Stuart Bunting

An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation.

Microsomes prepared from rabbit or pig aortas transformed endoperoxides (PGG2 or PGH2) to an unstable substance (PGX) that inhibited human platelet aggregation. PGX was 30 times more potent in this respect than prostaglandin E1. PGX contracted some gastrointestinal smooth muscle and relaxed certain isolated blood vessels. Prostaglandin endoperoxides cause platelet aggregation possibly through the generation by platelets of thromboxane A2. Generation of PGX by vessel walls could be the biochemical mechanism underlying their unique ability to resist platelet adhesion. A balance between formation of anti- and pro-aggregatory substances by enzymes could also contribute to the maintenance of the integrity of vascular endothelium and explain the mechanism of formation of intra-arterial thrombi in certain physiopathological conditions.

Arterial walls are protected against deposition of platelet thrombi by a substance (prostaglandin X) which they make from prostaglandin endoperoxides

Prostaglandin (PG) endoperoxides (PGG2 and PGH2) contract arterial smooth muscle and cause platelet aggregation. Microsomes from pig aorta, pig mesenteric arteries, rabbit aorta and rat stomach fundus enzymically transform PG endoperoxides to an unstable product (PGX) which relaxes arterial strips and prevents platelet aggregation. Microsomes from rat stomach corpus, rat liver, rabbit lungs, rabbit spleen, rabbit brain, rabbit kidney medulla, ram seminal vesicles as well as particulate fractions of rat skin homogenates transform PG endoperoxides to PGE- and PGF- rather than to PGX-like activity. PGX differs from the products of enzymic transformation of prostaglandin endoperoxides so far identified, including PGE2, F2alpha, D2, thromboxane A2 and their metabolites. PGX is less active in contracting rat fundic strip, chick rectum, guinea pig ileum and guinea pig trachea than are PGG2 and PGH2. PGX does not contract the rat colon. PGX is unstable in aqueous solution and its antiaggregating activity disappears within 0.25 min on boiling or within 10 min at 37degrees C. As an inhibitor of human platelet aggregation induced in vitro by arachidonic acid PGX was 30 times more potent than PGE1. The enzymic formation of PGX is inhibited by 15-hydroperoxy arachidonic acid (IC50 = 0.48 mug/ml), by spontaneously oxidised arachidonic acid (IC 50 less than 100 mug/ml) and by tranylcypromine (IC50 = 160 mug/ml). We conclude that a balance between formation by arterial walls of PGX which prevents platelet aggregation and release by blood platelets of prostaglandin endoperoxides which induce aggregation is of the utmost importance for the control of thrombus formation in vessels.

Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation

Fresh arterial tissue generates an unstable substance (prostablandin X) which relaxes vascular smooth muscle and potently inhibits platelet aggregation. The release of prostaglandin (PG) X can be stimulated by incubation with arachidonic acid or prostaglandin endoperoxides PGG2 or PGH2. The basal release of PGX or the release stimulated with arachidonic acid can be inhibited by previous treatment with indomethacin or by washing the tissue with a solution containing indomethacin. The formation of PGX from prostaglandin endoperoxides PGG2 or PGH2 is not inhibited by indomethacin. 15-hydroperoxy arachidonic acid (15-HPAA) inhibits the basal release of PGX as well as the release stimulated by arachidonic acid or prostaglandin endoperoxides (PGG2 or PGH2). Fresh arterial tissue obtained from control or indomethacin treated rabbits, when incubated with platelet rich plasma (PRP) generates PGX. This generation is inhibited by treating the tissue with 15-HPAA. A biochemical interaction between platelets and vessel wall is postulated by which platelets feed the vessel wall with prostaglandin endoperoxides which are utilized to form PGX. Formation of PGX could be the underlying mechanism which actively prevents, under normal conditions, the accumulation of platelets on the vessel wall.

The chemical structure of prostaglandin X (prostacyclin)

The chemical structure of prostaglandin X, the anti-aggregatory substance derived from prostaglandin endoperoxides, is 9-deoxy-6, 6alpha-epoxy-delta5-PGF1alpha. The stable compound formed when prostaglandin X undergoes a chemical transformation in biological systems in 6-keto-PGF1alpha. Prostaglandin X is stabilized in aqueous preparations by raising the pH to 8.5 or higher. The trivial name prostacyclin is proposed for 9-deoxy-6, 9alpha-epoxy-delta5-PGF1alpha.

A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation

Microsomal fractions from arterial walls of pigs and rabbits and fundus of rat stomach generate from prostaglandin endoperoxides (PGG2 or H2) an unstable substance, prostaglandin X (PGX) which is a potent inhibitor of platelet aggregation induced by several different substances. Other microsomal fractions including corpus of stomach, lung and ram seminal vesicles generate smaller amounts of PGX from PGG2 or PGH2. Incubation of microsomes from arterial wall or fundus of stomach with platelet-rich plasma under various conditions shows that the enzyme which generates PGX can utilize endoperoxides liberated from platelets or added to the cuvette, thereby preventing, interrupting or reversing the process of platelet aggregation. The generation of PGX is strongly inhibited (IC50 0.43 mug/ml) by 15-hydroperoxy arachidonic acid. These observations are important in the interpretation of vascular diseases such as atherosclerosis and thrombosis and provide a rational basis for the use of anti-oxidants in the prevention and treatment of these diseases.

Identification of an enzyme in platelet microsomes which generates thromboxane A2 from prostaglandin endoperoxides

The microsomal fraction of horse and human platelets contains an enzyme which converts prostaglandin cyclic endoperoxides (PGG2 or PGH2) to a substance which is much more potent in contracting strips of rabbit aorta. This substance has the same characteristics as thromboxane A2, and can be distinguished from other products of arachidonic acid metabolism by differential bioassay.

Imidazole: A selective inhibitor of thromboxane synthetase

Imidazole inhibits the enzymic conversion of the endoperoxides (PGG2 and PGH2) to thromboxane A2 by platelet microsomes (IC50: 22 MICRONG/ML; DETERMINED BY BIOASSAY). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of 14C-thromboxane B2 from 14C-PGH2. Several imidazole analogues and other substances were tested but only 1-methyl-imidazole was more potent than imidazole itself. The use of imidazole to inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.

Prostaglandin endoperoxide and thromboxane generating systems and their selective inhibition

Two enzyme systems and their selective inhibition are described. Microsomes from ram seminal vesicles (RSV) incubated with arachidonic acid at 22 degrees C generated a rabbit aorta contracting substance which, after rapid ether extraction, had characteristics similar to purified standard endoperoxides. Incubation of either purified endoperoxide or the product from RSV and arachidonic acid with horse platelet microsomes (HPM) yielded a more potent rabbit aorta contracting substance characterized as thromboxane A2, with a half life of 35.9 +/- 2.2 s at 37 degrees C after ether extraction. Two inhibitors, indomethacin and benzydamine exhibited selectivity for the two enzyme systems. The IC50 for benzydamine against thromboxane synthetase was 100 mug/ml and 250 mug/ml against RSV. Indomethacin showed a greater degree of selectivity with an IC50 of 5 mug/ml for the ram seminal vesicle cyclo-oxygenase compared to 100 mug/ml for thromboxane synthetase.

Polymorphonuclear leukocytes produce thromboxane A2-like activity during phagocytosis.

Homogenates of phagocytosing polymorphonuclear leukocytes obtained from rabbit peritoneum were incubated with the prostaglandin endoperoxides PGG2 or PGH2. After 2 min at 0degree C, incubation mixtures contained an increased rabbit aorta contracting activity. Ether extracts of incubation mixtures contained a substance which contracted the superfused strips of rabbit aorta and coeliac artery and had a half life which was similar to thromboxane A2. The generation of thromboxane A2-like activity from PG endoperoxides was prevented by boiling the homogenate prior to incubation, or by pretreatment with benzydamine, a drug which blocks thromboxane formation in platelets. Production of thromboxane A2-like material by leukocyte homogenates was compared with platelet microsomal thromboxane synthetase.

THE STRUCTURE OF PROSTAGLANDIN I 2

The chemical structure of prostaglandin I, the anti-aggretory substance derived from prostaglandin endoperoxides , is 9-deoxy-6, 9α-epoxy-Δ 5 -PGF 1 α .The stable compound formed when prostaglandin X undergoes a chemical transformation in biological systems is 6-keto-PGF 1 α. Prostaglandin I is stabilized in aqueous preparations by raising the pH to 8.5 or higher. The trivial name prostacyclin is proposed for 9-deoxy-6, 9α-epoxy-Δ 5 -PGF 1 α