John R. Vane

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.

Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis

Unlike arachidonic acid (eicosatetraenoic acid, C20:4omega-6, A.A.), eicosapentaenoic acid (C20:5omega-3, E.P.A.) does not induce platelet aggregation in human platelet-rich plasma (P.R.P.), probably because of the formation of thromboxane A3 (T.X.A3) which does not have platelet aggregating properties. Moreover, E.P.A., like A.A., can be utilised by the vessel wall to make an anti-aggregating substance, probably a delta17-prostacyclin (P.G.I3). This finding suggests that, in vivo, high levels of E.P.A. and low levels of A.A. could lead to an antithrombotic state in which an active P.G.I3 and a non-active T.X.A3 are formed. Eskimos have high levels of E.P.A. and low levels of A.A. and they also have a low incidence of myocardial infarction and a tendency to bleed. It is possible that dietary enrichment with E.P.A. will protect against thrombosis.

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.

New insights into the mode of action of anti-inflammatory drugs

The discovery of a second cyclooxygenase has provided fresh impetus to the search for new anti-inflammatory drugs. The second enzyme is effectively absent from healthy tissues but its levels rise dramatically during inflammation. It can be induced in migratory cells by bacterial lipopolysaccharide, cytokines and growth factors. The constitutive cyclooxygenase-1 (COX-1) can thus be considered a “housekeeping” enzyme, in contrast to cyclooxygenase-2 (COX-2) which is activated by tissue damage. Both enzymes have a molecular weight of around 70kDa and similar Km and Vmax values for their reaction with arachidonic acid. Several non steroid anti-inflammatory drugs which have more than 1,000 fold selectivity for COX-2 over COX-1 are in the early stages of drug development.

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.

Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo

E. coli lipopolysaccharide (LPS; 15 mg kg-1 i.v.) produced a long-lasting reduction in mean arterial blood pressure (MAP) in the anaesthetized rat. Inhibition of nitric oxide endothelium-derived relaxing factor (EDRF) synthesis with NG-monomethyl-L-arginine (MeArg, 1 mg kg-1 min-1 i.v. for 30 min) produced an increase in MAP and largely attenuated the LPS-induced hypotension; both effects were significantly reversed with L-arginine (6 mg kg-1 min-1 i.v.). When compared to MeArg, phenylephrine (300 mg kg-1 h-1 i.v.) produced a similar pressor response, but much less attenuation of the hypotensive response to LPS. Thus, a stimulation of EDRF release contributes to the LPS-induced hypotension in the anaesthetized rat.

Anti-inflammatory drugs and their mechanism of action.

Abstract. Nonsteroidal anti-inflammatory drugs (NSAIDs) produce their therapeutic activities through inhibition of cyclooxygenase (COX), the enzyme that makes prostaglandins (PGs). They share, to a greater or lesser degree, the same side effects, including gastric and renal toxicity. Recent research has shown that there are at least two COX isoenzymes. COX-1 is constitutive and makes PGs that protect the stomach and kidney from damage. COX-2 is induced by inflammatory stimuli, such as cytokines, and produces PGs that contribute to the pain and swelling of inflammation. Thus, selective COX-2 inhibitors should be anti-inflammatory without side effects on the kidney and stomach. Of course, selective COX-2 inhibitors may have other side effects and perhaps other therapeutic potential. For instance, COX-2 (and not COX-1) is thought to be involved in ovulation and in labor. In addition, the well-known protective action of aspirin on colon cancer may be through an action on COX-2, which is expressed in this disease. Moreover, NSAIDs delay the progress of Alzheimers disease. Thus, selective COX-2 inhibitors may demonstrate new important therapeutic benefits as anticancer agents, as well as in preventing premature labor and perhaps even retarding the progression of Alzheimers disease.

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.