When microsomes of sheep or bovine semi-nal vesicles are incubated with [acetyl-3H]aspirin (acetyl sali-cylic acid), 200 Ci/mol, we observe acetylation of a single protein, as measured by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The protein has a molecular weight of 85,000 and correspnds to a similar acetylated protein found in the particulate fraction of aspirin-treated human platelets. The aspirin-mediated acetylation reaction proceeds with the same time course and at the same concentration as does the inhibition of prostaglandin synthase (cyclo-oxygenase) (EC 1.14.99.1;8,11, Prostaglandin biosynthesis involves the initial conversion of arachidonic acid to cyclic endoperoxide intermediates designated PGG2 and PGH2 (1, 2). These relatively unstable compounds are then metabolized by other enzymes to form stable prostaglandins, or the endoperoxide intermediates are broken down to form malonaldehyde and other products (1,3). The initial conversion is catalyzed by a microsomal enzyme (or enzymes) contained in numerous tissues, including bovine and sheep seminal vesicles and human platelets (2-4). The microsomal enzyme that metabolize's arachidonic acid has been termed prostaglandin synthase or cyclo-oxygenase (EC 1.14.99.1; 8,11,14-eicosatrienoate, hydrogendonor:oxygen oxidoreductase). While the enzyme has been solubilized by neutral detergents (5-7), it has not been purified or characterized as to molecular weight or subunit structure.The anti-inflammatory effect (8) as well as the anti-platelet effect of aspirin (9) may be mediated through inhibition of prostaglandin synthesis. Recent experiments indicate that aspirin blocks conversion of arachidonic acid to cyclic endoperoxides (3, 10). Conversely, arachidonic acid prevents the inhibitory effect of aspirin on the enzyme, and the concentration of arachidonic acid that protects the enzyme correlates with the Michaelis constant of the fatty acid as cyclooxygenase substrate (11). Therefore, Rome and Lands have postulated that aspirin inhibits cyclo-oxygenase by competing with substrate for the active site (11). The inhibition by aspirin is not strictly competitive since it irreversibly inactivates cyclo-oxygenase in vitro.The mechanism by which aspirin inhibits cyclo-oxygenase is undefined. The drug is known to acetylate proteins such as albumin, immunoglobulins, and fibrinogen (12, 13). However, these reactions proceed slowly over hours and require 1-20 mM concentrations of aspirin. Aspirin inhibits cyclooxygenase under clearly different conditions; namely, within minutes at concentrations in the micromolar range (3,14).We have recently examined the relationship between protein acetylation and the inhibition of platelet function by aspirin (15). We demonstrated that aspirin acetylates a single 85,000 molecular weight protein of the particulate fraction of human platelets. The time course and aspirin concentration dependence of the acetylation reaction correlated well with the same parameters of aspirin's effect on platelet function (16)....

We provide evidence that the mechanism for arachidonate release from stimulated human platelets involves two enzymes: a phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) and a diglyceride lipase. After incubation of platelets with thrombin for 15 seconds, 1.2 nmol of 1-stearoyl-2-arachidonoyl diglyceride per 109 platelets was isolated. Arachidonate was released from this substrate by the action of diglyceride lipase located in the particulate fraction of platelets. The enzyme has a pH optimum of 7.0, is stimulated by calcium ions and reduced gutathione, and liberates 31 nmol of fatty acid per min per Ignofplatelet particulate protein. The diglyceride lipase has sufficient activity to account for the 5-10 nmol of arachidonate released per 109 platelets upon thrombin stimulation. That only arachidonate is released upon thrombin stimulation may be explained by the fact that the diglyceride substrate in platelets contains only arachidonate in the 2 position. The lipase activity found in platelet membranes can also hydrolyze the 1-position fhtty acid. Stearate is not released when intact platelets are stimulated with thrombin, and the fate of this fatty acid remains to be elucidated. Several tissues, including blood platelets, exhibit the so-called "inositol effect" when stimulated by appropriate agonists (1

A B S T R A C T Aspirin inhibits platelet frinction by permanently acetylating the cyclooxygenase that forms prostaglandins. We determined the sensitivity of platelets to aspirin in normal subjects by measuring [3H-acetyl]aspirin-susceptible cyclooxygenase in washed platelets obtained at various times after aspirin ingestion. A single 325-mg aspirin dose inactivated 89% of platelet cyclooxygenase. The inhibition persisted for 2 days suggesting that oral aspirin also inactivated megakaryocyte cyclooxygenase. Thereafter, active enzyme returned with a time-course reflecting platelet turnover (life-span 8.2+2 days). Single doses of 20-650 mg aspirin resulted in 34->95% inhibition after 24 h. Daily doses of 20-325 mg aspirin for brief periods produced 61->95% inactivation when measured 24 h after cessation of the drug. Platelet cyclooxygenase is more sensitive to inactivation by aspirin than enzyme in sheep seminial vesicles.

Since platelet cyclo-oxygenase is much more sensitive to inactivation by aspirin than is the enzyme in the arterial wall and low doses of aspirin may prevent thrombosis by blocking thromboxane synthesis, we conducted a randomized, double-blind trial of aspirin (160 mg per day) vs. placebo in 44 patients on chronic hemodialysis. The study was continued until there were 24 patients with thrombi and both groups had been under observation for a mean of nearly five months. Thrombi occurred in 18 of 25 (72 per cent) of patients given placebo and 16 of 19 (32 per cent) of those given aspirin (P less than 0.01). The incidence of thrombosis was reduced from 0.46 thrombi per patient month in the placebo group to 0.16 thrombi per patient month in the aspirin group (p less than 0.005). A dose of 160 mg of aspirin per day is an effective, nontoxic antithrombotic regimen in patients on hemodialysis.

A B S T R A C T Intact human platelets loaded with "PO, contain multiple phosphorylated proteins. Thrombin treatment of intact "PO-loaded platelets results in a 2-6-fold increase in phosphorylation of a platelet protein (designated "peak 7" protein) of approximately 40,000 mol wt as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-150. A similar increase in phosphorylation was observed in a platelet protein (designated "peak 9" protein) of approximately 20,000 mol wt. The time for half-maximal phosphorylation of peak 7 and peak 9 protein was 10-14 s. The concentration of thrombin at half-maximal phosphorylation was 0.25 U/ml for both proteins. Prior incubation of platelets with dibutyryl cyclic adenosine 3',5'-monophosphate or prostaglandin E1 inhibited thrombin-induced peak 7 and peak 9 protein phosphorylation. The erythroagglutinating phytohemagglutinin of Phaseolus vulgaris, a non-proteolytic release-inducing agent, induced peak 7 and peak 9 protein phosphorylation. Thus, the characteristics of peak 7 and peak 9 protein phosphorylation are similar to those of the platelet release reaction, suggesting that the phosphorylation of these proteins may play a role in the platelet release reaction.When platelet sonicates or the supernatant fraction from platelet sonicates were incubated with ['y-"P]ATP there was phosphorylation of both peak 7 and peak 9 proteins. This phosphorylation was unaffected by either added thrombin or adenosine 3',5'-cyclic monophosphate (cAMP) despite the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. Thus, the throm-