M. J. Silver

Bovine endothelial cells in culture produce thromboxane as well as prostacyclin.

Bovine aortic endothelial and smooth muscle cells in culture were incubated with arachidonic acid or prostaglandin H2. The amount of prostacyclin nd thromboxane A2 synthesized ws then determined by specific radioimmunoassay for 6-keto-prostaglandin F1 alpha and thromboxane B2. Although smooth muscle cells produced only 6-keto-prostaglandin F1 alpha and thromboxane B2 in a ratio of 5:1 to 10:1. The same ratio of these metabolites of arachidonic acid ws also found when prostaglandin production from endogenous arachidonic acid was stimulated in endothelial cells by the ionophore A23187. Cyclooxygenase inhibitors inhibited the production of both metabolites equally, whereas thromboxane synthetase inhibitors selectively inhibited the production of thromboxane B2. Cells in culture were also incubated with [1-14C]arachidonic acid and the extracted products were identified by two-dimensional thin-layer chromatography. 6-Keto-prostaglandin F1 alpha was the only metabolite produced by smooth muscle cells, but endothelial cells synthesized 6-keto prostaglandin F1 alpha, thromboxane B2, prostaglandin E2, and prostaglandin F2 alpha.

Formation of an Intermediate in Prostaglandin Biosynthesis and Its Association with the Platelet Release Reaction

A compound that could be converted to prostaglandin F(2alpha) by mild chemical reduction was formed by human platelets in response to arachidonic acid, collagen, or L-epinephrine. It was present in maximal amounts at about 1 min after addition of arachidonic acid or collagen to platelet-rich plasma. Its initial formation appeared to precede platelet aggregation by these agents and was closely correlated with the release of adenine nucleotides and radioactive 5-hydroxytryptamine from platelets. Moreover, the compound was itself found outside the platelets. This compound is probably an endoperoxide intermediate in prostaglandin biosynthesis and may be a trigger for the platelet release reaction.

Formation of Prostaglandins during the Aggregation of Human Blood Platelets

Prostaglandins E(2) and F(2alpha) were formed in response to ADP, L-epinephrine, or collagen by human platelets suspended in plasma containing citrate anticoagulant and stirred at 37 degrees C. The prostaglandins formed by platelets in response to collagen were rapidly released and the amounts formed were proportional to the amount of collagen added. The formation of the prostaglandins was associated with the single wave of aggregation induced by collagen or the second wave of aggregation induced by epinephrine. The above findings are discussed with reference to published studies on the biochemical changes occurring during platelet aggregation. It is suggested that the formation and release of prostaglandins is associated with the secretion of endogenous ADP and 5-hydroxytryptamine.

Prostaglandins as Inhibitors of Human Platelet Aggregation

Summary. The potencies of prostaglandins (PG) I2, PGD2 and PGE1 as inhibitors of human platelet aggregation induced by threshold concentrations of four aggregating agents were determined in platelet‐rich plasma from normal individuals who had not ingested aspirin. The order of activity against ADP, adrenaline and collagen was always PGI2 > PGD2 > PGE1. However, PGD2 and PGE1 were almost equipotent with PGI2 when tested against arachidonic acid (AA). The threshold inhibitory effects of PGD2, PGE1 and PGI2 could be overcome by increasing the concentrations of the aggregating agents AA, collagen or ADP. Adrenaline was found to be different from the other aggregating agents. It could overcome inhibition of platelet aggregation by PGD2 but could not overcome inhibition by PGI2 or PGE1. These facts support the hypothesis that platelet receptors for PGI2 and PGE1 are similar to each other and different from the receptor(s) for PGD2.

Persistence of thromboxane A2-like material and platelet release-inducing activity in plasma.

During the incubation of arachidonic acid with platelet-rich plasma, a persistent activity appeared that caused the release of [14C]5-hydroxytryptamine from indomethacin-treated platelets. By the time-course of its appearance and disappearance, this release-inducing activity could be dissociated from prostaglandin endoperoxides and associated with thromboxane A2-like material. This material persists in plasma because of its continued production and increased stability.

Fatty acids and the initial events of endothelial damage seen by scanning and transmission electron microscopy.

A method was developed for observing changes in the endothelial cells in rabbit ear veins in vivo by scanning electron microscopy. Injection of fatty acids into the ear vein caused damage to the endothelium. The first signs of damage seen were marked bulges in the nuclei and loss of the rhomboidal shape of the endothelial cells. More severe damage included loss of nuclei, leaving holes in the cytoplasm. Some parts of the damaged endothelium showed complete separation of cells from each other and exposure of sub-endothelial tissue to which platelets with pseudopodia were adhering. Damage to the endothelium was produced by arachidonic, linoleic, gamma-linolenic, 8,11,14-eicosatrienoic, 5,8,11,14,-eicosatetraenoic or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acids. The effect of arachidonic acid was not prevented by pre-treating the animals with aspirin. It appears that damage produced by the fatty acids is non-specific.

Uptake and inactivation of a-type prostaglandins by human red cells***

Incubation of A type prostaglandins with whole blood or washed red cells at 37 degrees C converted them to more polar products with negligible vasodepressor and smooth muscle-contracting activities. This conversion did not occur in platelet-rich plasma. Uptake of the prostaglandins by red cells was demonstrated at both 4 degrees C and 37 degrees C. The data suggest 1) that if PGA is released from tissues into the blood stream or is administered for therapeutic purposes, its biological activity would be diminished by human red cells, and 2) that development of an assay for PGA in blood should take into account its uptake and metabolism by human red cells.

Drug effects on platelet aggregation and prostaglandin formation.

In this paper, we intend to present our current understanding of the mechanisms of induction and inhibition of platelet aggregation and prostaglandin formation. We will try to show how this new understanding may help us rationally design effective antithrombotic therapies. We will not review the many drugs that inhibit platelet aggregation. For this, the reader is referred elsewhere (1).