGUI ALBA J.
DE
NESTOR J. FERNANDEZ DAMILANO
Instituto Nacional de Estudios sobre Virosis Hemorrágicas, 2700 Pergamino, Argentina
In a double-blind trial patients with Argentine hæmorrhagic fever treated with immune plasma within 8 days of the onset of the disease had a much lower mortality than those given normal plasma. Some patients treated with immune plasma developed late neurological complications.
Summary
INTRODUCTION
fevers produced by arenaviruses (Argentine, Bolivian, and Lassa fevers) are severe diseases associated with a high mortality, for which no specific preventive or therapeutic measures are yet available.’ The mortality of experimental infections with arenaviruses can be reduced with immune plasma,2-4 and clinical observations suggest that administration of immune plasma early in the course of the disease is useful. 5-7 However, the evidence for the beneficial effects of this form of treatment is generally considered inconclusive.8,9 We now report a double-blind study of the effects of immune plasma in the treatment of Argentine HÆMORRHAGIC
haemorrhagic fever (AHF). PATIENTS AND METHODS
All patients referred to our centre for treatment and study of AHF between 1974 and 1978 were evaluated by two experienced physicians. If they agreed on the clinical diagnosis of
AHF, the patient entered the study providing symptoms had
begun not more than 8 days before admission. 217 patients took part in the study. The patients were randomly allocated to receive intravenously 500 ml of either immune plasma (with antibodies against Junin virus, the aaiological agent of AHF) or normal plasma. The two types of plasma were distributed in identical plastic containers by independent observers, who also coded the units of plasma. They had no access to the patients, nor did the physicians know which plasma was being given to their patients. All the units of immune plasma used in this trial were obtained from donors convalescent from AHF, in whom a four-fold or greater rise in antibody titres had been demonstrated with complement-fixation tests. All units of normal plasma were obtained from donors without a history of AHF who were residents of Buenos Aires, a city located outside the endemic area of the disease. All units of plasma were tested serologically for Chagas’ disease, brucellosis, syphilis, and hepatitis B. To ensure a better evaluation of the effects of the two types of plasma, the rest of the treatment consisted of adequate hydration and few and uniform symptomatic measures. No steroids were used, and antibiotics were given only to patients with superimposed bacterial infections. For virological studies now in progress blood samples were drawn immediately before and 24 h after administration of plasma. Clinical signs and results of routine laboratory tests were recorded daily for each
patient. RESULTS
The diagnosis of AHF was confirmed in 188 cases by serological conversion in the survivors and by the isolation of Junin virus strains from the blood or necropsy specimens in patients who died. The results in these 188 patients show that immune plasma significantly reduces the mortality of AHF when it is given before the ninth day of the disease (table i). In a further 29 patients the clinical diagnosis of AHF was not confirmed virologically. 18 of these were treated with immune plasma and 11 with normal plasma. There were no deaths among the 18 receiving immune plasma, but 3 of the 11 treated with normal plasma died.
Consiglio Nazionale delle Ricerche, Rome, and Istituto di Patologia Medica II dell’Università di Firenze, Florence, Italy
for at least 2 weeks, were investigated before and after a single dose of aspirin (2, 2-5, 3-5, 5, 8, and 10 mg/kg). Subjects were allotted to the various dosage groups of aspirin, by means of a list of random numbers. The subjects did not know which dose of aspirin they were taking. One to three tablets of 320 mg effervescent aspirin (Bayer) were dissolved in 1 dl of tapwater. Then a volume of solution, calculated according to the subjects’ weight, was made up to 1 dl with tap-water. PGI2 production, platelet aggregation, and malondialdehyde (MDA) formation were investigated before and 2, 12, 24, 48, and 72 h after aspirin administration.
PGI2Assay The effect of aspirin
ingestion on platelet aggregation, malondialdehyde (MDA) formation, and prostacyclin (PGI2) production by the blood-vessel wall was investigated in twenty-five healthy young volunteers. PGI2 production induced by ischæmia in the arm was assayed in venous blood. MDA formation and platelet aggregation induced by adenosine disphosphate (ADP), collagen, and adrenaline were inhibited by doses of aspirin smaller than those inhibiting prostacyclin production. The doses which inhibited 50% of platelet aggregation (ID 50) were 3·2 mg/kg for adrenaline, 3·4 for ADP, and 3·2 for collagen, whereas the ID 50 for prostacyclin production was 4·9 mg/kg. Inhibition of platelet aggregation increased only slightly with increasing doses, whereas inhibition of PGI2 production increased linearly up to 8 mg/kg. Inhibition of platelet aggregation and MDA formation was still present after 72 h, whereas prostacyclin inhibition reversed within 24 h in all subjects after 2, 3·5, and 5 mg/kg and in 6 out of 9 subjects after 8 and 10 mg/kg. These findings indicate that inhibition of platelet cyclo-oxygenase occurs with smaller doses of aspirin and lasts longer than inhibition of vessel-wall cyclo-oxygenase. 3·5 mg/kg is the dose of aspirin most likely to produce a consistent inhibition of platelet aggregation and only a slight inhibition of prostacyclin production. Summary
Introduction ASPIRIN strongly inhibits platelet aggregation by blocking cyclo-oxygenase.1-3 Endothelial cells in the vessel wall synthesise prostacyclin (PGI2),4-6 a substance which causes vasodilatation and potently inhibits platelet aggregation. Since synthesis of prostacyclin is also cyclo-oxygenase dependent, the use of aspirin as an antiaggregating agent for prevention of thrombosis might not be without risk, because aspirin inhibits not only thromboxane Az formation, but also prostacyclin production. Ischsemia of 3 min duration can induce PGIz release in circulating blood.7 Prostacyclin production by the vessel wall is measured in this way. We compared the effects of different doses of aspirin on platelet aggregation and PGIz production by vessel wall after
ischxmia.
Prostacyclin was assayed in venous blood by Vane’s "superfusion" technique3modified by Ferreira and De Souza Costa9 and by Bult et al.’° with bovine coronary artery (BCA), rat stomach strip (RSS), and chick rectum (CR) as test tissues. The tissues were connected to Harvard muscle transducers and contractions were recorded on a multichannel Rikadenki pen recorder. A solution containing indomethacin (1µg/ml), which blocked endogenous generation of endoperoxides and prostaglandins by the tissues, and a mixture of antagonists which rendered the tissues insensitive to acetylcholine, hydroxytryptamine, catecholamines, and histamine was used. The ratio of antagonist solution to test blood was 1/10. The tissues were first perfused with Krebs’ solution at a rate of 0-7ml/min until they were stabilised. Then the tissues were perfused with venous blood from the subject under study. As soon as the tissues had again reached a baseline, prostacyclin produced by the vessel wall after 3 minutes’ ischmmia of the forearm was assayed. Ischoemia was produced by inflating the cuff of the sphygmomanometer to 20 mm Hg above the systolic pressure of the subject. Prostacyclin production was expressed as PGI2 output (concentration in ng/ml multiplied by blood-flow in ml min-’ 100 g’ tissue). Blood-flow was measured by straingauge plethysmography. Measurements were made every 15 s and the total flow of the first minute was calculated by measuring the area under the curve. Blood supply to the test tissues was maintained at a rate of 0-7 ml/min by an infusion pump. This rate enables the draining of venous blood for 60-90 min without discomfort to the subject. The interval between the withdrawal of the blood from the vein and its arrival on the first tissue of the cascade was 60 s, this permitted the detection of highly unstable substances such as prostacyclin. Synthetic prostacyclin (kindly supplied by Dr J. R. Vane, Wellcome Research Laboratories, Beckenham, Kent) was used as a standard reference and infused in Krebs’ solution, in a concentration which produced a response in the test tissues to that observed in experimental procedures. The infusion of standard prostacyclin was maintained for one minute since prostacyclin is released almost exclusively during the first minute of post-ischoeniic hyperæmia.7
equivalent
Platelet Aggregation Platelet-rich plasma (PRP) was obtained from venous blood anticoagulated with trisodium citrate 3-8% (1/10) and centrifuged at 180 g for 10 min. Platelet aggretation was investigated in a Born apparatus. PRP was aggregated by ADP (2 µmol/l), by adrenaline (10 µmol/l), and by collagen (Stago) (5 Variations of optical density and percentage aggregacalculated. The occurrence of the second wave of aggregation was recorded.
pLg/ml).
tion
Materials and Methods
,
were
Subjects
Malondialdehyde Formation
Twenty-five young healthy volunteers (fourteen men and eleven women) aged 18-30 years, who had not taken aspirin
MDA production by platelet buttons from 1 ml of PRP stimulated by thrombin (5 U/ml) was assayed according to the method of Okuma et al." Prostacyclin, platelet aggregation and malondialdehyde were assayed by three different observers who did not compare results until the study was completed.
*Preliminary results were presented at the 5th International Congress on Thromboembolism, Bologna, May 29-June 2, 1978.
1214 Results
Effects of Aspirin on Platelet Aggregation and MDA Production MDA production was completely inhibited 2 h after the administration of 2 mg/kg of aspirin. Adrenaline and collagen-induced platelet aggregation were partially inhibited (23 and 30%) and ADP-induced aggregation was only slightly affected (table I). A dose of 2.5 mg/kg reduced ADP, adrenaline, and collagen induced platelet aggregation, by 25%, 35.6% and 35% respectively. The second wave of aggregation by ADP was inhibited in two out of four patients, and the second wave of aggregation by adrenaline was inhibited in all patients. The inhibition of platelet aggregation was almost maximum for all three agents 2 h after the administration of 3.5 mg/kg of aspirin. The doses necessary to inhibit platelet aggregation by 50% (ID 50) were 3.2 mg/kg for adrenalin, 3.4 mg/kg for ADP, and 3.5mg/kg for collagen (fig. 1). Further increase in the dose (5, 8, and 10 mg/kg) only provoked a slight increase in inhibition, which was not proportional to the increase in dose (fig. 1). Whatever dosage was used, aspirin never completely inhibited platelet aggregation induced by any of the aggregating agents. At a dosage of 3.5mg/kg the second wave of aggregation induced by adrenaline or ADP was inhibited in all patients. The duration of aspirin-induced inhibition of platelet aggregation varied with dose and
1—Effect of increasing doses of aspirin on prostacyclin production and platelet aggregation 2 h after administration.
Fig.
Mean values in different patient groups treated with doses of aspirin are given (SD shown in table I and II).
increasing
aggregating agent. Adrenaline-induced aggregation was longer than ADP and collagen induced aggregation (table I). 72 h after a 3.5mg/kg dose of aspirin, adrenaline-induced aggregationwas still inhibited by 35%, ADP-induced aggregation by 22.5%, and collageninduced aggregation by 18%. 72 h after a 5 mg/kg dose of aspirin, platelet aggregation was similarly inhibited (table i and fig. 2). 3.5mg/kg and 5 mg/kg of aspirin inhibited
TABLE I-EFFECTS OF INCREASING DOSES OF ASPIRIN ON PLATELET AGGREGATION AND MDA FORMATION
(PERCENTAGE ASPIRIN)
INHIBITION COMPARED WITH VALUES BEFORE
inhibited the second wave of adrenaline-induced aggreand 72 h (two out of four patients) and the second wave of aggregation induced by ADP for 24 and 48 h (two out of four subjects). MDA production was still markedly inhibited 72 h after 3.5-10 mg/kg (table
gation for 48
I).
Hours Fig.
2-Duration of effect of one dose of aspirin (5 mg/kg) on platelet aggregation induced by collagen, adrenaline, and ADP, and on production of prostacyclin and malondialdehyde
(MDA). Mean values of 4 cases
(SD in table i).
1215 TABLE II-EFFECTS OF INCREASING DOSES OF ASPIRIN ON OUTPUT IN
PROSTACYCLIN PRODUCTION INDUCED BY
ng/min)
(PROSTACYCLIN
3 MINUTES’
ISCHEMIA IN ARM
Effects of Aspirin on Prostacyclin Production by Ischæmia
Prostacyclin production induced by ischoemia was affected by aspirin only at doses higher than 2.5mg/kg. A dose of 2 mg/kg completely inhibited MDA production, but did not affect prostacyclin production (table n). 2 h after a dose of 3.55 mg/kg production of prostacyclin was inhibited by 29-61%, a dose of 5 mg/kg produced an average inhibition of 50-96%. Almost complete inhibition (90-07%) of PGIz production occurred after 8 mg/kg. The inhibition of prostacyclin is short-lasting. 24 h after 2, 3 - 5, and 5 mg/kg aspirin, PGIz had reverted to normal in all subjects (table i). Nevertheless, in three out of nine subjects treated with 8 and 10 mg/kg of aspirin, prostacyclin was still inhibited by 81-7%, 70%, and 65 - 8% (data not shown). However, 48 h after aspirin, prostacyclin production had returned to pretreatment values. Discussion
Aspirin inhibition of platelet aggregation
was
propor-
tional to doses in the range 2-5 mg/kg, but not for larger doses (8 and 10 mg/kg). These results differ from the observations in the rat12 but they accord with other studies in man. 13 Production of prostacyclin by the vessel wall, MDA formation by platelets, and platelet aggregation were differently affected by increasing doses of aspirin. Platelet cyclo-oxygenase proved particularly sensitive to the action of aspirin; MDA formation was inhibited by 100% and 95%, 2 and 24 h after the administration of 2 mg/kg of aspirin. At this dose platelet aggregation was poorly inhibited and PGI2 production by the vessel wall was unaffected. The different dosage of aspirin needed to produce a 50% inhibition of platelet aggregation and
prostacyclin production after ischsemia reflects the different effects of aspirin on platelet and vessel-wall cyclooxygenase-i.e., 3.2 mg/kg for adrenalin-induced aggregation, 3-4 mg/kg for ADP-induced aggregation, 335 mg/kg for collagen-induced aggregation, and 5 mg/kg for prostacyclin production. The degree of platelet cyclooxygenase inhibition obtained in the present study, as judged by MDA production, resembled that reported by Burch et al.14 who measured cyclo-oxygenase acetylation in platelets and found an inhibition of 82% 24 hours after a 160 mg dose of aspirin. However, the explanation for differences in inhibition of platelet and endothelial cyclo-oxygenase by aspirin may lie in drug distribution. Nevertheless, there is evidence that the aspirin sensitivity of cyclo-oxygenase varies with the tissues of origin. In cell cultures, platelet cyclo-oxygenase proved more sensitive to aspirin than vessel-wall cyclo-oxygenase.15 Prostaglandin synthesis in intact platelets in vitro was sixfold more sensitive than human synovium to aspirin. 16 Halushka et al.17 demonstrated that large doses (30 mg/kg intravenously) of aspirin in dogs completely block platelet aggregation and platelet arachidonic-acid metabolism but have no effect on myocardial synthesis of immunoreactive prostaglandin-E-like material. O’Grady and Moncada 18 demonstrated that low doses of aspirin prolong the bleeding-time in man; this accords with the view that low doses affect platelets without inhibiting prostacyclin formation. Burch et al.14 demonstrated that platelet cyclo-oxygenase is nearly 30 times more sensitive to aspirin than the cyclo-oxygenase from sheep seminal vesicles. The durations of inhibition of vessel-wall cyclo-oxygenase (assayed by the production ofPGI2), and of platelet cyclo-oxygenase (assessed by MDA production) were also remarkably different. Vessel-wall cyclo-oxygenase activity usually returned to normal 24 h after aspirin administration, whereas platelet cyclo-oxygenase was still inhibited 72 h after aspirin administration. Our results accord with the slow recovery of platelet cyclo-oxygenase observed by Roth et al. after aspirin, and with the findings of Moncada and Korbut’9 in rabbits, that vessel-wall cyclo-oxygenase quickly returns to normal. As the acetylation of cyclo-oxygenase by aspirin is irreversiblethe longer duration of inhibition of platelet cyclo-oxygenase compared with that of endothelial cyclo-oxygenase is likely to be due both to the inability of platelets to synthesise cyclo-oxygenase3and probably to the action of aspirin on megakaryocyte cyclo-oxygenase.’" Our results confirm that the metabolism of arachidonic acid via cyclo-oxygenase is not essential to the which can occur during complete inhibition of MDA. The arachidonic-acid pathway may modulate the reactions involved in platelet aggregation by modifying the threshold of aggregating stimuli or by potentiating the effect of aggregating agents via the release reaction caused by thromboxane formation. 14.20 Doses of 3-5 mg/kg of aspirin, given at 3-day intervals, seem to be able to induce maximum in-vivo inhibition of platelet aggregation without significantly affecting prostacyclin production by the vessel wall. Higher doses significantly inhibit PGIz prodution but only slightly increase inhibition of platelet aggregation. The identification of a dose of aspirin capable of giving the maximum of antiaggregating action and the minimum
platelet aggregation
1216
of prostacyclin inhibition could be useful in the prevenr tion of thrombosis. The administration of doses much greater than the optimum one could be the explanation for the poor results sometimes achieved in the prophylaxis of arterial thrombosis with aspirin. We thank the Wellcome Research Laboratories (Beckenham, Kent) for providing the standard prostacyclin. Requests for reprints should be addressed to G.M., Istituto di Patologia Medica II, Viale Morgagni 85, 50134 Firenze, Italy. REFERENCES 1. Smith JB, Willis AL. Aspirin selectively inhibits prostaglandin production in human platelets. Nature 1971; 231: 235-37. 2. Roth GJ, Stanford N, Majerus PW. Acetylation of prostaglandin synthetase by aspirin. Proc Natn Acad Sci U.S.A. 1975; 72: 3073-76. 3. Roth GJ, Majerus PW. The mechanism of the effect of aspirin in human platelets. J Clin Invest 1975; 56: 624-32. 4. Moncada S, Gryglewski RJ, 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. 5. Gryglewski RJ, Bunting S, Moncada S, Flower RJ, Vane JR. Arterial walls are protected against deposition of platelet thrombi by a substance (Prostaglandin X) which they make from prostaglandin endoperoxides. Prosta-
glandins 1976; 12: 685-711. 6. Moncada S, Higgs EA, Vane JR. Human arterial and venous tissues generate prostacyclin (prostaglandin X) a potent inhibitor of platelet aggregation. Lancet 1977; i: 18-20. 7. Neri Serneri GG, Masotti G, Poggesi L, Galanti G. Release of PGI2 in humans after local blood flow changes (post-ischemic hyperemia and venous stasis). Fifth International Congress on Thromboembolism,
JR. The use of isolated organs for detecting active substances in the circulating blood. Br J Pharm 1964; 23: 360-73. 9. Ferreira SH, De Souza Costa F. A laminar flow superfusion technique with much increased sensitivity for the detection of smooth muscle stimulatingsubstance. Eur J Pharmac 1976; 39: 379-81. 10. Bult H, Parnham MJ, Bonta IL. Bioassay by cascade superfusion using a highly sensitive laminar flow technique. J Pharm Pharmac 1977; 29: 8. Vane
369-70. 11. Okuma M, Steiner M, Baldini M. Studies on lipid peroxides in platelets. I. Method of assay and effect storage. J Lab Clin Med 1970; 75: 283-96. 12. Seuter F. Inhibition of platelet aggregation by acetylsalicylic acid on other inhibitors. Hæmostasis 1976; 5: 85-95. 13. Stuart RK. Platelet function studies in human beings receiving 300 mg of aspirin per day. J Lab Clin Med 1970; 75: 463-71. 14. Burch JW, Stanford N, Maherus PW. Inhibition of platelet prostaglandin synthetase by oral aspirin. Clin Invest 1978; 61: 314-19. 15. Baenziger NL. Dillender MJ, Majerus PW. Cultured human skin fibroblasts and arterial cells produce a labile platelet-inhibitory prostaglandin. Biochem Biophys Res commun 1977; 78: 301. 16. Patrono C, Ciabattoni G, Greco F, Grossi-Belloni D. Comparative evaluation of the inhibitory effects of aspirin like drugs on prostaglandin production by human platelets and synovial tissue. Adv Prostaglandin Throm-
boxaneRes 1976; 1: 125-31. 17. Halushka PV, Daniell HB, Miller WL, Thiboudeaux H. Increased coronary sinus prostaglandin E-like material (PGE) during myocardial infarction (MI): beneficial effects of aspirin (ASA). Clin Res 1977; 25: 225A. 18. O’Grady J, Moncada S. Aspirin: a paradoxical effect on bleeding time. Lancet 1978; ii: 780. 19. Moncada S, Korbut R. Dipyridamole and other phosphodiesterase inhibitors act as antithrombotic agents by potentiating endogenous prostacyclin. Lancet 1978; i: 1286-89. 20. Majerus PW. Why aspirin? Circulation 1976; 54: 357-59.
Bologna, May 29-June 2, 1978, p. 45 (Abstract).
Preliminary Communications EFFICACY OF IMMUNE PLASMA IN TREATMENT OF ARGENTINE HÆMORRHAGIC FEVER AND ASSOCIATION BETWEEN TREATMENT AND A LATE NEUROLOGICAL SYNDROME
JULIO I. MAIZTEGUI ALBA J.
DE
NESTOR J. FERNANDEZ DAMILANO
Instituto Nacional de Estudios sobre Virosis Hemorrágicas, 2700 Pergamino, Argentina
In a double-blind trial patients with Argentine hæmorrhagic fever treated with immune plasma within 8 days of the onset of the disease had a much lower mortality than those given normal plasma. Some patients treated with immune plasma developed late neurological complications.
Summary
INTRODUCTION
fevers produced by arenaviruses (Argentine, Bolivian, and Lassa fevers) are severe diseases associated with a high mortality, for which no specific preventive or therapeutic measures are yet available.’ The mortality of experimental infections with arenaviruses can be reduced with immune plasma,2-4 and clinical observations suggest that administration of immune plasma early in the course of the disease is useful. 5-7 However, the evidence for the beneficial effects of this form of treatment is generally considered inconclusive.8,9 We now report a double-blind study of the effects of immune plasma in the treatment of Argentine HÆMORRHAGIC
haemorrhagic fever (AHF). PATIENTS AND METHODS
All patients referred to our centre for treatment and study of AHF between 1974 and 1978 were evaluated by two experienced physicians. If they agreed on the clinical diagnosis of
AHF, the patient entered the study providing symptoms had
begun not more than 8 days before admission. 217 patients took part in the study. The patients were randomly allocated to receive intravenously 500 ml of either immune plasma (with antibodies against Junin virus, the aaiological agent of AHF) or normal plasma. The two types of plasma were distributed in identical plastic containers by independent observers, who also coded the units of plasma. They had no access to the patients, nor did the physicians know which plasma was being given to their patients. All the units of immune plasma used in this trial were obtained from donors convalescent from AHF, in whom a four-fold or greater rise in antibody titres had been demonstrated with complement-fixation tests. All units of normal plasma were obtained from donors without a history of AHF who were residents of Buenos Aires, a city located outside the endemic area of the disease. All units of plasma were tested serologically for Chagas’ disease, brucellosis, syphilis, and hepatitis B. To ensure a better evaluation of the effects of the two types of plasma, the rest of the treatment consisted of adequate hydration and few and uniform symptomatic measures. No steroids were used, and antibiotics were given only to patients with superimposed bacterial infections. For virological studies now in progress blood samples were drawn immediately before and 24 h after administration of plasma. Clinical signs and results of routine laboratory tests were recorded daily for each
patient. RESULTS
The diagnosis of AHF was confirmed in 188 cases by serological conversion in the survivors and by the isolation of Junin virus strains from the blood or necropsy specimens in patients who died. The results in these 188 patients show that immune plasma significantly reduces the mortality of AHF when it is given before the ninth day of the disease (table i). In a further 29 patients the clinical diagnosis of AHF was not confirmed virologically. 18 of these were treated with immune plasma and 11 with normal plasma. There were no deaths among the 18 receiving immune plasma, but 3 of the 11 treated with normal plasma died.
