SUMMARY1. Adenosine diphosphate (ADP) and adrenaline caused the aggregation of human platelets suspended in plasma containing citrate anticoagulant and stirred at 370 C. The aggregation occurred in two phases and the second phase was associated with the appearance in the plasma of up to 30 % of the ATP and 55 % of the ADP present in the platelets. The concentration of ADP appearing in the plasma was up to 7 times the concentration added.2. Radioactivity was released by ADP and by adrenaline from platelets labelled with radioactive 5-hydroxytryptamine; this release was closely correlated with the second phase of aggregation and with the release of nucleotides.3. Acid phosphatase, ,-glucuronidase and adenylate kinase were released to a small extent during second phase aggregation by ADP or adrenaline; thrombin and collagen particles caused significantly greater release of fi-glucuronidase than of either acid phosphatase or of adenylate kinase.4. Morphological changes indicating degranulation of the platelets were observed during the second phase of aggregation produced by adrenaline and by ADP.5. The second phase of aggregation, degranulation of platelets, and the release of nucleotides, of labelled 5-hydroxytryptamine and of enzymes, were all inhibited by concentrations of amitriptyline which did not inhibit aggregation.
Clopidogrel, like the homologous thienopyridine derivative tidopidine, selectively inhibits platelet aggregation induced by ADP. We have previously described two nucleotide-binding sites on platelets related to ADPmediated platelet responses. The first is a high-affinity binding she for 2-methylthio-ADP (2-MeSADP) that is linked to the inhibition of stimulated adenylate cyclase. The second is the 100-kd exofacial membrane protein aggregin, which is labeled by the reactive ADP analogue 5'-p-fluorosulfonyibenzoyl adenosine (FSBA) that is related to shape change and aggregation. We set out to determine if either of these sites is blocked in vivo by clopidogrel or its active metabolite. Six subjects were given clopidogrel (75 mg/day for 10 days) in a double-blind crossover experiment All of the subjects developed prolonged bleeding times while taking the drug. The rate of onset of the effect on bleeding time varied among subjects. Platelet aggregation induced by ADP or thrombin was significantly impaired by the drug treatment, but no effect was detected on shape change. The incorporation of [ 3 H]FSBA into aggregin was also unaffected. Inhibition of adenylate cyclase by ADP or by 2-MeSADP was greatly reduced in all subjects, and in the case of 2-MeSADP, there was evidence for a noncompetitive effect Inhibition of adenylate cyclase by epinephrine was unaffected. In the three subjects for whom binding measurements were made, the number of binding sites for [32 P]2-MeSADP was reduced from 534±44 molecules per platelet during control and placebo periods (11 determinations) to 199±78 molecules per platelet during drug treatment (three determinations). There was no consistent change in the binding affinity. The inhibition of platelet function by clopidogrel is associated with a selective reduction in the number of functional receptors mediating the inhibition of stimulated adenylate cyclase activity by ADP.
SUMMARY1. Adrenaline at concentrations too low to cause aggregation of human platelets potentiates the aggregation by adenosine diphosphate. Noradrenaline has the same effect but is less active than adrenaline; isopropylnoradrenaline is inactive or inhibitory.2. The potentiation of adenosine diphosphate by catecholamines is blocked by the adrenergic ac-receptor antagonists phentolamine and dihydroergotamine but not by 2-halogenoethylamines or by adrenergic fl-receptor antagonists.3. Both the first and second phases of adenosine diphosphate aggregation are potentiated by catecholamines but the second phase more than the first.4. The release from the platelets of adenine nucleotides which is associated with the second phase of aggregation is also increased by adrenaline.
1. The involvement of intracellular 3':5'-cyclic AMP in the inhibition of platelet aggregation by prostaglandin E(1), isoprenaline and adenosine has been examined by a radiochemical technique. Platelet-rich plasma was incubated with radioactive adenine to incorporate (14)C radioactivity into platelet nucleotides. Pairs of identically treated samples were taken, one for the photometric measurement of platelet aggregation induced by ADP, the other for estimation of the radioactivity of 3':5'-cyclic AMP. 2. Theophylline, papaverine, dipyridamole and 2,6-bis-(diethanolamino)-4-piperidinopyrimido[5,4d]pyrimidine (compound RA233) were found to inhibit 3':5'-cyclic AMP phosphodiesterase from platelets. At concentrations of 3':5'-cyclic AMP greater than 50mum the most active inhibitor was dipyridamole; at 3':5'-cyclic AMP concentrations less than 19mum, papaverine and compound RA233 were more active than dipyridamole. 3. In the presence of compound RA233 (50mum), the effectiveness of prostaglandin E(1) as an inhibitor of platelet aggregation was increased tenfold. Compound RA233 also increased the stimulation by prostaglandin E(1) of the incorporation of radioactivity into 3':5'-cyclic AMP. 4. Compound RA233 (50mum) increased the effectiveness of both adenosine and 2-chloroadenosine as inhibitors of aggregation by 70-100-fold, and in the presence of compound RA233 both adenosine and 2-chloroadenosine stimulated the incorporation of radioactivity into 3':5'-cyclic AMP; the extent of the stimulation was proportional to the logarithm of the nucleoside concentration. 5. Compound RA233 (100-500mum) inhibited platelet aggregation by itself and caused small increases in the radioactivity of 3':5'-cyclic AMP. Partial positive correlations were found between the radioactivity of 3':5'-cyclic AMP in platelets measured at the time of addition of the aggregating agent (ADP) and the extent to which the aggregation was inhibited. 6. The results are interpreted as indicating that adenosine, 2-chloroadenosine, isoprenaline, prostaglandin E(1) and drugs that inhibit platelet 3':5'-cyclic AMP phosphodiesterase all inhibit aggregation by a common mechanism involving intracellular 3':5'-cyclic AMP.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.