Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes.

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.     

Effect of synthetic enantiomeric cannabinoids on platelet aggregation.

The effect of a synthetic pair of enantiomeric cannabinoids on platelet function was evaluated. The nonpsychotropic enantiomer, the 1,1-dimethylheptyl homolog of (+)-(3S,4S)-7-hydroxy-delta-6-tetrahydrocannabinol (HU-211), was found to be more active in inhibiting ADP-induced platelet aggregation than the highly psychotropic (-)-enantiomer (HU-210). The related (+)-(3R,4R) cannabinoid, HU-213, which lacks the 7-hydroxy moiety, exerted its inhibitory effect within a wider range of concentrations. The results indicate a differentiation between psychotropic activity and inhibition of platelet aggregation in the cannabinoid group of compounds.     

Effect of verapamil on platelet aggregation

The influence of the calcium channel blocker verapamil on the aggregation of human blood platelets was studied in vitro in comparison with the calcium channel blocker diltiazem and with the 5-HT antagonist cyproheptadine. Verapamil inhibited the 5-HT-potentiated. ADP-induced aggregation more effectively than the aggregation induced by adrenaline, ADP and collagen. Verapamil antagonized the 5-HT effect in a noncompetitive manner. The same was true of cyprohepatadine which was by more than one order of magnitude more potent than verapamil in inhibiting the 5-HT-induced aggregation. Diltiazem was much less effective than verapamil.     

New carbamoylpiperidines as human platelet aggregation inhibitors

A series of 3-carbamoylpiperidines (nipecotamides) are designed, synthesized and tested for their inhibitory action against adenosine diphosphate (ADP)-induced aggregation of human platelets. A structure-activity analysis of the bis(nipecotamido)aralkane type showed that a substituent on the piperidine ring should preferably be an amide and that the electronegativity of the carbonyl oxygen and the orientation of the amide group affected activities. Based on the knowledge of factors influencing platelet activation and aggregation, a nitric ester moiety which could release nitric oxide (NO) in situ, is incorporated into the nipecotamide structure. These compounds exhibit increased activity compared to those having no -ONO2 function. They also show stereoselectivity, with the meso isomer being approximately twice as potent as the synthetic diastereomeric mixture. Replacement of the -ONO2 function with hydroxyl, ester or alkyl groups considerably diminishes aggregation-inhibitory potential. Nipecotamides are shown here to inhibit the basal and collagen-induced rise in platelet inositol trisphosphate (IP3) levels, as well as phosphoinositide turnover. A comprehensive mechanism of action is proposed taking earlier results into consideration.     

The effect of ADP, calcium and some inhibitors of platelet aggregation on protein phosphokinases from human blood platelets.

A protein phosphokinase (ATP: protein phosphotransferase EC 2.7.1.37) which is stimulated by 3',5'-cyclic adenosine monophosphate (cyclic AMP) has been partially purified from both the cytoplasmic and membrane fractions of human platelets. The kinetics of both enzymes preparations are similar in respect to cyclic AMP, ATP, ADP and AMP. 5-10-minus 7 M cyclic AMP stimulated both preparations by approximately 100%. Both ADP and AMP at a concentration of 5-10-minus 5 M inhibited protein phosphokinase activity of the soluble and membrane preparation by between 50% and 70%. The response of the two enzyme preparations to calcium differed. 10 mM Ca-2+ inhibited soluble protein phosphokinase activity approximately 80% both in the presence and absence of 5-10 minus 7 M cyclic AMP whereas the same concentrations of Ca-2+ inhibited the membrane-bound enzyme by approximately 60% in the presence of 5-10-minus 7 M cyclic AMP and 40% in the absence of cyclic AMP. This observation may be of importance in understanding the mechanism of platelet aggregation.     

Thienopyrimidine-based P2Y12 platelet aggregation inhibitors

Herein we describe the design and synthesis of a novel series of potent thienopyrimidine P2Y₁₂ inhibitors and the negative impact protein binding has on the inhibition of platelet aggregation.     

Anticoagulants and inhibitors of platelet aggregation derived from leeches

Increased life expectancy is associated with aging populations in the developed countries, and we can expect an increased incidence of cardiovascular and inflammatory diseases and cancers. A priority for medical research is to reduce such morbidity. Leeches have been demonstrated to be a useful source of drugs to treat cardiovascular diseases, as they have evolved highly specific mechanisms to feed on their hosts by blocking blood coagulation. Powerful molecules acting at different points in the coagulation cascade or in the inhibition of platelet aggregation have been purified from these animals. Moreover, clinical trials confirm their potential to treat cardiovascular diseases.     

Effect of heparin on platelet aggregation in vitro

Heparin added to citrated platelet rich plasma influences shape change and aggregation of platelets in different ways. In the presence of heparin neither ADP nor collagen induces shape change, while shape change after thrombin or arachidonic acid remains unaltered. Heparin potentiates the first aggregation step induced by ADP and epinephrine but inhibits aggregation induced by thrombin and ristocetin. The second phase of aggregation and the release reaction are not directly influenced by heparin no matter which aggregation agent is used.     

Effect of heparin and heparinoids on platelet aggregation

Biological effectiveness of bovine lung heparin and porcine mucosal heparin was tested in vitro and compared with the effectiveness of 12 semisynthetic heparinoids obtained by sulfation of waste heparin mucopolysaccharides and other biomolecules. Equieffective concentrations of these substances were determined in the sense of prolongation of the thrombin time and the APTT, inhibition of the thrombin- and collagen-induced aggregation, and potentiation of the primary ADP-induced aggregation. The influence of sulfation was proved on the biological effectiveness as well as the significance of the proper choice of the parent structure. Some polycondensates of the polysaccharide type were effective altogether with the sulfated waste heparin mucopolysaccharides. On the contrary, protein structures and low molecular weight glycosides exhibited little or no activity. The effective substances were related to heparin not only by the anticoagulant activity but also by the inhibitory action on the thrombin- and collagen-induced platelet aggregation. Contrary to heparin, higher concentrations of the studied heparinoids strongly potentiated the ADP-induced aggregation response. Strong inhibition of the collagen-induced aggregation was proved after administration of heparin to patients with end-stage renal failure on days without haemodialysis. Less significant changes in the secondary aggregation were observed also after administration of S-heparin (one of the studied heparinoids) to volunteers in the form of the rectal suppositories.     

Polymeric inhibitors of platelet aggregation. Synergistic effects and proposals for a new mechanism

We have studied the inhibition of ADP-induced platelet aggregation in sheep platelet-rich plasma by water-soluble polymers bound to the prostaglandin analogue 5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin ('BW 245' C, (I). The use of unambiguous modes of binding this antiplatelet drug to polymers has enabled us to study some structural features which influence inhibitory activity. Evidence is adduced which indicates that the chemical mechanisms responsible for inhibition by free and coupled BW 245 are similar. The most important observation is a remarkable synergism demonstrated by the greatly enhanced activity of a mixture of a polymer coupled to BW 245 with the uncoupled parent polymer. In some cases (e.g., with high-molecular-weight dextran) the effect may reach (and possibly exceed) two orders of magnitude. The influence of polymer molecular weights and 'cross-polymer' effects have both been examined. A mechanism has been proposed to account for these phenomena, involving adsorption of the added (inactive) polymer on to the platelet membranes, facilitating interaction of the polymer-bound drug with receptors, made more accessible by alteration to the surface geometry. This mechanism is based on physical processes, unlike other explanations of synergistic behaviour, e.g., that of prostaglandins used in conjunction with non-polymeric drugs. The observed dependences of synergistic effects upon polymer molecular weight and type and distribution of drug molecules along chains are typical 'polymer' phenomena which are all consistent with the proposed mechanism.     

Polymeric inhibitors of platelet aggregation. II. Copolymers of dipyridamole and related drugs with N-vinylpyrrolidone

A study has been made of the behaviour of the drugs dipyridamole and mopidamol (RA 233) attached to poly(N-vinylpyrrolidone). The inhibitory activities towards platelet aggregation induced by ADP or platelet activation factor (PAF) in sheep plasma have been examined and found to exceed the activities of the parent drugs, by factors up to 20. At the same time the abilities of the polymer-bound drugs in potentiating prostaglandin-type anti-aggregatory activities are much lower than those of the unattached drugs. The observations are explained in terms of polymer adsorption on to the platelet membranes, producing a high surface concentration of the drugs with consequent high inhibitory action. Intracellular action, such as the inhibition of phosphodiesterase, is reduced by the difficulty experienced by the polymeric drug in passing through the membrane, so that potentiation of prostaglandin activity, particularly against PAF, becomes small. A terpolymer containing units of dipyridamole and the prostaglandin analogue 5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin (245C) shows a degree of ‘self-potentiation’.     

Effects of adrenergic blockers on platelet aggregation.

The action of orciprenaline, tolazoline, propanolol and inpea on platelet aggregation induced by ADP epinephrine and norepinephrine was studied in vitro in human platelet-rich plasma. Orciprenaline did not significantly affect aggregation induced by ADP. Tolazoline inhibits the aggregation induced by epinephrine and norepinephrine more intensely than the beta-blockers. Inpea blocks the platelet aggregation induced by epinephrine and norepinephrine to a greater extent than propanolol at similar concentrations. The beta-blockers inhibit platelet aggregation non-specifically.