Inhibition Of ADP-induced Platelet Aggregation Research Articles

Inhibition of platelet aggregation with intravenous and oral administration of a carboprostacyclin analogue, 15-cyclopentyl-ω-pentanor-5(E)-carbacyclin (ONO 41483) in man

Intravenous and oral administration of a chemically stable carboprostacyclin analogue, 15-cyclopentyl-ω-pentanor-5(E)-carbacyclin (ONO 41483), resulted in ex-vivo inhibition of ADP-induced platelet aggregation in man. The maximum tolerated intravenous dose was 2.5 ng/kg/min for 1 hour and this produced a mean of 27.1% inhibition in 3 volunteers. For oral administration the tolerated single dose was 200 μg. At this dose, there was 56.3% inhibition of aggregation (mean of 3 results). High oral (400 μg) and intravenous doses (5 and 10 ng/kg/min for 1 hour) of ONO 41483, which caused marked inhibition of aggregation (ranging 39–100%), was accompanied by flushing of face and extremities, headache and phlebitis. However, none of the doses tested produced significant changes in arterial blood pressure or heart rate.

Inhibition of platelet aggegation and reversal of vasopressin-induced ECG changes by a carboprostacyclin analogue, ono 41483, in primates

15-cyclopentyl-ω-pentanor-5(E)-carbacyclin (ONO 41483), a chemically stable carboprostacyclin analogue, was 3.3 times less active than prostacyclin but was 2.6 times more active than carboprostacyclin in inhibiting aggregation of ADP-induced baboon platelet in vitro. On human platelets in vitro, ONO 41483 was 9.4 times less active than prostacyclin and 12.7 times more active than carboprostacyclin. ONO 41483 was 3.7 times less active than prostacyclin but was 2.2 times more active than carboprostacyclin in producing a fall in arterial blood pressure in anaesthetised baboons. Intravenous and oral administration of ONO 41483 in baboons produced ex vivo inhibition of ADP-induced platelet aggregation at doses that did not affect blood pressure or heart rate. In addition, bolus intravenous doses (3 to 10 μg/kg) of ONO 41483 reversed vasopressin-induced ECG changes in the monkey, suggesting an ability of the compound to relieve coronary spasm.

Inhibition of Human and Rat Platelet Aggregation by Extracts of Mo-er (Auricularia auricula)

Hot water extracts of Mo-er (1 gm by 15 ml of water), an oriental food (Auricularia auricula), inhibit strongly both human and rat platelet ADP-induced aggregation. HPLC analysis of two varieties of Mo-er, A. auricula and A. polytricha (a black tree fungus), shows that they contain adenosine (Ado), 133 and 154 micrograms per gram of dry fungus, respectively. The inhibition of ADP-induced platelet aggregation by Mo-er extracts and by Ado was compared. Mo-er extracts caused a more rapid onset and a longer duration of inhibition that produced by equivalent amounts of Ado. Furthermore, Mo-er extract treated with adenosine deaminase to degrade the Ado retained the capacity to inhibit platelet aggregation. The inhibitory effects of Mo-er extracts of ADP-induced human platelet aggregation are greatly potentiated by the inhibitors of cyclic AMP phosphodiesterase such as oxagrelate (phthalazinol) and papaverine. The inhibition of platelet aggregation is only partially blocked by 2',5'-dideoxy-adenosine (DDA), an inhibitor of platelet adenylate cyclase and 5'-deoxy, 5'-methylthioadenosine (MTA), an antagonist of ADO receptors. ADP-induced rat platelet aggregation is strongly inhibited by Mo-er extracts, but not by Ado. This inhibition is not reversed by either DDA or MTA. These findings indicate that Mo-er extracts contain an agent (or agents) in addition to Ado, that blocks platelet aggregation by a mechanism that does not involve the platelet cyclic AMP system.

Effect of cAMP Phosphodiesterase Inhibitors on ADP-Induced Shape Change, cAMP and Nucleoside Diphosphokinase Activity of Rabbit Platelets

The effects of cAMP phosphodiesterase inhibitors on ADP-induced shape change and cAMP concentrations have been studied. Caffeine (10 mM), theophylline (8 mM), dipyridamole (0.2 mM), or papaverine (0,05 mM) prevented the shape change of washed rabbit platelets induced by 0.4 microM ADP. At these concentrations, none of these cAMP phosphodiesterase inhibitors increased 14C-cAMP in platelets in which the cytoplasmic adenine nucleotides had been labelled with 14C-adenine. By a protein binding assay, only papaverine by itself increased platelet cAMP above its basal level. These results indicate that two pools of cAMP may exist in platelets. Both methods showed that stimulation of platelet adenylate cyclase with PGE1 (1 microM) resulted in an increase in platelet cAMP and all these cAMP phosphodiesterase inhibitors potentiated this increase caused by PGE1. By themselves, some of these compounds may act through mechanisms that do not involve platelet cAMP. The effects of these cAMP phosphodiesterase inhibitors on platelet nucleoside diphosphokinase (NDK) activity were also investigated. At concentrations that prevented ADP-induced shape change, papaverine and dipyridamole had no effect on the formation of 14C-ATP from 14C-ADP by washed rabbit platelets. The methylxanthines partially inhibited NDK activity of washed rabbit platelets and of isolated platelet membranes, probably due to the structural similarity between the adenine ring of ADP and these substances. However, adenine (8 mM) inhibited ADP-induced shape change and platelet NDK activity but was a less effective inhibitor of ADP-induced platelet aggregation. Thus it seems unlikely that interference with platelet NDK or the ADP receptor is the major mechanism by which the methylxanthines inhibit platelet functions.

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure.

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.

Inhibition of platelet aggregation with intravenous and oral administration of carboprostacyclin in man

Intravenous infusion of carboprostacyclin, a chemically stable analogue of prostacyclin (PGI 2) resulted in ex-vivo inhibition of ADP-induced platelet aggregation at doses that did not produce significant changes in blood pressure or heart rate. Oral administration of relatively large doses of this compound also inhibited ex-vivo ADP-induced platelet aggregation but this was accompanied by headache, facial flush, tachycardia and changes in blood pressure.

Platelet and other effects of carboprostacyclin - A stable prostacyclin analogue

Carboprostacyclin, a chemically stable analogue of prostacyclin (PGI 2) is 12.5 and 25.0 times respectively weaker than PGI 2 in inhibiting ADP-induced aggregation of baboon and human platelets in vitro. Carboprostacyclin was also 28.5 times weaker as a vasodepressor than PGI 2 in anaesthetised baboons. Intravenous and oral administration of carboprostacyclin in baboons resulted in ex-vivo inhibition of ADP-induced platelet aggregation at doses that did not produce changes in blood pressure or heart rate. Like PGI 2, carboprostacyclin contracted guinea pig tracheal chain preparation in vitro but decreased histamine induced lung resistance in anaesthetised guinea pigs. Both PGI2 and carboprostacyclin relaxed the human respiratory tract smooth muscle in vitro.

The effects of vincristine on platelet aggregation studied by a filter loop technique in the rat.

1 A method for measuring aggregation of platelets of adenosine diphosphate (ADP) is described using a filter inserted into the flowing aortic blood in the rat. 2 Repeated infusions of ADP resulted in a fall in the calculated aggregation index without significant changes in the platelet count. 3 Vincristine (0.05 mg/kg) intravenously caused significant inhibition of ADP-induced platelet aggregation. 4 Infusion of ADP caused some peripheral vasodilatation though it is unlikely that this contributed to the effects seen to any great extent.

A Circulating Inhibitor of Platelet Aggregation in Bartter's Syndrome

Aggregation studies were performed on platelets from five patients with Bartter's syndrome. Epinephrine failed to induce aggregation in all five patients. Adenosine 5'-diphosphate (ADP) produced a single reversible phase of aggregation, and there was depressed sensitivity to collagen. Response to ristocetin was normal. There was a dose-related inhibition of ADP-induced platelet aggregation when plasma from the patients was addeded to normal platelet-rich plasma. This inhibition was diminished or absent when patients were receiving aspirin. Washed platelets from two patients who were no longer undergoing aspirin therapy, showed a normal response to epinephrine in normal platelet-poor plasma. Bleeding time was reduced from 23 minutes to 12 minutes in one patient while on aspirin therapy. These studies suggest that a circulating inhibitor of platelet aggregation, probably of prostaglandin origin, is present in the plasma of patients with Bartter's syndrome.

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 (PGI 2) at 15 to 150 ng/kg/min reversed and prevented the blockage of the coronary arteries. The efficacy of PGI 2 in preventing blockage correlated with inhibition of ADP-induced platelet aggregation in platelet rich plasma prepared from blood samples withdrawn from the dogs during PGI 2 infusion. Other coronary vasodilators, nitroglycerin and PGE 2, that have no antiaggregatory effects, failed to prevent blockage whereas PGE 1 and indomethacin, which do block aggregation, also prevented blockage of the vessels. PGI 2 or its precursor, PGH 2, dripped topically on the obstructed site prevented the blockage of the artery. This local effect of PGI 2 could be obtained with amounts too small to cause systemic inhibition of platelet aggregation. The results show that PGI 2 prevents blockage of partially obstructed coronary arteries and this effect correlates with inhibition of platelet aggregation. Furthermore, the data suggest that locally produced PGI 2 may have a local antiaggregatory effect without inhibiting platelet aggregation in the general circulation.

Selective Binding Site for [3H]Prostacyclin on Platelets

Prostacyclin (PGI(2)) is the most potent, naturally occurring inhibitor of platelet aggregation known. To determine whether PGI(2) is bound by platelets, high specific activity [9-(3)H]PGI(2) was synthesized by iodination and subsequent base treatment of the labeled precursor [9-(3)H]prostaglandin (PG)F(2alpha) methyl ester. Binding experiments were performed at room temperature with normal citrated human platelet-rich plasma that contained [(14)C]sucrose or [(14)C]PGF(1alpha) as an internal marker for the extracellular space. Binding of [(3)H]PGI(2) plateaued within 2 min and this bond radioactivity could be displaced rapidly by excess nonradioactive PGI(2). Scatchard analysis of concentration-dependent binding yielded a hyperbolic plot which appeared to be caused by the existence of two classes of binding sites. The higher affinity class has a dissociation constant of 12.1+/-2.7 nM and a capacity of 93 (+/-21)sites per platelet. The lower affinity class had a dissociation constant of 0.909+/-.236 muM and a capacity of 2,700+/-700 sites per platelet. The relative ability of PGI(2), PGE(1), PGE(2), and 6-keto-PGF(1alpha) to displace [(3)H]PGI(2) initially bound to the higher affinity class of sites were 100:5:<0.3: <0.3. These relative abilities parallel the relative potencies of these compounds as inhibitors of ADP-induced platelet aggregation in vitro. However PGD(2), which is more potent than PGE(1) as an inhibitor of aggregation, did not displace bound [(3)H]PGI(2). The higher affinity binding site for PGI(2) appears to be the specific receptor through which PGI(2) exerts its effect on platelets.

Synthesis and use of a new spin-labeled analogue of ADP with platelet-aggregating activity.

A new spin-labeled derivative of ADP, 2-(4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl)thioadenosine-5'-diphosphate, has been synthesized. The compound causes both the reversible and irreversible phases of aggregation of human blood platelets at concentrations similar to those required for similar phases of aggregation by ADP itself. The spin-labeled ADP also rivals ADP as a substrate for pyruvate kinase. The interaction of intact human blood platelets and of isolated platelet membranes with the platelet-aggregating spin-labeled derivatives of ADP has been studied. The dramatic decrease in the ESR signal of the spin label is primarily due to chemical reduction of the nitroxide, rather than immobilization of the label. When platelets and spin-labeled ADP are mixed, a rapid burst of nitroxide reduction occurs, followed by a much slower reduction similar in time course to that seen for other spin labels. The rapid burst of reduction, but not the slow reduction, is inhibited by adenosine, an inhibitor of ADP-induced platelet aggregation, and by sulfhydryl-blocking agents. Experiments conducted with Ellman's reagent and platelet membranes or washed platelets revealed a 10 to 30% increase in the number of reactive membrane sulfhydryl groups when ADP was present. These results indicate that there is an increase in the number of reactive sulfhydryl groups on the platelet surface when platelets or membranes are stimulated by ADP.

Inhibition of ADP-Induced Platelet Aggregation by Dipyrone in Patients with Acute Myocardial Infarction

ADP induced platelet aggregation was investigated in 48 patients within three days of the first signs of acute myocardial (AMI). Thirty six of them received 1 gram of dipyrone. Twelve patients who did not receive dipyrone served as controls. Platelet aggregation was found severely inhibited in 11 patients who had received dipyrone up to 12 hours before investigation and moderately inhibited among 25 patients who were given the drug 12-24 hours prior to the investigation. All the patients with AMI who did not receive dipyrone, exhibited a state of hyperaggregability evidenced by the presence of a second phase of aggregation even with 0.5 microM ADP. The inhibitory activity of dipyrone on the second phase of platelet aggregation resembles that of other non steroidal anti-inflammatory drugs.

Anti-platelet action of intravenous infusion of prostacyclin in man

Sodium salt of prostacyclin in 0.1 M Tris buffer pH 9.0 was infused intravenously at doses of 2, 5, 10, 20 and 50 ng/kg/min into six healthy men, aged 26–46 years. Begining with a dose of 5 ng/kg/min prostacyclin caused a dose-dependent inhibition of ADP-induced platelet aggregation in plasma and dissipation of circulating platelet aggregates in blood. At a dose of 20 ng/kg/min template bleeding time and concomitant blood loss were doubled. Prostacyclin did not influence partial thromboplastin time, prothrombin time and euglobulin lysis time. The anti-platelet action of prostacyclin was accompanied by a moderate fall in diastolic blood pressure, increase in heart rate, vasodilation in the regions of face, neck and palms, as well as by arterialization of venous blood. All of the pharmacological effects of prostacyclin disappeared briefly after termination of the infusion. This is the first report on antiplatelet action of prostacyclin in man and it is concluded that prostacyclin may be a valuble drug in the treatment of thromboembolic diseases.

Discrimination between platelet prostaglandin receptors with a specific antagonist of bisenoic prostaglandins

Platelet aggregation and secretion induced by PGG 2 and by analogues of PGH 2 and PGE 2 were inhibited by NO164. Inhibition was apparently competitive (K i ∼ 20 μM). Responses to ADP, vasopressin and arachidonic acid were unaffected. Inhibition of ADP-induced platelet aggregation by PGD 2 and PGE 2 (but not by PGE 1) was antagonised by NO164.

ChemInform Abstract: PLATELET AGGREGATION INHIBITORS. II ‐ N‐HETEROCYCLIC ALDOXIME METHIODIDES

On the basis of an hypothesis according to which suitable nucleophilic agents may convert adenosine diphosphate (ADP) into adenosine monophosphate (AMP) and adenosine, well known inhibitors of ADP-induced platelet aggregation, some N-heterocyclic aldoxime methiodides were tested as inhibitors of ADP-induced rabbit platelet aggregation. Several 1-aryl-2-hydroxyiminomethly-3-methylimidazolium iodides significantly inhibit in vitro and in vivo-in vitro ADP-induced rabbit platelet aggregation.

The activity of γ-linolenate and dihomo-γ-linolenate methyl esters in vitro and in vivo on blood platelet function in non-human primates and in man

γ-linolenate and dihomo-γ-linolenate methyl esters have been shown to be potent inhibitors of adenosine diphosphate-(ADP) induced platelet aggregation in vitro and in vivo . Using fresh human plasma, dihomo-γ-linolenate methyl ester appeared to be approximately twice as potent as the γ-linolenate methyl ester in vitro . Both compounds were shown to be potent inhibitors of ADP-induced platelet aggregation after oral administration to the baboon. At 4mg/kg (p.o.) γ-linolenate methyl ester was inactive when administered daily for 5 days. A potent inhibitory effect was observed with this compound after acute administration at 25 mg/kg by the same route. Dihomo-γ-linolenate methyl ester inhibited ADP-induced platelet aggregation after oral administration at 2 mg/kg for 5 days. Combined with 0.7g Naudicelle oil,∗ dihomo-γ-linolenate methyl ester inhibited platelet aggregation at oral doses of 15, 4 and 1.5 mg/kg respectively, 3 hours after dosing. In man, dihomo-γ-linolenate methyl ester inhibited platelet aggregation at dose levels of 1.5 mg/kg when administered as a single oral dose or daily over a period of 5 days. In the acute study inhibition was observed up to 4 hours after dosing and had normalised after 6 hours. In the chronic study a progressive inhibition was achieved over the 5 day dose period with inhibition 24 hours after dosing on the 3rd day. A possible sex difference in the efficacy of dihomo-γ-linolenate methyl ester was observed in the baboon and in human volunteers, the material being less effective in females.

Inhibition of ADP-Induced Platelet Aggregation by Adenosine Tetraphosphate

In contrast to previous reports, highly purified adenosine tetraphosphate (AP4) does not induce the aggregation of platelets but inhibits the aggregation and release reaction in platelet-rich plasma promoted by ADP. The inhibitory action of AP4 on the aggregation by ADP is compared with that of AMP and ATP. The data presented suggest a competitive manner of inhibition of the ADP-induced aggregation by AP4.

Biological Properties of Dialysable Peptides Derived from Plasmin Digestion of Bovine Fibrinogen Preparations

Dialysable peptides (M. W. less than 12,000) were obtained by plasmin digests of purified bovine fibrinogen. The biological effects of these peptides were studied in rats in three different test systems: ADP-induced platelet aggregation, isolated atria contractility and vascular permeability. The effects induced by the peptides were: inhibition of ADP-induced platelet aggregation, increase in the frequency of isolated atria contractions and local increase in vascular permeability. All these activities were concentration dependent. Six micrograms of the peptides were still effective in increasing vascular permeability; in the in vitro systems the smallest effective dose ranged between 165 and 650 mug/ml. Following elution through a Sephadex G-25 gel with bidistilled water, four fractions were obtained. The second fraction (M.W. about 5,000) was the most active, followed by the first and then the third one; the fourth fraction was inactive. These data suggest that local accumulation of peptides in vivo may be of clinical relevancy.

6-methyl-1,2,3,5-tetrahydroimidazo(2,1-b)quinazolin-2-one, a potent inhibitor of ADP-induced platelet aggregation.

6-methyl-1,2,3,5-tetrahydroimidazo(2,1-b)quinazolin-2-one, a potent inhibitor of ADP-induced platelet aggregation.