Decreased serum levels of a factor stimulating prostacyclin synthesis in acute myocardial infarction

ProstaglandinsLeukotrienesand Medicine 16: 389-394,1984

DECREASED SERUM LEVELS OF A FACTOR STIMULATING PROSTACYCLIN SYNTHESIS IN ACUTE MYOCARDIAL INFARCTION Yoshiki Yui, Yoshiki Takatsu, Ryuichi Hattori, Keiji Sakaguchi, Takashi Susawa, Natsuko Ikeda, Chuichi Kawai Third Division, Department of Internal medicine, Faculty of Medicine, Kyoto University, Kyoto 606, Japan (Reprint requests to CK) Abstract To investigate the role of a factor in serum stimulating prostacyclin synthesis in acute myocardial infarction, we compared the activities of the factor among 7 patients with acute myocardial infarction(2.540.8 hrs and 80+17 hrs after the onset of symptoms), 12 patients with angina pectoris, and 7 normal subjects. In this study, we found that in patients with acute myocardial infarction, this activity is much lower immediately after infarction (2.5iO.8 hrs) than 80+17 hrs later, or in patients with stable angina pectoris, or in healthy volunteers. Since prostacyclin is antiaggregating and vasodilating, the deficiency in this ability during the very early phase of acute myocardial infarction may be related to the development of the thrombosis of acute myocardial infarction. Introduction Prostacyclin(PG12) is a biologically active prostaglandin which has platelet antiaggregating and vasodilating properties(l,2). It is synthesized in the vessel wall, but the regulatory mechanisms controlling production of prostacyclin in vivo are not known.

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Recently, some investigators reported that unknown circulating factor(s) in serum or plasma might regulate prostacyclin production through the stimulation of the vascular prostacyclin synthesis(3,4,5). A marked decrease in this activity in plasma has been found in the hemolytic uremic syndrome(3) and in sickle cell anemia(4). In this report, we present data demonstrating that during the very early phase of myocardial infarction, patients have a marked decrease in a factor in the serum necessary for stimulating vascular prostacyclin production. Methods Patient PopuLation The study group consisted of seven patientsfaged 4755 years) with acute myocardial infarction. The diagnosis of acute myocardial infarction was based on the acute onset of chest pain lasting for more than 30 minutes and persistent ST segment elevation lasting for more than 30 minutes and progression to new Qwaves of greater than 0.04 second duration in the standard 12 lead electrocardiogram. Diagnosis was subsequently confirmed by electrocardiographic evolutionary changes and positive creatine kinase-MB determinations. The first sampling time from the onset of symptoms was 2.520.8 hrs. The diagnosis of angina pectoris was made by both clinical symptoms and coronary arteriograms which revealed more than 50% reduction in luminal diameter in one or more major coronary arteries. Serum samples were obtained from the pulmonary arteries through a Swan-Ganz catheter. Samples from healthy volunteers and patients with stable angina pectoris were obtained from the antecubital vein. Evaluation of Serum Stimulation of Vascular Prostacyclin Synthesis(6) Wistar rats weighing about 200g were sacrificed by a blow to the neck. The aorta was quickly dissected, trimmed of fat and connective tissues and cut into rings 2 mm in width under a binocular microscope. These aortic rings were incubated in 30 ml of 50 mM Tris-HCl buffer at pH 7.4 for 30 minutes; the buffer was then renewed. This incubation was repeated six times for three hours.

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The "exhausted" vascular rings were then incubated for one hour at 37OC with 200 ul serum. Normal saline was used as a control. Under these conditions, human serum stimulated the "exhausted" vascular rings to generate prostacyclin. Prostacyclin concentration was determined by the radioimmunoassay of 6-keto-prostaglandin Fl . Sample purification was performed by the reverse p Rase column. After the preconditioning of a Sep-Pak column (Waters C-18 reverse phase column) by 10 ml of distilled water and 20 ml of ethanol, 1.0 ml of diluted sample acidified by 100 ul of 2 N HCl was applied to the column. Step-wise elution was performed by the following procedures: washing with 5 ml of distilled water, washing with 5 ml of 5% ethanol, washing with 5 ml of petroleum ether and the final elution with 5 ml of ethyl acetate. The final eluent was centrifuge-evaporated to dryness at 50°C and resuspended in 150 ~1 of 0.1 M phosphate-buffered saline(0.9%), pH 7.4, with gelatin (0.1%); 100 ul of this solution was incubated with antiserum and tritiated 6-keto-prostaglandin Fla. The final assay volume was 0.5 ml. The mixture was incubated for 16 hrs at 4OC. Antibody-bound 6-keto-prostaglandin Fdpfwas separated from the ml of dextran-coated charcoal unbound compound with (mixture of 3.75 mg of dextran and 37.5 mg of charcoal per ml) by centrifugation at 1,000 x g for 10 minutes, and the amount of antibody-bound 6-keto-prostaglandin F in the supernatant was determined. Validation Ola this assay was obtained by dilution and recovery studies. All assays had a 80% recovery. The sensitivity of the assay was 100 pg per ml of sample. The cross-reactivity of this antibody was as follows: 6-ketoprostaglandin F lalOO%, prostaglandin A,, A2, ~a:O~~~2~:o~~~~;a~~~~~~ ~h~~~b~~~~~a~?andin E 1.2%, p ostaglandin Fl 1.9% and prostaglandin2F2, 2.6%. I 5HI 6-keto-prospaglandin F 2a (120-180 Ci/mmol) was obtained from New England Nut ear Corporation. Authentic 6-keto-prostaglandin Fl was supplied by the Ono Pharmnaceutical Company. The &ugs used were of the highest grade. Statistics Values are presented as mean+SD. A multiple comparison test

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with one way analysis of variance was used. A p value less than 0.05 was considered significant. Result Figure 1 shows that prostacyclin generation in the very early phase of acute myocardial infarction(l5+15 ng/mg/hr 6-keto-PG F,,) was significantly lower than 80217 hrs after the onset(68+20 ng/mg/hr). Values at 80+17 hrs were not significantly different from those of patients with stable angina pectoris(61+31 ng/mg/hr), but both levels were significantly lowere than those of volunteers(ll7+48 ng/mg/hr).

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Figure 1: Ability of prostacyclin production by serums among control(C), acute myocardial infarction (AMI), and angina pectoris. Prostacyclin generation was expressed as 6-keto-prostaglandin F production (ng/mg wet weight of ring/hr I”. (#, P
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Discussion Recently, for the treatment of acute myocardial infarction, a percutaneous transluminal coronary reperfusion technique has been employed(7) and coronary artery thrombosis and vasospasm have been found to play an important role in the pathogenesis of acute myocardial infarction. In the vessel wall, arachidonic acid is converted to prostacyclin, an unstable vasodilator and antiaggregating substance(l). Prostacyclin is the natural and main defence of the vessel wall against deposition of platelet aggregates. On the other hand, in platelets, arachidonic acid is converted to thromboxane A2, an unstable vasoconstrictor and platelet aggregating substance. Prostacyclin and thromboxane A2 are believed to constitute an important homeostatic mechanism for the regulation of platelet aggregability and coronary arterial tone. The manipulation of this balance may affect thrombus formation and vasospasm. Therefore, the administration of selective thromboxane A2 inhibiters(8) or intravenous prostacyclin infusion(9) is now under investigation. Several workers have reported the existence of unknown factor(s) in the serum or plasma which might regulate the synthesis of prostacyclin in the vascular endothelium(3,4,5). In the hemolytic uremic syndrome(3) and in sickle cell anemia(41, a significant decrease of this factor in plasma has been reported. The microvacular and thrombotic complications seen in these disorders may be closely related to this decrease. In this study, we found that in the very early phase of myocardial infarction, the activity of stimulating prostacyclin synthesis in serum was significantly decreased. The significant decrease of such a factor for prostacyclin production in serum may help to explain coronary arterial thrombus formation and spasm. Acknowledgment We express our appreciation to Dr.Alice S. Cary for her help with the preparation of the manuscript. References (I) Vane JR. Prostaglandins and the cardiovascular system. Br Heart J 49:405,1983 (2) Yui Y, Nakajima H, Kawai C, Murakami T. Prostacyclin therapy in patients with congestive heart failure. Amer J cardiol 50:320,1982

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(3) Remuzzi G, Marchesi D, Mecca G, Misiani R, Livio M, De gaetano G. Hemolytic-uremic syndrome: deficiency of plasma factor(s) regulating prostacyclin activity(?). Lancet ii:871,1978 (4) Stuart MJ, Sills RH. Deficiency of plasma prostacyclin or PGI2 regenerating ability in sickle cell anemia. Br J Hematol 48:545,1981 (5) Seid JM, Jones PBB, Russell GG. The presence in normal plasma, serum and platelets of factors that stimulate the production of prostacyclin(PGI2) by cultured endothelial cells. Clinical Science 64:387, 1983 (6) Senzaki S, Sugiyama T, Kanaji K, Oukuma M, Uchino Y. Prostacyclin-producing factor in plasma. Clinical Hematology (abstract in Japanese) p.233,1983 (7) Rentrop P, Blanke H, Karsch KR, Kaiser H, Kostering H, Leitz K. Selective intracoronary thrombolysis in acute myocardial infarction and unstable angina pectoris. Circulation 63:307,1983 (8) Yui Y, Hattori R, Takatsu_ Y, Nakajima H, Wakabayashi A, Kawai C, Kayama N, Hiraku S, Inagawa T, Tsubojima M, Naito J. Intravenous infusion of a selective inhibitor of thromboxane A2 synthetase in man: Influence on thromboxane B2, 6-keto-prostaglandin FICX' and platelet aggregation. Circulation, in press(October issue),1984 (9) Chierchia S, Patron0 C, Crea F, Ciabattoni G, De Caterina R, Cinotti GA, Distante A, Maseri A. Effects of intravenous prOStaCyClin in variant angina. 65:470,1982 Circulation

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