R. Korbut

On the mechanism of antithrombotic action of flavonoids

Flavonols (quercetin and rutin) and flavanes (cyanidol and meciadonol) were studied for their effect on non-enzymatic lipid peroxidation, lipoxygenase and cyclo-oxygenase activities, binding to albumin and platelet membranes. These biochemical properties of four flavonoids were compared with respect to their antithrombotic action in vivo and their efficacy at influencing the platelet-endothelium interaction in vitro. All four flavonoids inhibited the ascorbate-stimulated formation of malondialdehyde by boiled rat liver microsomes (quercetin greater than rutin approximately cyanidol approximately meciadonol) and inhibited platelet lipoxygenase activity (quercetin greater than cyanidol greater than meciadonol greater than rutin) whereas only flavonols, but not flavanes, stimulated cyclo-oxygenase and were bound to platelet membranes. The same two flavonols dispersed platelet thrombi which were adhering to the rabbit aortic endothelium in vitro (EC50 for quercetin was 80 nM and for rutin 500 nM) and prevented platelets from aggregation over blood-superfused collagen strip in vivo (ED50 for quercetin was 5 nmol/kg and for rutin 33 nmol/kg i.v.). Cyanidol and meciadonol were not effective as anti-thrombotic agents. It is concluded that activated platelets adhering to vascular endothelium generate lipid peroxides and oxygen-free radicals which inhibit endothelial biosynthesis of prostacyclin and destroy endothelium-derived relaxing factor (EDRF). Flavonols are anti-thrombotic because they are selectively bound to mural platelet thrombi and owing to their free radical scavenging properties resuscitate biosynthesis and action of endothelial prostacyclin and EDRF. Thus, flavonols release the thrombolytic and vasoprotective endothelial mediators only in these vascular segments which are covered by a carpet of aggregating platelets.

Lungs as a generator of prostacyclin--hypothesis on physiological significance.

SummaryIn vivo anti-platelet de-aggregatory activity of exogenous prostacyclin is enhanced after its passage through the pulmonary circulation of anaesthetized cats, probably because of a concomitant generation of endogenous prostacyclin by the lungs. Evidence is also presented that perfused lungs of guinea pigs and rats spontaneously release considerable amounts of prostacyclin. It is therefore postulated that a continuous biosynthesis of prostacyclin by pulmonary endothelium is a general physiological phenomenon, while the generation of thromboxane A2 by lungs occurs in response to pathological stimuli. Coronary and cerebral arteries are supposed to benefit from this hormonal function of the lungs.

In vivo method for quantitation of anti-platelet potency of drugs

SummaryAortic strips from atherosclerotic rabbits or Achilles tendons from healthy rabbits were superfused with blood (3 ml/min) from anaesthetized and heparinized cats, while blood was returned to the venous system of animals. The superfused tissues gained in weight because of deposition of platelet thrombi on their surface. This gain in weight was continuously monitored and quantified. Forty minutes after intravenous administration of indomethacin (14 mg/kg), aspirin (7 mg/kg) or nictindole (2 mg/kg) the formation of platelet deposits was reduced by half. Three hours after i.v. administration of each drug at a dose of 20 mg/kg the remaining anti-platelet activities were 92% for aspirin, 59% for indomethacin and 18% for nictindole as compared to their antithrombotic action, which was recorded 40 min after their administration. Thrombogenesis was also prevented by a direct infusion of nictindole (50 ng/ml) or indomethacin (2000 ng/ml) into a stream of superfusing blood. Thereby our method enables us to quantify in vivo anti-aggregating potency of drugs, to estimate the duration of this action, and to compare their in vitro and in vivo aggregation-inhibitory activities.

De-aggregatory action of prostacyclin in vivo and its enhancement by theophylline

In the mixed venous blood of anaesthetized, heparinized cats prostacyclin de-aggregated platelet thrombi, which were formed on the surface of blood-superfused collagen strips or on the surface of blood-superfused aortic strips from atherosclerotic rabbits. The reversal of platelet aggregation by prostacyclin was still achieved 3 hrs after the formation of platelet clumps. After an intravenous injection of prostacyclin the ID50 for its de-aggregatory action was 7.5 microgram/kg. Theophylline ethyl-diamine (aminophylline), at a dose of 3 mg/kg i.v., did not reverse platelet aggregation but it enhanced the duration of the de-aggregatory action of prostacyclin; it had little effect on the hypotensive action of prostacyclin. It is concluded that prostacyclin disintegrates platelet clumps long after they are formed in heparinized blood in vivo and that its anti-platelet action, but not hypotensive action, is selectively potentiated by a phosphodiesterase inhibitor. The above experimental data indicate the possibility of the combined use of theophylline and prostacyclin in arterial thrombosis.

Reversal of platelet aggregation by prostacyclin

Summary Platelet thrombi were formed on the surface of blood superfused collagen strips. Mixed-venous blood from right atrium of heparinized and anaesthetized cats superfused (3 ml/min) a piece of the tendon of Achilles from a rabbit. An increase in weight of the superfused strip was continuously recorded and this gain in weight represented the deposition of platelet clumps. The maximal deposition of the clumps (400 – 600 mg) occured after 30 – 40 min of the superfusion. The pretreatment of the collagen strips with prostacyclin (0.5 – 5 ng/ml) inhibited the deposition of the platelet clumps. The treatment of the preformed platelet clumps with prostacyclin (1 – 20 ng/ml) resulted in a dispersion of the clumps and a reversal of platelet aggregation. A similar deaggregatory effect of prostacyclin occured after an intravenous injection of the hormone, ID 50 for the in vivo de-aggregatory activity of prostacyclin was 7.5 μg/kg. The in vivo reversal of platelet aggregation by prostacyclin was observed even 3 hours after the platelet clumps were formed. Thus for the first time the de-aggregatory action of prostacyclin in vivo was demonstrated and quantified.

Endogenous Mechanisms which Regulate Prostacyclin Release

When infused intravenously into anaesthetized cats angiotensin II (1--4 micrograms/kg) released into the circulation an unstable substance that caused de-aggregation of platelet clumps, relaxed a strip of bovine coronary artery, and its release was blocked by aspirin and indomethacin. Because of these characteristics this substance is likely to be prostacyclin. Catecholamines and phenylephrine did not induce the release of prostacyclin. It is suggested that a chemical modification of the molecule of angiotensin II may render a peptide with little hypertensive properties which will be an activator of prostacyclin biosynthesis.

Beta-pyridylcarbinol (RonicolR) releases a prostacyclin-like substance into arterial blood of patients with arteriosclerosis obliterans

Beta-pyridylcarbinol (Ronicol) when injected (1,2-1,4 mg/kg i.v.) into four patients with arteriosclerosis obliterans caused a release of an unstable disaggregatory substance into arterial blood of those patients. This release was not observed in other two patients who had been pretreated with aspirin (1,8 g p.o.daily). It is postulated that some of pharmacological properties of nicotinic acid derivatives are mediated through the release of endogenous PGI2-like substance.

A biological method for studying the interaction between platelets and vascular endothelium

A segment of fresh rabbit thoracic aorta (RbA) was turned inside out and superfused (1.5 ml/min) with citrated (3.8%) or heparinized (10 U/ml) blood of rabbit and the superfusate was discarded. RbA gained in weight due to deposition of thrombi on its endothelial surface. These thrombi were mainly composed of platelets. The interaction between platelets and endothelium was augmented in RbAs from animals with atherosclerosis and in RbAs pretreated with aspirin (110 microM) or 15-HPETE (150 microM) or by the enzymatic system generating oxygen free radicals (xanthine:xanthine oxidase - 100 microM: 0.1 U/ml). On the other hand, this interaction was impaired by superoxide dismutase (20 U/ml) or catalase (0.2 U/ml). Finally, the dissipation of thrombi by thromboxane A2-synthetase inhibitor--dazoxiben was found to be related to an increase in endothelial generation of prostacyclin.

The influence of hydrocortisone and indomethacin on the release of prostaglandin-like substances during circulatory shock in cats, which was induced by an intravenous administration of rabbit blood

Summary An intravenous injection of fresh rabbit blood into cats caused a circulatory shock appearing in three distinct phases. Using the blood-bathed organ technique it was found that the first hypotensive phase was correlated with an appearance of histamine and kinin-like substances in mixed venous blood the second hypertensive phase was associated with catecholaminemia, while the last fatal hypotensive phase was accompanied by a release of prostaglandin-like substances. Indomethacin 20 mg/kg i.v. or hydrocortisone 50 mg/kg i.v. abolished the release of prostaglandin-like substances, prevented the fatal hypotension and thereby protected cats against immediate death. Pretreatment with propranolol 0.5 mg/kg and phenoxybenzamine 1 mg/kg i.v. abolished the second and the third phases of the shock, but did not influence the survival times of the animals. It is concluded that pharmacological inhibition of prostaglandin release either by steroidal or non-steroidal anti-inflammatory drugs protects against circulatory shock, which is induced by an injection of foreign species blood.

Almitrine increases plasma fibrinolytic activity through the release of prostacyclin from lungs into circulation.

Summary Almitrine (Vectarion R ) — a potent respiratory stimulant induced the release of a PGI 2 -like substance into circulation of rabbits. This release was accompanied by activation of euglobulin fibrinolytic system. Fibrinolytic effect of almitrine could be matched by an intravenous infusion of authentic prostacyclin. Pretreatment of rabbits with indomethacin inhibited almitrine-induced fibrinolytic activity. It is postulated that chemoreceptor stimulants activate plasma fibrinolytic systems via the release of endogenous prostacyclin. The release of endogenous prostacyclin by drugs presents an alternative approach to the therapy of thrombotic disorders with synthetic prostacyclin.