Melvin J. Silver

Metabolism of [14C]arachidonic acid by human platelets

A time dependent incorporation of [1-14C] arachidonic acid into platelet phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine was observed in platelet-rich plasma. When platelets, so labelled, were washed and treated with thrombin, there was a major decrease in the radioactivity of phosphatidylcholine and phosphatidylinositol. This decrease was accounted for by the appearance of several previously identified (Hamberg and Samuelsson (1974) Proc. Natl. Acad. Sci. U.S. 71, 3400) 14C-labelled oxygenated products of arachidonic acid.

Selective release of archidonic acid from the phospholipids of human platelets in response to thrombin.

Arachidonic acid is unique amongst human platelet fatty acids in that it is the precursor of prostaglandins and thromboxanes. Since a number of these oxygenated products of arachidonic acid have potent effects on platelet function, an understanding of the metabolsim of their precursor is important. Human platelets have a mechanism for incorporating arachidonic acid from plasma into their phospholipids and, in response to thrombin, they reveal mechanisms for hydrolyzing this arachidonic acid from platelet phosphatidylcholine and phosphatidylinositol. This report deals with the specificity of these mechanisms. The present studies show that human platelets contain phospholipase A2 activities that preferentially release arachidonic acid. One of these activities specifically utilizes 1-acyl-2-arachidonyl-phosphatidyl-choline. Another utilizes platelet phosphatidylinositol and/or phosphatidylserine, both of which are highly enriched with arachidonic acid.

Arachidonic acid-induced human platelet aggregation and prostaglandin formation☆

Abstract Arachidonic acid and several of its salts caused aggregation of platelets in platelet-rich plasma. Other fatty acids did not cause aggregation when tested under similar conditions. Platelet aggregation induced by arachidonic acid was inhibited by adenosine, β naphthol, non-steroidal anti-inflammatory agents, unsaturated fatty acids and albumin. At concentrations too low to cause aggregation, arachidonic acid enhanced aggregation induced by collagen, ADP or epinephrine. Arachidonic acid caused platelets to form PGE 2 and PGF 2α . However, it failed to induce prostaglandin synthesis or platelet aggregation in plateletrich plasma obtained one hour after the ingestion of a therapeutic dose of aspirin. This effect of aspirin persisted for several days. The significance of these findings in relation to hemostasis and thrombosis is discussed.

Arachidonic Acid Causes Sudden Death in Rabbits

Injection of sodium arachidonate (1.4 milligrams per kilogram) into the marginal ear veins of rabbits caused death within 3 minutes. Histological examination showed platelet thrombi in the microvasculature of the lungs. Rabbits were protected from the lethal effects of arachidonic acid by pretreatment with aspirin. Fatty acids closely related to arachidonic acid did not cause death.

Prostaglandin D2 inhibits the aggregation of human platelets.

Abstract Prostaglandin D2, heretofore considered to be biologically inactive, was found to be more than twice as potent as prostaglandin E1 as an inhibitor of aggregation in human citrated platelet-rich plasma. It was much less potent than prostaglandin E1 in rabbit or rat platelet-rich plasma. This prostaglandin, or a derivative of it, may prove to be of greater value than prostaglandin E1 in maintaining the viability of stored platelets and as an antithrombotic agent.

Selective binding site for [3H]prostacyclin on platelets.

Prostacyclin (PGI(2)) is the most potent, naturally occurring inhibitor of platelet aggregation known. To determine whether PGI(2) is bound by platelets, high specific activity [9-(3)H]PGI(2) was synthesized by iodination and subsequent base treatment of the labeled precursor [9-(3)H]prostaglandin (PG)F(2alpha) methyl ester. Binding experiments were performed at room temperature with normal citrated human platelet-rich plasma that contained [(14)C]sucrose or [(14)C]PGF(1alpha) as an internal marker for the extracellular space. Binding of [(3)H]PGI(2) plateaued within 2 min and this bond radioactivity could be displaced rapidly by excess nonradioactive PGI(2). Scatchard analysis of concentration-dependent binding yielded a hyperbolic plot which appeared to be caused by the existence of two classes of binding sites. The higher affinity class has a dissociation constant of 12.1+/-2.7 nM and a capacity of 93 (+/-21)sites per platelet. The lower affinity class had a dissociation constant of 0.909+/-.236 muM and a capacity of 2,700+/-700 sites per platelet. The relative ability of PGI(2), PGE(1), PGE(2), and 6-keto-PGF(1alpha) to displace [(3)H]PGI(2) initially bound to the higher affinity class of sites were 100:5:<0.3: <0.3. These relative abilities parallel the relative potencies of these compounds as inhibitors of ADP-induced platelet aggregation in vitro. However PGD(2), which is more potent than PGE(1) as an inhibitor of aggregation, did not displace bound [(3)H]PGI(2). The higher affinity binding site for PGI(2) appears to be the specific receptor through which PGI(2) exerts its effect on platelets.

Detection of thromboxane B2 in peripheral blood of patients with Prinzmetal's angina

The plasma levels of thromboxane B2 (TxB2) were determined by radioimmunoassay in 6 consecutive patients with Prinzmetals angina and in 9 healthy volunteers. In the normal group TxB2 was not detectable (= 0.5 pmoles/ml), while in patients with variant angina TxB2 was consistently detected (1.5-140 pmole/ml).

Regulation of arachidonate-induced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid

The lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was biosynthesized, purified and incubated with washed human platelet. It inhibited arachidonic acid, azo-prostaglandin H2 or U-46619-induced aggregation and secretion in a concentration-dependent fashion (IC50 = 2-3 microM). Collagen-induced aggregation and secretion were also inhibited (IC50 = 6 microM). 12-HPETE inhibited malondialdehyde and thromboxane B2 formation in platelets stimulated with arachidonic acid or thrombin. While thrombin-induced aggregation was unaffected by 50 microM 12-HPETE, thrombin-induced secretion was inhibited. Inhibiton of secretion by 12-HPETE was observed in platelets from untreated as well as aspirin-treated donors, indicating that 12-HPETE inhibits secretion independently of its ability to inhibit prostaglandin formation. Aggregation of washed human platelets by arachidonic acid yielded a bell-shaped concentration-response curve. Diminished aggregation at higher concentrations was associated with an increase in the ratio of lipoxygenase products to thromboxane B2. The data suggest that 12-HPETE formation may regulate platelet aggregation and secretion and that its primary effect, at low concentrations, is inhibition of endoperoxide-induced responses. At higher concentrations 12-HPETE also inhibits arachidonic acid metabolism. Thus, the combined inhibitory effects on endoperoxide-induced aggregation and thromboxane formation would explain the diminished aggregation observed in response to high concentrations of arachidonic acid.

Duration of inhibition of platelet prostaglandin formation and aggregation by ingested aspirin or indomethacin.

Abstract Platelet prostaglandin production was abolished in platelet-rich plasma taken from 4 adult subjects one and 6 hours after ingestion of aspirin or indomethacin. The second wave of epinephrine-induced platelet aggregation was also abolished. Sodium salicylate did not produce these effects. While the effects of aspirin persisted for as long as 2 to 3 days those of indomethacin lasted less than 24 hours.

Prostaglandin endoperoxides and thromboxane A2 can induce platelet aggregation in the absence of secretion

PLATELET aggregation and secretion are widely studied not only because they are believed to reflect the major in vivo platelet function, formation of the primary haemostatic plug, but also because they involve fundamental cellular regulatory mechanisms. The synthesis of biologically active prostaglandin (PG) intermediates (endoperoxides and thromboxanes) seems to be an important step in the regulation of platelet function1,2, but the physiological roles and mechanism(s) of these compounds are unknown. It is claimed by Malmsten et al.3 and Samuelsson et. al.4 that PG endoperoxides and thromboxane A2 cause platelet aggregation only by inducing platelets to secrete ADP that in turn causes aggregation. In view of observations that platelets deficient in releasable ADP (storage pool-deficient platelets or platelets depleted of storage granules) still aggregate in response to arachidonic acid5–7, the precursor of the intermediates, the validity of this claim has been open to question. We have investigated platelet stimulation by PG endoperoxides (PGG2 and PGH2), an endoperoxide analogue ((15S)-hydroxy-11α, 9α-(epoxymethano) prosta-5Z, 13E-dienoic acid; U-46619), thromboxane A2-like material and arachidonic acid, using a new instrument that simultaneously monitors aggregation and secretion in the same platelet suspension8, permitting careful analysis of the relationships between aggregation and secretion. We report here that although high concentrations of the endoperoxide analogue, the natural endoperoxides and thromboxane A2 induce both platelet aggregation and secretion, low concentrations cause aggregation without detectable secretion.