LG 82-4-00 and LG 82-4-01, Specific Inhibitors of Thromboxane Synthetase in Human Platelets without Intrinsic Activity

SummaryPlatelet aggregation, secretion of 5-hydroxy tryptamine and production of thromboxane B2 were monitored simultaneously in human platelet suspensions in the absence and presence of cyclooxygenase or thromboxane synthetase inhibitors. Aggregation, secretion and thromboxane B2 formation in response to either sodium arachidonate or epinephrine were blocked by aspirin or by 1-N-butyl imidazole suggesting that thromboxane biosynthesis was an essential requirement for platelet activation by these agents. In contrast, thrombin and collagen could apparently induce aggregation and secretion via two pathways: at low doses involving thromboxane production, but at higher doses by a direct mechanism independent of thromboxane biosynthesis. In the case of ADP, inhibition of thromboxane production blocked secretion but had little effect on aggregation, indicating that secretion was probably dependent on thromboxane biosynthesis which probably occurred as a result of aggregation. Thus it appears that although the processes of thromboxane production, release of dense granule constituents and aggregation may often be intimately linked, each process can occur independently of the other, depending upon the stimulus used.

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Selective Inhibition of Platelet Agglutination and Aggregation by Aromatic Amidino Compounds

10.1055/s-0039-1680564 ◽

1977 ◽

Author(s):

J. D. Geratz ◽

R. R. Tidwell ◽

K. M. Brinkhous ◽

S. F. Mohammad

Keyword(s):

Selective Inhibition ◽

Inhibitory Effect ◽

Human Platelets ◽

The Other ◽

Future Studies ◽

Bovine Plasma ◽

Inhibitory Activities ◽

High Concentrations ◽

Ristocetin Cofactor ◽

Specific Inhibitors

A series of aromatic amidino compounds were investigated for their inhibitory effect on platelet agglutination and platelet aggregation. Agglutination of fresh or fixed human platelets was produced by bovine plasma or by human plasma in combination with ristocetin, while aggregation of fresh platelets was induced by ADP, thrombin or collagen. Highly effective inhibitors were found for both types of platelet clumping, but there was no parallelism between the inhibitory activities in the two test systems.5-(5-Amidino-2-benzimidazolyl)-2-(4-hydroxybenzene)benzimidazole suppressed agglutination exclusively. Pentamidine, on the other hand, strongly blocked the aggregation reactions, but did not interfere with agglutination, even at high concentrations. Compounds which inhibited aggregation also prevented the liberation of serotonin from the platelets.This investigation has led to the identification of new specific inhibitors of platelet agglutination and aggregation which can serve an important role in future studies of the two processes. The exact mode of interaction between ami dines and platelets is still being explored.. In the case of agglutination, inhibition most likely occurred at the level of binding of ristocetin cofactor to the platelet membrane. In the case of aggregation, however, interference could have taken place at the membranes or in the cytoplasm and could have been enzymatic or non-enzymatic in nature.

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SQ 22536, an Adenylate-Cyclase Inhibitor, Prevents the Antiplatelet Effect of Dazoxiben, a Thromboxane-Synthetase Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661037 ◽

1984 ◽

Vol 51 (01) ◽

pp. 125-128 ◽

Cited By ~ 15

Author(s):

Vittorio Bertelé ◽

Anna Falanga ◽

Marian Tomasiak ◽

Chiara Cerletti ◽

Giovanni de Gaetano

Keyword(s):

Adenylate Cyclase ◽

Platelet Rich Plasma ◽

Thromboxane A2 ◽

Human Platelets ◽

Selective Inhibitor ◽

Prostaglandin D2 ◽

Plasma Samples ◽

Thromboxane Synthetase ◽

Camp Levels ◽

Platelet Thromboxane

SummaryThis study shows that dazoxiben, a selective inhibitor of thromboxane A2-synthetase in human platelets, inhibited arachidonic acid-induced platelet aggregation in platelet-rich plasma samples from four out of 16 healthy volunteers. In these four “responder” samples, the anti-aggregating effect of dazoxiben was prevented by the compound SQ 22536, a 9-substituted adenine analogue, endowed with an inhibitory activity on adenylate-cyclase. The compound SQ 22536 also counteracted the antiaggregating effect of prostaglandin D2, a known activator of platelet adenylate-cyclase. When platelet thromboxane A2-syn- thetase was blocked by dazoxiben, a marked increase of prostaglandin D2 was concomitantly observed both in “responder” and “non responder” samples. The compound SQ 22536 blunted the increase in platelet cAMP caused by either dazoxiben and sodium arachidonate or prostaglandin D2. It is suggested that the antiaggregating effect of dazoxiben is mediated by newly synthesized prostaglandin D2. The latter acts by stimulating adenylate-cyclase and increasing cAMP levels. The compound SQ 22536 prevents both phenomena. In “non responder” samples some factors - still to be defined - might counteract similarly to the compound SQ 22536 the antiaggregating activity of PGD2.

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Evidence for Two Separate Receptors Mediating The Stimulation of Platelet Adenylate Cyclase By Prostaglandin I2 (PGI2), PGD2 and PGE1

10.1055/s-0038-1665817 ◽

1979 ◽

Author(s):

D. C. B. Mils ◽

D. E. Macfarlane

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Partial Agonist ◽

Human Platelets ◽

Intrinsic Activity ◽

High Dose ◽

Prostaglandin I2 ◽

Subsequent Response ◽

High Concentrations ◽

Stimulation Of

Prostacyclin (PGI2), and prostaglandins D2 and E1, all inhibit aggregation of human platelets by stimulating adenylate cyclase. in platelets prelabelled with l4C adenine, PGI2 has a higher intrinsic activity than either PGD2 or PGE1, as well as a higher apparent affinity. PGE1 but not PGD2, inhibits the action of high concentrations of PGI2, both when added simultaneously with PGI2 and when added 3 min later. in the latter case the slow spontaneous fall in intracellular cyclic AMP is accelerated by PGE. but not by PGD2. PGF1α, at concentrations up to 100 μM neither stimulated the cyclase 1 by itself, nor did it inhibit the effects of PGI2, PGE1, or PGD2. PGF2α, which did cause a small increase in cyclic AMP, inhibited PGD2 strongly, PGE1 not at all, and PGI2 slightly at high concentrations. N0164, an inhibitor of aggregation induced by bis-enoic prostaglandins, inhibited cyclase stimulation by PGD2 but not by PGE1, PGE2, PGD1 or PGI2, though it reduced PGI2-induced inhibition of platelet aggregation. Preaddition of PGE1, but not of PGI2 or PGD2, at submaximal concentrations, inhibited subsequent response to high dose PGI2. The results suggest that PGE1 and PGD2 probably act on the same enzyme, but through a different receptor. PGE1 acts as a partial agonist for the receptor for PGI2, but in addition causes a tachyphylaxis not seen with PGI2 or with PGD2.

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Platelet Function in Alcoholics Treated with Disulfiram

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/107602969600200110 ◽

1996 ◽

Vol 2 (1) ◽

pp. 51-54

Author(s):

Maria Luigia Randi ◽

Ilia Zanella ◽

Piero Pujatti ◽

Barbara Soini ◽

Antonio Girolami

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Bleeding Time ◽

Alcohol Intoxication ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Thromboxane Synthetase ◽

Group A ◽

Group B ◽

Normal Controls

Disulfiram is usually used in alcoholics as aversion therapy. It binds to various enzymes and pro teins in blood and in tissues. In particular, it inhibits the thromboxane synthetase in human platelets and, for this reason, it has been surmised that disulfiram has a possible effect on platelet function. So far, disulfiram failed to confirm this hypothesis in healthy volunteers. However, it appears able to decrease the threshold collagen concen tration for platelet aggregation. Our aim was to evaluate the effect of disulfiram on the platelet function of alco holics. In this study, 24 alcoholics, divided in group A (12 abstinent patients with disulfiram 200 mg/day), group B (12 abstinent patients without treatment), and 17 normal controls, are reported. Different tests were performed at time 0 (acute alcohol intoxication), time 2, and time 15 after the beginning of abstinence. A significant increase was observed in bleeding time (BT) of group B and in platelet count of both groups. No modification was seen in prothrombin time. In group A, a significant increase of platelet aggregation under adenosine diphosphate (ADP; 2 μ M) stimulus was observed. Whereas no difference was seen in platelet 5-hydroxytryptamine (5-HT), serum 5-HT increased significantly at time 15 in group A. We con clude that the increase in serum 5-HT was probably due to the inhibition of monoamine oxidase (MAO) promoted by disulfiram, followed by an activation of MAO induced by disulfiram.

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The Effects of Platelet-Derived Contractile Agents on Human Digital Arteries

Clinical Science ◽

10.1042/cs0660443 ◽

1984 ◽

Vol 66 (4) ◽

pp. 443-451 ◽

Cited By ~ 20

Author(s):

R. F. W. Moulds ◽

V. Iwanov ◽

R. L. Medcalf

Keyword(s):

Platelet Aggregation ◽

Blood Vessels ◽

Human Blood ◽

Coronary Spasm ◽

Prostaglandin F2α ◽

Therapeutic Benefit ◽

Human Platelets ◽

Contractile Responses ◽

Transient Ischaemic Attacks ◽

Thromboxane Synthetase

1. The contractile responses of spiral strips of human digital arteries to samples of a suspension of human platelets aggregated by thrombin have been studied at different time intervals after aggregation. 2. Platelets added to the arterial strips 1 min after aggregation of the platelets produced contractile responses which were significantly greater than those produced by corresponding platelets added 20 min after aggregation. 3. When platelets were aggregated in the presence of indomethacin or the thromboxane synthetase antagonist 1-benzylimidazole, contractile responses produced by the platelets 1 min after aggregation were significantly reduced. They were then not significantly different from those produced by addition of the aliquots 20 min after aggregation, which were unaffected. 4. Pretreatment of the arteries with the serotonin antagonist ketanserin nearly abolished the contractile responses produced by addition of the platelets 20 min after aggregation, and significantly reduced those produced by addition of the platelets 1 min after aggregation. 5. Ketanserin did not affect the contractile responses of the arteries to potassium chloride, prostaglandin F2α, or the endoperoxide analogue U-46619, but antagonized the contractile effects of exogenous serotonin. 6. Combination of pretreatment of the arteries with ketanserin and aggregation of the platelets in indomethacin or 1-benzylimidazole virtually abolished contractile responses to platelets added both 1 min and 20 min after aggregation. 7. Tensions developed to different dilutions of platelets added 1 min after aggregation to arteries pretreated with ketanserin were not significantly different from those obtained to the same dilutions added 20 min after aggregation to arteries not pretreated with ketanserin. 8. It is concluded that there are two components to the contractile responses of human blood vessels produced by the aggregation of human platelets. One is labile, not produced when aggregation occurs in indomethacin or 1-benzylimidazole and is probably produced by thromboxane A2. The other is stable, markedly reduced by ketanserin, and probably produced by serotonin. Each of the components makes an approximately equal contribution to the contractures of human blood vessels produced by platelet aggregation. 9. It is also concluded that serotonin antagonists, in combination with a thromboxane synthetase inhibitor, may be of therapeutic benefit in clinical syndromes possibly mediated by platelet aggregation, such as cerebral transient ischaemic attacks and coronary spasm.

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Calmodulin stimulates thromboxane synthesis in human platelets: Studies with thromboxane synthetase inhibitors

Progress in Lipid Research ◽

10.1016/0163-7827(81)90080-1 ◽

1981 ◽

Vol 20 ◽

pp. 447-452 ◽

Cited By ~ 3

Author(s):

Patrick Y-K Wong ◽

Wai Yiu Cheung

Keyword(s):

Human Platelets ◽

Thromboxane Synthetase

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Failure of Preactivated Human Blood Platelets to Restore Defective Thromboxane Synthesis despite Prolonged Incubation in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651045 ◽

1989 ◽

Vol 62 (03) ◽

pp. 1016-1022 ◽

Cited By ~ 2

Author(s):

Marcus Stockschläder ◽

Rüdiger E Scharf

Keyword(s):

Blood Platelets ◽

Human Platelets ◽

Autologous Plasma ◽

Prolonged Incubation ◽

Platelet Storage ◽

Thromboxane Synthetase ◽

Responsible Mechanism ◽

Short Time ◽

Storage Pool Disease

SummaryAcquired platelet storage pool disease has been shown to be associated with reduced platelet thromboxane synthesis. However, the mechanisms for this dysfunction are incompletely understood. The present experiments were designed to evaluate some of the possible defects which may account for impaired thromboxane formation in human platelets previously exposed to thrombin in vitro. Washed platelets pretreated with 0.5 U/ml thrombin for 20 sec and subsequently recovered as single degranulated platelets were incapable of forming normal amounts of thromboxane upon a second stimulation with thrombin (as compared to Tyrode-pretreated control platelets). In contrast, thrombin-degranulated platelets released additional amounts of thromboxane in response to arachidonate, or collagen, indicating that short-time exposure to thrombin does not irreversibly inactivate platelet cyclooxygenase or thromboxane synthetase. Upon incubation of the thrombin-pretreated platelets in autologous plasma in the presence of 14C-arachidonate, the label became associated with the platelets to the same extent as with control platelets. However, the platelets did not recover their ability to synthesize normal amounts of thromboxane upon restimulation with thrombin, and only about half of the label was lost from the thrombin-pretreated platelets as compared to control platelets in response to thrombin. The ability of collagen to cause loss of 14C-arachidonate and formation of thromboxane was the same regardless of whether or not the platelets had been pretreated with thrombin. Incubation of platelets in plasma in the presence of added arachidonate for 90 min resulted in complete refractoriness to a second stimulation with thrombin but not with collagen. However, the control platelets also lost most of their ability to synthesize thromboxane when incubated with arachidonate for 90 min and thereafter stimulated with thrombin. Thus, the presence of added arachidonate affects the thrombin-inducible thromboxane synthesis after prolonged incubation of human platelets in plasma. Our observations suggest that depletion of endogenous arachidonate is not a major cause for the defective thrombin-induced thromboxane synthesis in thrombin-pretreated platelets. It is more likely that impaired mobilization of endogenous arachidonic acid explains this dysfunction. Defective mobilization of arachidonate in thrombin-degranulated platelets may be due to agonists-specific receptor desensitization, but the responsible mechanism has not been identified.

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Platelets Provide A Source Of Thromboxane B2 (TXB2) During Endotoxemia

10.1055/s-0038-1652039 ◽

1981 ◽

Author(s):

Marie J Stuart

Keyword(s):

Arachidonic Acid ◽

Acid Metabolism ◽

Thromboxane A2 ◽

Endotoxin Shock ◽

Human Platelets ◽

Thromboxane B2 ◽

Arachidonic Acid Release ◽

E Coli ◽

Thromboxane Synthetase ◽

Acid Release

Thromboxane B2 the stable end product of Thromboxane A2 plays a central role in endotoxin shock in the experimental animal (Cook et al, JCI 65: 227, 80). Plasma Thromboxane B2 is elevated following the infusion of endotoxin, and animals pretreated with a thromboxane synthetase inhibitor are protected from its deleterious effects. The tissue source of Thromboxane A2 during endotoxemia has not however been delineated. Since one of the end products of platelet Ara- chidonic Acid metabolism is Thromboxane B2, we studied the effects of endotoxin (E. Coli, 1 μg/ml) on platelet Arachidonic Acid release and conversion to Thromboxane B2. In paired experiments, in the presence of endotoxin, the addition of thrombin (0.5 U/ml) caused human platelets to release 29.1 ± 3.4% (1S.E.) of 14C Arachidonic Acid. This value was increased (p<0.02) when compared to Arachidonic Acid release in the absence of endotoxin (21.9 ± 3.6%). Similarly, Thromboxane B2 production was elevated (p <0.01) when platelets were preincubated with endotoxin prior to the addition of thrombin (6.1 ± 0.6), compared to Thromboxane B2 formed in its absence (3.4 ± 0.5). Endotoxin thus facilitates the release of Arachidonic Acid from platelet phospholipids, and enhances conversion of the released Arachidonic Acid to Thromboxane B2 i.e. one of the sources of Thromboxane B2 during endotoxemia is the platelet. Since Thromboxane A2 is a potent aggregator, this study also provides a mechanism for the thrombocytopenia observed in endotoxemia.

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