James W. Aiken

Prevention of blockage of partially obstructed coronary arteries with prostacyclin correlates with inhibition of platelet aggregation

Coronary arteries (circumflex or left anterior descending) of anesthetized dogs were partially obstructed to approximately 5% of the normal lumen size by fitting a plastic cylinder around the vessel. Under these conditions, blood flow in the artery was not maintained but, instead, gradually declined over a few minutes until the vessel was completely blocked. Shaking the plastic obstructor restored blood flow temporarily, however, flow gradually declined again to zero. Sometimes flow was spontaneously restored by immediate increases that occurred at irregular intervals while, on other occasions, blood flow had to be restored by shaking the obstructor every time the rate declined to near zero. Intravenous infusion of prostacyclin (PGI2) at 15 to 150 ng/kg/min reversed and prevented the blockage of the coronary arteries. The efficacy of PGI2 in preventing blockage correlated with inhibition of ADP-induced platelet aggregation in platelet rich plasma prepared from blood samples withdrawn from the dogs during PGI2 infusion. Other coronary vasodilators, nitroglycerin and PGE2, that have no antiaggregatory effects, failed to prevent blockage whereas PGE1 and indomethacin, which do block aggregation, also prevented blockage of the vessels. PGI2 or its precursor, PGH2, dripped topically on the obstructed site prevented the blockage of the artery. This local effect of IGI2 could be obtained with amounts too small to cause systemic inhibition of platelet aggregation. The results show that PGI2 prevents blockage of partially obstructed coronary arteries and this effect correlates with inhibition of platelet aggregation. Furthermore, the data suggest that locally produced PGI2 may have a local antiaggregatory effect without inhibiting platelet aggregation in the general circulation.

Comparison in anesthetized dogs of the anti-aggregatory and hemodynamic effects of prostacyclin and a chemically stable prostacyclin analog, 6a-carba-PGI2 (carbacyclin)

The intravascular anti-aggregatory and systemic and hemodynamic effects of prostacyclin and carbacyclin were compared by intravenous infusion in pentabarbital anesthetized dogs. Ten times as much carbacyclin was needed to produce comparable inhibition of platelet aggregation in the lumen of partially obstructed circumflex coronary arteries. These doses of carbacyclin caused similar decreases in total peripheral resistance as equi-effective anti-aggregatory doses of prostacyclin. There was a trend for the decrease in blood pressure with carbacyclin to be less than that produced by equi-effective anti-aggregatory doses of prostacyclin because carbacyclin caused somewhat greater increases in cardiac output. Changes in heart rate were similar with both substances. During carbacyclin and prostacyclin infusion resistance in normal (unobstructed) coronary arteries decreased. Both substances had comparable effects on pulmonary vascular resistance, right atrial pressure and left ventricular dp/dt at equivalent anti-aggregatory doses both before and after atropine (1 mg/kg) and hexamethonium (5 mg/kg). During 5 to 6 hour infusions of carbacyclin there was no evidence of desensitization of dog platelets to the anti-aggregatory activity. These results show that carbacyclin has a similar spectrum of activity as prostacyclin and is about one-tenth as potent.

Inhibition of platelet thromboxane A2 synthase activity by sodium 5-(3′-pyridinylmethyl)benzofuran-2-carboxylate

This report outlines the activity of a new thromboxane synthase inhibitor sodium, 5-(3-pyridinylmethyl)-2-benzofurancarboxylate, (U-63557A). U-63557A is a potent inhibitor of the thromboxane synthase in human platelets in vitro, as well as in rhesus monkey platelets ex vivo. A single oral dose of 3.0 mg/kg U-63557A inhibits the platelet thromboxane synthase in rhesus monkeys approximately 80% for at least 12 hrs. U-63557A has been administered to monkeys twice a day, (10 mg/kg) for 14 days, without evidence of drug tachyphylaxis or rebound. U-63557A does not inhibit thrombin-stimulated PGI2 biosynthesis in human endothelial cells, the 5-lipoxygenase in human neutrophils, or the cyclo-oxygenase in a variety of test systems. In anesthetized dogs, U-63557A injected i.v. at 0.1 to 5 mg/kg prevented the blockage of stenosed coronary arteries caused platelet aggregation. Similar effects were obtained by oral administration of 1-5 mg/kg. The thromboxane synthase inhibitor was more efficacious than cyclooxygenase inhibitors and equal to PGI2 in efficacy. Under appropriate conditions the protective effects of U-63557A could be reversed by i.v. cyclooxygenase inhibitors suggesting that its efficacy depended in part on endogenous PGI2 formation. Due to its specificity, oral activity, and extended duration of action, U-63557A is a promising compound for the evaluation of the role of thromboxane synthase in a variety of pathophysiological states.

Modulation of autonomic neurotransmission by PGD2: Comparison with effects of other prostaglandins in anesthetized cats

Experiments with anesthetized cats were done to study possible roles of different prostaglandins (PGs) in modulating sympathetic neuroeffector transmission. We recorded contractions of the nictitating membrane (n.m.), blood flow in the carotid artery, heart rate and blood pressure, both under control conditions and while stimulating the cut cervical sympathetic nerve. Intra-carotid arterial injection (i.a.) of PGD2 depressed sympathetic transmission to the n.m. without depressing the effects of exogenous norepinephrine (NE). In contrast, PGE2 enhanced the effects of nerve transmission or exogenous NE on the stimulated n.m. PGI2 had similar but shorter effects to PGE2. PGF2 alpha or a stable PGH2 analog, contracted the n.m. smooth muscle with no detected effect on nerve transmission. Carotid blood flow was increased by PGD2, PGE2 and PGI2. PGD2 and PGI2 caused bradycardia that could be blocked by atropine. This ability of PGD2 to modulate autonomic nerve activity is of particular interest because of recent reports that nerve tissue synthesizes PGD2.

6-Keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent

A direct comparison of the relative potencies of the two antiaggregatory prostaglandins PGI2 and 6-keto-PGE1 showed PGI2 was at least 20 times more potent than 6-keto-PGE1 when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI2 and 6-keto-PGI2 to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI2 was equivalent to 300 ng of 6-keto-PGE1. PGI2 was also more potent (10-20 times) than 6-keto-PGE1 as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine. PGI2 was also more active than 6-keto-PGE1 as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE1 was at least 10 times less potent thatn PGI2, the exact difference could not be determined because 6-keto-PGE1 caused significant falls in blood pressure before anti-platelet activity could be detected. PGI2 is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE1, but these data do not rule out the possibility that some of the activities attributed to PGI2 could be the result of the conversin of PGI2 and/or 6-keto-PGF1 alpha to 6-keto-PGE1.

Effects of prostaglandin D3 on nerve transmission in nictitating membrane of cats.

In anesthetized cats, intra-arterial injection of PGD3 toward the nictitating membrane caused long-lasting, dose-related decreases in the response of the nictitating membrane to sympathetic nerve stimulation. During peak depression of nerve transmission the response of the nictitating membrane to intra-arterial norepinephrine was not depressed suggesting that PGD3 suppressed the release of norepinephrine. PGD3 was as potent as PGD2 for modulating sympathetic nerve transmission but was less effective in activating a vagally mediated bradycardia. The results show that the PGD3 can modulate autonomic nerve transmission.

Rat aortic strip as a bioassay tissue for thromboxane A2 and rabbit aorta contracting substance (RCS) released from guinea pig lung by bradykinin or anaphylaxis.

Abstract Rat aortic strips and rabbit aortic strips were superfused in series with Krebs solution. Comparison of the sensitivity of the tissues to thromboxane A 2 (TXA 2 ), generated by mixing prostaglandin (PG)H 2 with human platelet microsomes (HPM), indicated that the rat aorta was just as sensitive an assay tissue as the rabbit aorta. Furthermore, it was equally selective in that it did not respond to low levels of the other metabolites of arachidonic acid. When the two tissues were used simultaneously to assay aortic contracting activity released from perfused guinea pig lung by bradykinin, both tissues detected activity that could be matched with similar known amounts of TXA 2 . However, when ovalbumin was used to release aortic contracting activity from sensitized guinea pig lung, the amounts of TXA 2 needed to match the responses of the assay tissues often differed by 2–3 fold. This suggested that other substances, as well as TXA 2 , released during anaphylaxis can affect the aortic strips and thus influence the bioassay of TXA 2 . This discrepancy in assay of TXA 2 can be detected only when more than one assay tissue is used. In the series of experiments in which we used the assay tissues to detect TXA 2 released by bradykinin, we noted that bradykinin released more TXA 2 from unsensitized lungs than from sensitized ones. Although the significance of this observation remains unclear, it suggests that there are quantitative differences in the PG biosynthetic pathways induced by the sensitization process.

Actions on indomethacin and prostaglandins E2 and D2 on nerve transmission in the nictitating membrane of the cat

The effects of PGE2, PGD2 and cyclooxygenase inhibitors on sympathetic nerve transmission in the nictitating membrane of anesthetized cats were studied to further characterize the actions of these prostaglandins and to define their possible role as neuromodulators. PGD2 depressed neurotransmission throughout a broad range of stimulus free quencies, apparently by presynaptic inhibition of norepinephrine release. PGE2 enhanced the effects of both nerve stimulation and exogenous norepinephrine in intact preparations but only depressed the effects of exogenous norepinephrine in the chronically denervated nictitating membrane, suggesting that part of the effect of PGE2 on neurotransmission was presynaptic. When norepinephrine reuptake was blocked by desipramine, PGE2 still enhanced neurotransmission. Indomethacin, but not other cyclooxygenase inhibitors (aspirin, ibuprofen, flurbiprofen, meclofenamic acid), inhibited the response of the nictitating membrane to nerve stimulation without depressing the effects of exogenous norepinephine. Curiously, indomethacin, but again not other cyclooxygenase inhibitors, specifically antagonized the ability of PGE2 to enhance nerve transmission. These results further characterize the pharmacological effects of PGE2 and PGD2 at the nictitating membrane. The lack of effect of cyclooxygenase inhibitors suggests that neither endogenous PGE2 or PGD2 play a functional role at this synapse. The effects of indomethacin appear to be unrelated to inhibition of prostaglandin synthesis.

Pharmacological Analysis of Factors Influencing Platelet Aggregation in Stenosed Coronary Arteries of Dogs

In vitro studies on platelets, mostly carried out using an aggregometer: have led to the conclusion that a number of naturally occurring substances promote platelet aggregation. These include epinephrine, serotonin, thromboxane A, (TXA,), prostaglandin Hz, adenosine 5’-diphosphate (ADP), collagen, thrombin, and platelet activating factor (AGEPC). However, the relative importance in vivo of each of these factors alone or in combination with each other (or with other physical influences, such as shear) is difficult to assess. Our laboratory has used an in vivo experimental approach536 to analyze, pharmacologically, the importance of some of these substances as promoters of coronary artery thrombosis in stenosed vessels in dogs. In the process of this evaluation, the data accumulated pointed to the conclusion that prostacyclin (PGIz) may play a role as an endogenous inhibitor of platelet aggregation, especially when its activity is enhanced by other pharmacologic means, and that serotonin is of considerable importance as an endogenous promoter of coronary artery thrombosis in this model.