Acetylation by Aspirin (Prostaglandin Synthase/Prostacyclin/Thromboxane A2/Human Platelets/Acetylsalicylic Acid) JOHN W

Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 5181-5184, October 1978 Medical Sciences Sensitivity of fatty acid cyclooxygenase from human aorta to acetylation by aspirin (prostaglandin synthase/prostacyclin/thromboxane A2/human platelets/acetylsalicylic acid) JOHN W. BURCH, NANCY LEWIS BAENZIGER, NANCY STANFORD, AND PHILIP W. MAJERUS* Division of Hematology-Oncology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 Communicated by P. Roy Vagelos, July 28, 1978 ABSTRACT The rate of acetylation of fatty acid cyclooxy- from Nu Chek Prep, Elysian, MN. Nonidet P40 was purchased genase (prostaglandin synthase, EC 1.14.99.1) by [acetyl-3H- from Particle Data Laboratories, Elmhurst, IL. aspirin was measured in microsomes from human aortas and Indomethacin; coronary arteries and intact and disrupted human platelets. We bovine hemoglobin, type I; reduced glutathione; and epi- also measured the inhibition by aspirin of prostacyclin gener- nephrine bitartrate were purchased from Sigma Chemical Co., ation from exogenous arachidonic acid in shredded human St. Louis, MO. All other chemicals were reagent grade. aorta. Cyclooxygenase in human aorta and coronary artery Acetylation of Vascular Microsomes. Frozen aortas or microsomes is approximately 1/250th as sensitive to aspirin as coronary arteries were shredded with a hand-held enzyme in intact platelets, and 1/60th as sensitive to aspirin as vegetable enzyme measured in a platelet microsomal preparation. On the shredder or crushed with a hammer. Care was taken to keep basis of the in vitro data presented, we predict that small oral the tissues frozen (i.e., in contact with dry ice) during these doses of aspirin are sufficient to inhibit platelet prostaglandin procedures. The shredded or broken tissue was homogenized production but are not sufficient to substantialFy affect aorta in ice-cold 0.154 M KCl, 10 ml per g of tissue, with a Polytron or coronary artery prostaglandin production. homogenizer (five 1-min bursts at setting 7). The resulting homogenate was centrifuged 5 min at 4400 g. The supernate Inhibition of prostaglandin and thromboxane synthesis in was filtered cheesecloth 'and a platelets interferes with platelet function, as measured in Vvo through particulate fraction (1) or in vitro (2). This finding has led to clinical trials to de- containing microsomes was prepared by centrifugation at termine whether inhibitors of fatty acid cyclooxygenase 180,000 X g (max) for 30 min. The pellet of microsomes was (prostaglandin synthase, EC 1.14.99.1), the enzyme responsible rinsed with distilled water and resuspended by brief homoge- for the conversion of arachidonic acid to prostaglandin G2, will nization in phosphate buffer,t pH 6.5, or in the phosphate alter the mortality rate after myocardial infarction. Several trials buffer supplemented with 50 ,tg of hemoglobin per ml, 5 mM using aspirin and sulfinpyrazone in men who have survived one epinephrine bitartrate, and 3 mM reduced glutathione. One myocardial infarction suggest that these drugs decrease the milliliter of buffer was added for microsomes from each 0.5 death rate (3-6). The wisdom of this therapeutic approach, to 1 g of shredded whole tissue. The resulting microsomal sus- however, has been challenged (7, 8). The major product of pension (1 ml) was incubated at 370 with [acetyl-3H]aspirin. arachidonic acid transformation via cyclooxygenase in arterial Reactions were stopped at various times by adding 8 ml of cold tissue, prostacyclin (PGI2), is postulated to be a naturally oc- buffer (40) and placing the samples on ice. The microsomal curring antithrombotic agent (8). Accordingly, inactivation of suspensions were collected by centrifugation as described above cyclooxygenase in blood vessels could promote thrombosis. and solubilized in 0.5% Nonidet P40. The radioactivity incor- Aspirin blocks prostaglandin production (9, 10) by covalently porated into cyclooxygenase was determined after acetone acetylating fatty acid cyclooxygenase (11). The enzyme in precipitation of the Nonidet P40-solubilized protein, solubili- human zation in sodium dodecyl sulfate, and polyacrylamide gel platelets is extremely sensitive to small doses of the drug electrophoresis in the presence of sodium dodecyl sulfate as in vivo and in vitro (12). We now demonstrate that fatty acid described previously (12). In experiments utilizing indo- cyclooxygenase from human thoracic aorta and human coro- methacin, ibuprofen, or flurbiprofen, these drugs were incu- nary artery is much less sensitive to aspirin in vitro, as measured bated with the microsome suspensions for 5-10 min at room by acetylation of the enzyme with [acetyl-3H]aspirin or by temperature prior to addition of aspirin. Experiments utilizing bioassay of PGI2 produced by aorta after aspirin treatment. mixtures of aortic microsomes and platelet particulate fractions MATERIALS AND METHODS were also incubated for 5-10 min at room temperature prior to the addition of aspirin. Human ascending aortas and coronary arteries were obtained In experiments using vascular microsomes, "nonspecific" at autopsies performed less than 12 hr after death. The speci- acetylation by aspirin was measured as radioactivity incorpo- mens were cleaned of adventitia, immediately frozen in liquid rated into the cyclooxygenase peak from [acetyl-3H]aspirin in nitrogen, and stored at -80°. Washed human platelets and a the presence of a large excess of unlabeled aspirin. Nonspecific platelet particulate fraction were prepared (12). Prostacyclin, acetylation was measured at a single time in each experiment flurbiprofen, and ibuprofen were gifts from Upjohn, Kalama- and was assumed to be linear with time (11). In two experiments zoo, MI. [Acetyl-3H]aspirin, specific activity 177 Ci/mol, was frozen shredded aorta was thawed in aspirin-containing solution synthesized (11). Unlabeled aspirin was purchased from Merck, and the enzyme was thereby acetylated before microsome West Point, PA, and stored as a 100 mM solution in ethanol. preparation. These experiments gave the same results, indi- ['4C]Serotonin, 40-60 Ci/mol, was purchased from New En- gland Nuclear, Boston, MA. Arachidonic acid was purchased Abbreviation: PGI2, prostacyclin. * To whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page t Buffers used in the present experiments. Phosphate buffer, pH 6.5: charge payment. This article must therefore be hereby marked "ad- 4.3mM K2HPO4/4.3 mM Na2HPO4/24.4 mM NaH2PO4/113 mM vertisement" in accordance with 18 U. S. C. §1734 solely to indicate NaCl/5.5 mM glucose. Tris buffers: 15 mM Tris-HCl (pH 7.4 or 8.6 this fact. at 25°)/139 mM NaCI/5.5 mM glucose. 5181 Downloaded by guest on September 27, 2021 5182 Medical Sciences: Burch et al. Proc. Natl. Acad. Sci. USA 75 (1978) cating that microsome preparations did not affect sensitivity to aspirin. Acetylation of Platelets or Platelet Particulate Fractions. Washed intact platelets were suspended in phosphate buffer, pH 6.5, or in Tris buffer,t pH 7.4, and were incubated with [acetyl-3H]aspirin at 370. The platelet particulate fractions were resuspended by brief sonication in either of the above buffers E containing 50 ,tg of hemoglobin per ml, 5 mM epinephrine 0. bitartrate, 3 mM reduced glutathione, and 5 mM NaOH (to CT 80 neutralize the epinephrine bitartrate). They were also incubated with [acetyl-3H]aspirin at 370. At intervals, 2 X 108 platelets or the equivalent amount of particulate fraction was removed, 40 - added to sufficient sodium dodecyl sulfate/2-mercaptoethanol solution to result in respective final concentrations of 5% and 0.1 M, and placed on ice. Subsequently, the samples were boiled for 5 min and radioactivity incorporated-into cyclooxygenase 0.1 10 20 30 40 was determined by sodium dodecyl sulfate/polyacrylamide Gel slice gel electrophoresis (11). FIG. 1. Sodium dodecyl sulfate/polyacrylamide gel electropho- PGI2 Synthesis in Aortic Tissues. Frozen shredded aortic resis ofhuman aorta microsomes. Microsomes from 1 g ofhuman aorta tissue was suspended in Tris buffer, pH 7.4, with or without 200 were incubated with 200 jAM [acetyl-3H]aspirin for 90 min. ,uM aspirin (0.2% ethanol final concentration in either case), incubated at 370 at a concentration of 0.2-0.8 g wet weight/ml, death (31-88) or the time from death to autopsy. There were and then chilled. Tissue pellets were re-isolated by centrifu- no marked differences in the degree of atherosclerosis in the gation at 40 for 15 min at 14,500 g, washed once by resuspension various samples. The time course of acetylation obtained in in Tris buffer,t pH 8.6, and re-isolated. To generate PGI2, several experiments using microsomes from human aortas or pellets were then suspended in Tris buffer, pH 8.6, (0.8-1.2 g human coronary arteries, or using intact human platelets, is wet weight/ml) containing 25 ,gM arachidonic acid, stirred 8 shown in Fig. 2. Note that the rapid acetylation of cyclooxy- minutes at 37°, then chilled, and the tissues were collected by genase in platelets was obtained at 1/10th the aspirin concen- centrifugation as above. Supernates from this step were frozen tration used with aorta and coronary artery. The reactions with in liquid nitrogen and stored at -80°. The PGI2 thus generated aspirin under these conditions are pseudo-first-order, because was measured by the inhibition of thrombin-induced [14C]- they are carried out in the presence of a large excess of aspirin serotonin release in washed human platelets as described pre- relative to enzyme. The negative slope of each line equals the viously (13). In this assay, incubation of platelets with 200-M,1 apparent rate constant, kapparent, which is directly proportional aliquots of supernate increased the thrombin concentration to aspirin concentration (11). We have defined a "potency" required for serotonin release; the resultant shift in the thrombin term as kapparent divided by aspirin concentration to compare dose-response curve, AT5o, is defined as the dose of thrombin the ease of acetylation by aspirin of cyclooxygenase from dif- required for 50% serotonin release in the presence of sample ferent tissues.

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