The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE1

After oral administration, ticlopidine specifically inhibits ADP-induced platelet aggregation, prolongs the bleeding time and prevents thrombosis in man. Its mechanism of action is not well known. Ticlopidine inhibits ADP-induced binding of fibrinogen to platelet glycoprotein GP IIb-IIIa but not shape change and increases deaggregation. Ticlopidine has no direct effect on the GP IIb-IIIa complex. We studied the effects of ticlopidine (500 mg/day for 8 days) in four healthy male volunteers on washed platelet aggregation induced by 5 microM ADP or thrombin (0.1 units/mL) and potentiated by 1 microM adrenaline (Adr), on basal and 1 microM PGE1-stimulated cAMP levels and on elevation of cytosolic free Ca2+ concentration ([Ca2+]i). We found that: (i) ticlopidine inhibits aggregation by ADP but not the potentiation by Adr of ADP-induced aggregation; (ii) ADP, Adr or thrombin decreases cAMP levels raised by PGE1, an effect inhibited by ticlopidine only for ADP and not for Adr or thrombin; and (iii) Ca2+ influx and Ca2+ mobilization from internal stores were not affected. These results suggested that ticlopidine or a metabolite impairs the coupling mechanism of the ADP aggregation pathway at an unknown level.     

The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man

The thienopyridines, ticlopidine and PCR 4099, inhibit ex vivo aggregation in response to ADP and other agonists. It has been shown that ticlopidine induces a functional defect in the binding of fibrinogen to its platelet membrane receptor. We have studied the effects on platelet functions of PCR 4099 in rat and in man. The aim of the study was to check the possibility of a direct modification of the fibrinogen binding site on the GP IIb-IIIa complex. Washed platelet suspensions were used for aggregation and fibrinogen binding studies. Platelet lysates were submitted to SDS-polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and immunoprecipitation. We found that administration of PCR 4099 inhibited selectively and irreversibly ADP-induced aggregation. Although the effect of ADP on aggregation was blocked, PCR 4099 did not modify ADP-induced shape change. Only the effects of low concentrations of thrombin on platelet aggregation were inhibited. Fibrinogen binding was dramatically inhibited in rat and in man when platelets were stimulated with ADP and low concentrations of thrombin. At high concentration of thrombin there still remained a part of fibrinogen binding inhibition although aggregation was not impaired. Electrophoretic and immunoelectrophoretic studies showed no difference before and after treatment by PCR 4099. In particular, the GP IIb-IIIa-complex was not dissociated, its electrophoretic mobility was not changed and three monoclonal anticomplex antibodies recognized it in the same manner before and after treatment. We conclude that PCR 4099 selectively inhibits the ADP aggregation pathway and that the inhibition of fibrinogen binding is probably not due to a direct modification of the GP IIb-IIIa complex.     

In-vitro and Ex-vivo Inhibition of Blood Platelet Aggregation by Naftazone

Because of the considerable interest in the role of platelets and antiplatelet therapy in cardiovascular disease, including the aggregation of platelets to each other during arterial thrombosis and atherogenesis, we have studied the effect of naftazone (Etioven), an original vasculotropic drug on platelet aggregation. Rat and human platelets were prepared and incubated in-vitro with different concentrations of naftazone. We found that naftazone inhibited both platelet secretion and aggregation in platelet-rich plasma (PRP) and washed platelets after stimulation with thrombin or ADP. Rats were also treated intraperitoneally for five days with various naftazone doses (0.125-10 mg kg^?1) and ex-vivo platelet aggregation compared, at various times after the last injection, with that of control animals. Inhibition by naftazone was dose-dependent in both PRP and isolated platelets. The inhibition was transient, a maximum value (～ 50%) being obtained about 3–6 h after the last injection, with a return to near-control values after 24 h. Naftazone also facilitated platelet deaggregation after in-vitro stimulation with thrombin or ADP. In another series of experiments, rats were treated intraperitoneally for five days with 10 mg kg^?1 of aspirin, ticlopidine, dipyridamole or naftazone. Platelets were prepared and tested for aggregation 90 min after the last injection. Thrombin-induced aggregation in PRP and washed platelets was significantly reduced after in-vivo treatment with ticlopidine and naftazone. Except for dipyridamole, all the drugs inhibited ex-vivo ADP-induced aggregation in PRP. In isolated platelet preparation, only naftazone induced a significant inhibition of ADP-or thrombin-stimulated aggregation. We conclude that naftazone inhibits platelet aggregation in-vitro and ex-vivo.     

ADP receptor antagonists as antiplatelet therapeutics

With the cloning of the P2Y12 receptor, the molecular basis for ADP-induced platelet aggregation is seemingly complete. Two platelet-bound ADP receptors, P2Y1 and P2Y12, operate through unique pathways to induce and sustain platelet aggregation via the glycoprotein (GP)IIb-IIIa integrin. P2Y1 operates via a glycoprotein q (Gq) pathway, activates phospholipase C, induces platelet shape change and is responsible for intracellular calcium mobilisation. P2Y12 inhibits adenylyl cyclase through a glycoprotein i (Gi)-dependent pathway, and is the target of the clinically used thienopyridines, ticlopidine (Ticlid, F. Hoffman-La Roche) and clopidogrel (Plavix, Bristol-Myers Squibb/Sanofi-Synthelabo). In addition, the receptor is targeted by the ADP analogue AR-C66096, which is currently in Phase IIb clinical trials, as well as other non-nucleoside-based preclinical leads.     

Cardiotoxin from Naja naja atra snake venom: A potentiator of platelet aggregation

Cardiotoxin, isolated from Naja naja atra snake venom, potentiates platelet aggregation induced by ADP, thrombin, collagen and venom phospholipase A₂. The malondialdehyde formation caused by ADP, thrombin and venom phospholipase A₂ were also increased in the presence of cardiotoxin. Both potentiation of aggregation and increase in malondialdehyde were blocked by indomethacin or Ca²⁺ (5 mM or 0.05 mM). Cardiotoxin did not potentiate thrombin-induced aggregation of p-bromophenacyl bromide-modified platelets. Thromboxane B₂ formation induced by thrombin or collagen was also increased by cardiotoxin, while that by arachidonate was not affected. As a membrane-active polypeptide, cardiotoxin might augment the Ca²⁺-flux during the activation of the platelet membrane by aggregation inducers and then increase the activation of endogenous phospholipase A₂.     

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca²⁺ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca²⁺ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.     

Acetal phosphatidic acids: novel platelet aggregating agents

1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.

2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 μM for human platelets and 0.25-0.5 μM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.

3 PGAP-induced irreversible aggregation of [¹⁴C]-5-hydroxytryptamine ([¹⁴C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0±2.4% (s.e.) of the platelet [¹⁴C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [¹⁴C]-5-HT-labelled sheep platelets was dose-dependent.

4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A₂ inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E₁ (PGE₁); these agents abolished the second, but not the first phase of ADP-induced aggregation.

5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 μM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 μM.

6 It is concluded that the acetal phosphatidic acids induce platelet aggregation per se by direct action at the platelet membrane, and that the acetal function is of primary importance in their potent platelet-stimulating activity. Moreover, as the acetal phosphatidic acids are the major components of the smooth muscle-contracting acidic phospholipid tissue extract `Darmstoff' (Vogt, 1949), their potent platelet-aggregating properties may be of physiological or pathological significance.

     

Effects of phosphate-modified analogs of adenosine 5′-diphosphate and adenosine 5′-triphosphate at P2T-purinoceptors mediating human platelet activation by ADP

Adenosine 5′-diphosphate (ADP) induces human platelet aggregation and inhibits stimulated adenylate cyclase by an action at P_2T-purinoceptors. Both of these effects of ADP are inhibited competitively by ATP. Structure-activity relationships for phosphate-modified analogs of ADP and adenosine 5′-triphosphate (ATP) were studied by testing their effects on human platelet activation. Of the ADP analogs, only β,γ-imido-ADP (AMPNHP) induced platelet aggregation, but was a weak partial agonist (pA₅₀ 4.53). ADP-induced platelet aggregation was antagonized noncompetitively by β,γ-methylene-ADP (AMPCP) (pA₂ 3.2), β,γ-ethylene-ADP (AMPCCP) (pA₂ 4.42), and β,γ-difluoromethylene-ADP (AMPCF₂P) (pA₂ 4.77), and competitively by β,γ-dichloromethylene-ADP (AMPCCl₂P) (pA₂ 4.68). None of the ADP analogs inhibited prostaglandin E₁ (PGE₁)-stimulated adenylate cyclase, and ADP-induced inhibition of PGE₁-stimulated adenylate cyclase was unaffected by AMPNHP, AMPCP, or AMPCCP (100 μM), but was antagonized by AMPCF₂P (pA₂ 4.36) and AMPCCl₂P (4.24). ADP-induced platelet aggregation was antagonized competitively by the ATP analogs β,γ-difluoromethylene-ATP (AMP-PCF₂P) (pA₂ 4.55), β,γ-dichloromethylene-ATP (AMP-PCCl₂P) (pA₂ 4.42), and β,γ-imido-ATP (AMP-PNHP) (pA₂ 4.32) and non-competitively by 2-methylthio-β,γ-methylene-ATP (2-MeS-AMP-PCP), 2-methylthio-β,γ-difluoromethylene-ATP (2-MeS-AMP-PCF₂P), and 2-methylthio-β,γ-dichloromethylene-ATP (2-MeS-AMP-PCCl₂P). In summary, agonist activity at the human platelet P_2T-purinoceptor was extremely sensitive to alterations to the diphosphate chain of ADP, and only AMPNHP induced platelet aggregation. Increasing the electronegativity of the methylene group by halogen substitution increased the antagonist potency of the ADP analog AMPCP but resulted in little or no change in the antagonist potencies of the ATP analogs AMP-PCP and 2-MeS-AMP-PCP. © 1996 Wiley-Liss, Inc.     

Effect of an inhibitor of platelets aggregation, ticlopidine of energy transduction of rat liver mitochondria in vitro.

The new platelet aggregation inhibitor, ticlopidine (5-(2-chloro-benzyl)-4-5-6-7-tetrahydrothieno (3-2-c) pyridine is an inhibitor of energy transduction in isolated rat liver mitochondria. Results show that the compound below concentrations of 100 nmoles/mg of mitochondrial proteins stimulates state 4 oxidation, inhibits state 3 oxidation (except with ascorbate as substrate), decreases ADP-O ratio and respiratory control, and stimulates the latent ATPase activity. This potent stimulation. ten and a half times greater than control, is inhibited by oligomycin (6 μg/mg protein) and is dependent on exogenous Mg²⁺. The inhibition of state 3 oxidation, with glutamate-malate and succinate as substrates. is not reversed by 2–4 DNP and ticlopidine inhibits in a same extent the ADP and DNP-stimulated respiration. In the presence of succinate uncoupled respiration is more susceptible to inhibition by ticlopidine with sonic submitochondrial particles (I₅₀ = 370 nmoles/mg protein) than with intact mitochondria (I₅₀ = 75 nmoles/mg protein). Ticlopidine could act as an uncoupler and as an inhibitor of the electron transport chain, between NADH-dehydrogenase and cytochrome c, and as an inhibitor of the succinate translocation. It is suggested that the blocking effect on mitochondrial energy might play some part in the ticlopidine anti-aggregating properties.     

Effects of 2-methylthioadenosine 5′-β,γ-methylenetriphosphonate and 2-ethylthioadenosine 5′-monophosphate on human platelet activation induced by adenosine 5′-diphosphate

Adenosine 5′-diphosphate (ADP) induces human platelet aggregation, increases intracellular levels of free calcium, and inhibits stimulated adenylate cyclase. These effects of ADP are mediated by P_2T-purinoceptors that are inhibited specifically and competitively by adenosine 5′-triphosphate (ATP). Inhibition of ADP-induced aggregation and increases in calcium by 2-alkylthio analogs of ATP and of adenosine 5′-monophosphate (AMP) are also specific, but the inhibition is non-surmountable. To examine further the nature of inhibition of ADP-induced platelet activation by 2-alkylthio analogs, the effects of 2-methylthioadenosine 5′-β,γ-methylenetriphosphonate (2-MeS-AMP-PCP) and 2-ethylthioadenosine 5′-monophosphate (2-EtS-AMP) were tested on ADP-induced platelet aggregation and inhibition of adenylate cyclase. 2-MeS-AMP-PCP inhibited platelet aggregation induced by ADP but not by epinephrine, arachidonic acid, 5-hydroxytryptamine (5-HT), platelet activating factor (PAF), or 11α,9α-epoxymethano-prostaglandin H₂ (U46619). Inhibition of ADP-induced platelet aggregation by 2-MeS-AMP-PCP was non-surmountable, and it achieved only 50% inhibition of ADP (5 μM)-induced aggregation. 2-MeS-AMP-PCP achieved 100% inhibition of ADP (5 μM)-induced inhibition of prostaglandin E₁-stimulated adenylate cyclase, and Schild analysis showed the inhibition to be potent (pA₂ 7.3) and competitive (slope 1.12). 2-MeS-AMP-PCP inhibited platelet aggregation induced by adenosine 5′-O-2-thiodiphosphate (ADP-β-S), which inhibited stimulated adenylate cyclase activity, but did not inhibit aggregation induced by adenosine 5′-O-1-thiodiphosphate (ADP-α-S), which does not inhibit stimulated adenylate cyclase. 2-EtS-AMP behaved similarly to 2-MeS-AMP-PCP. These results suggest that ADP may induce aggregation by interacting with two forms of the calcium-mobilizing P_2T-purinoceptor, only one of which is coupled to inhibition of adenylate cyclase and at which 2-alkylthio analogs of ATP and AMP are specific competitive antagonists. © 1996 Wiley-Liss, Inc.     

Components of traditional Chinese medicine inhibit platelet aggregation by blocking ADP receptor subtypes P2Y_1 and P2Y_(12)

Platelets aggregation and thrombosis formation are major reasons of cardiovascular and cerebral vascular diseases.To develop new generative,potent and safe agents for inhibiting platelet aggregation and preventing above diseases are urgently required.Some traditional Chinese medicines of″Houxue Huayu″have been shown to inhibit platelet aggregation potently.In the present study the mechanisms and the molecular targets of puerarin,salvianolic acid B and the analogue of 3-n-butylphthalide,dl-PHPB were investigated and compared with ticlopidine.Four platelet aggregation inducers,ADP,arachidonic acid,collagen and thrombin were used in the study.It was found that puerarin and dl-PHPB specifically inhibited ADP induced platelet aggregation like ticlopidine did.However,salvianolic acid B inhibited both ADP and collagen induced platelet aggregations with similar potency.Due to existing two ADP receptor subtypes on platelets,P2Y1 and P2Y12,we studied the action of above compounds on the receptors and the signaling pathways.It was found that dl-PHPB decreased IP1 accumulation produced by ADP,but had no effect on IP1 level induced by m-3M3 FBS,an activator of PLC.M-3M3 FBS might attenuate the inhibitory effect of dl-PHPB on ADP-induced platelet aggregation.In addition,dl-PHPB did not affect cyclic AMP formation in platelets by ADP,which is different from P2Y12 antagonist ticlopidine.Puerarin showed the similar effects of dl-PHPB.Therefore,the actions of dl-PHPB and puerarin might be through P2Y1receptor-PLC-βpathway.Salvianolic acid B did not reduce the IP1 accumulation stimulated by ADP.It might act on the receptor subtype P2Y12.Our results suggest that components of Chinese herb medicine might be a resource for development of novel anti-platelet drugs.     

虎杖晶Ⅳ号对家兔血小板聚集的影响

Polydatin is 3,4′, 5-trihydroxystilbene-3-β-mono-D-glucoside isolated from Polygonum cuspidatum Sieb et Zucc. The actions of polydatin 0.6, 2.6 and 10.4 μg/ml in vitro and 5 mg/kg in vivo on platelet aggregation were studied in rabbits. Polydatin was shown to inhibit ADP, arachidonic acid, Ca²⁺ and clonidine-induced platelet aggregation significantly. In vitro, the rates of inhibition were 18～36% (ADP-induced), 26～58% (arachidonic acid-induced) and 42～64% (Ca²⁺-induced). However, the inhibition of thrombin-induced aggregation was not statistically significant. In in vivo experiments, 5, 30 and 60 min after iv polydatin, the rates of inhibition were 52～63% (ADP-induced), 59～76% (arachidonic acidinduced) and 10～16% (Ca²⁺-induced) respectively.     

Platelet P2Y12 receptors are involved in the haemostatic effect of notoginsenoside Ft1, a saponin isolated from Panax notoginseng

BACKGROUND AND PURPOSE

Saponins isolated from Panax notoginseng (Burk.) F.H. Chen have been shown to relieve thrombogenesis and facilitate haemostasis. However, it is not known which saponin accounts for this haemostatic effect. Hence, in the present study we aimed to identify which saponins contribute to its haemostatic activity and to elucidate the possible underlying mechanisms.

EXPERIMENTAL APPROACH

Platelet aggregation was analysed using a platelet aggregometer. Prothrombin time, activated partial thromboplastin time and thrombin time were measured using a blood coagulation analyser, which was further corroborated with bleeding time and thrombotic assays. The interaction of notoginsenoside Ft1 with the platelet P2Y₁₂ receptor was determined by molecular docking analysis, cytosolic Ca²⁺ and cAMP measurements, and phosphorylation of PI3K and Akt assays.

KEY RESULTS

Among the saponins examined, Ft1 was the most potent procoagulant and induced dose-dependent platelet aggregation. Ft1 reduced plasma coagulation indexes, decreased tail bleeding time and increased thrombogenesis. Moreover, it potentiated ADP-induced platelet aggregation and increased cytosolic Ca²⁺ accumulation, effects that were attenuated by clopidogrel. Molecular docking analysis suggested that Ft1 binds to platelet P2Y₁₂ receptors. The increase in intracellular Ca²⁺ evoked by Ft1 in HEK293 cells overexpressing P2Y₁₂ receptors could be blocked by ticagrelor. Ft1 also affected the production of cAMP and increased phosphorylation of PI3K and Akt downstream of P2Y₁₂ signalling pathways.

CONCLUSION AND IMPLICATIONS

Ft1 enhanced platelet aggregation by activating a signalling network mediated through P2Y₁₂ receptors. These novel findings may contribute to the effective utilization of this compound in the therapy of haematological disorders.     

Effect of 2(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) on human platelets in vitro

The effect on human platelets of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) was tested in vitro by measuring cyclic adenosine monophosphate (cAMP) level, cytosolic Ca⁺⁺, [¹²⁵I]fibrinogen binding as well as aggregation induced by several agonists. AP155 dose-dependently inhibited aggregation both in platelet rich plasma (PRP) and in washed platelets (WP), exerting its maximal power in the presence of collagen, ADP and platelet activating factor (PAF). It specifically inhibited the activity of cAMP high affinity phosphodiesterase (PDE), resulting in a sufficient increase in cAMP levels to activate cAMP-dependent protein kinase. AP155 was able to inhibit aggregation, the increase in cytosolic Ca⁺⁺ induced by thrombin, and fibrinogen binding to ADP or thrombin-stimulated platelets. Thus, this new pyridopyrimidine derivative exerts its antiplatelet activity by increasing cAMP intracellular concentration.     

Inhibition in vitro of platelet aggregation and arachidonic acid metabolism by flavone

The effects of flavone on platelet aggregation and arachidonic acid (AA) metabolism were tested in vitro. When incubated at a concentration of 50 μM, flavone completely suppressed platelet aggregation induced by 150 μM AA in thirty-six out of forty-three subjects tested. A lower concentration (10 μM) was effective in about 50% of the donors. Flavone also inhibited the second wave of aggregation induced by epinephrine and ADP. Platelet thromboxane formation, estimated both by radioimmuno-assay measurements and by studies of ¹⁴C-labeled AA metabolism, was depressed by flavone. Flavone-treated platelets preferentially utilized [¹⁴C]AA for the lipoxygenase pathway while cyclo-oxygenase activity was depressed. Adenosine 3':5'-cyclic monophosphate (cAMP) was measured in flavone-treated and control platelets. While their baseline levels were similar, flavone-treated platelets showed a lower stimulation of cAMP induced by prostacyclin (PGI₂) than did controls. Phosphodiesterase activity was not affected by flavone as judged from the decay rates of PGI₂-stimulated cAMP levels. From these findings we conclude that the antiaggregating activity of flavone is not a consequence of changes in platelet cAMP but is due to inhibition of cyclo-oxygenase.     

Ticlopidine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in platelet-dependent disease states

Ticlopidine is an inhibitor of platelet action that has been used in the treatment of a variety of disease states in which platelets play a prominent role. Studies in animals and man have demonstrated that ticlopidine is a potent inhibitor of platelet aggregation induced by adenosine diphosphate (ADP), and variably inhibits aggregation due to collagen, adrenaline (epinephrine), arachidonic acid, thrombin, and platelet activating factor. Inhibition of platelet aggregation is both dose- and time-related, with its onset of activity being 24 to 48 hours, its maximal activity occurring after 3 to 5 days, and its activity still being present 72 hours after a final dose. Ticlopidine also inhibits the release reaction of platelets, prolongs bleeding time, reduces plasma levels of platelet factor 4 and beta-thromboglobulin in patients in whom these proteins are elevated, and may also inhibit platelet adhesion, increase red cell filtrability and decrease whole blood viscosity. In a large number of animal models, ticlopidine markedly inhibits thrombus formation or graft occlusion. Ticlopidine is well absorbed after oral administration. It is extensively metabolised and at least one of its metabolites is pharmacologically active. Therapeutic trials in patients with chronic arterial occlusion due to thrombangitis obliterans or arteriosclerosis obliterans, post-myocardial infarction, cerebrovascular thromboembolic disease, subarachnoid haemorrhage, vascular shunts or fistulas for haemodialysis, and sickle cell disease have shown promise for the use of ticlopidine. However, trials of patients with intermittent claudication, angina pectoris, diabetes mellitus with microvascular disease, aortocoronary bypass grafts, and vascular prostheses have had conflicting results or have shown an unfavourable side effect profile. Further studies are clearly required to establish the role of ticlopidine in many of these areas, some of which are already in progress. Overall, side effects occur in 10 to 15% of patients receiving ticlopidine. The most common side effects are gastrointestinal disturbances and skin rashes. Neither of these necessarily require discontinuation of therapy in most patients. Agranulocytosis, thrombocytopenia, and cholestatic jaundice have also been reported. Bleeding is infrequent except possibly in patients receiving ticlopidine prior to some surgical procedures.     

In vitro adrenaline and collagen-induced mobilization of platelet calcium and its inhibition by naftopidil, doxazosin and nifedipine

Aims The aim of the study was to obtain further information regarding the modes of action of doxazosin, naftopidil and nifedipine on platelet function.Methods We conducted an in vitro study of drug influences on adrenaline and collagen-induced mobilization of platelet calcium.‘fn2\Results In the presence of fibrinogen (300 μg ml⁻¹ ) both collagen (5 μg ml⁻¹ ) and adrenaline (16 μm ) stimulated the aggregation of washed platelets. Collagen induced a transient rise (+4.97±0.63 μm ) in platelet Ca²⁺ concentration, [Ca²⁺]_i, as measured using the photoprotein aequorin, which coincided with the onset of aggregation. Adrenaline induced a smaller rise (+3.6±0.96 μm ) which, however, occurred after the onset of aggregation. Naftopidil, an α₁-adrenoreceptor antagonist produced a concentration-dependent inhibition of collagen-induced Ca²⁺ mobilization, maximum inhibition (22.9±4%, P<0.05) occurring with 40 μm naftopidil. The inhibition of Ca²⁺ mobilization was not reflected by a concentration-dependent inhibition of platelet aggregation, although 40 μm naftopidil produced statistically significant inhibition (23.3±11.7%, P<0.05). The adrenaline-induced rise in [Ca²⁺]_i was inhibited dose dependently by naftopidil (e.g. 40 μm naftopidil, 100±0%, P<0.05), as was aggregation (40 μm naftopidil, 100±0%, P<0.05). Doxazosin, another α₁-adrenoreceptor blocker, inhibited Ca²⁺ mobilization induced by collagen to similar extents as for naftopidil (30 μm doxazosin, 17.4±2.5%, P<0.05), but did not inhibit platelet aggregation. It also inhibited the adrenaline-induced rise in [Ca²⁺]_i in a concentration-dependent manner (30 μm doxazosin, 37.6±13.7%, P<0.05), significant inhibitions of platelet aggregation also being produced (30 μm, 49.6±17.2%, P<0.05). As expected, the calcium channel blocker nifedipine produced concentration-dependent inhibitions of both collagen-induced Ca²⁺ mobilization (e.g. 28 μm nifedipine, 47.8±2.7%, P<0.05) and aggregation (28 μm, 55.1±9.2%, P<0.05).Conclusions These data indicate that the α₁-adrenoreceptor blockers, naftopidil and doxazosin, inhibit Ca²⁺ mobilization, this mechanism being possibly the means whereby these drugs inhibit platelet aggregation.     

Adenosine analogs and human platelets. Effects on nucleotide pools and the aggregation phenomenon.

The interaction between human blood platelets and adenosine and various adenosine analogs was examined. Effects were found both on the pools of nucleotides, as examined by the technic of high pressure liquid chromatography, and on the phenomenon of ADP-induced platelet aggregation. Although the normal ratio of ATP and ADP in platelets is about 1.5:1, after incubation with adenosine-8-¹⁴C, ATP and ADP were labeled in a ratio of about 7:1. This is consistent with a distribution of the nucleotides among storage and metabolic pools, with the adenosine-8-¹⁴C entering principally the metabolic pool. After 2 hr of incubation with 0–5 mM 2-fluoroadenosine (F-Ado), the concentration of F-ATP was approximately 12 μmoles/10¹¹ platelets. The ratio of F-ATP to F-ADP was approximately 7:1, indicating that it entered primarily the metabolic nucleotide pool. Also, during the first hr of incubation, as the F-ATP concentration increased, the ATP concentration fell. When F-ATP-containing platelets were treated with thrombin, an aggregator and storage granule releaser, the nucleotides released into the medium consisted principally of ATP and ADP in a ratio of about 0.8:1, with very little 2-fluoroadenine-containing nucleotides. After thrombin treatment, the washed platelet pellet contained most of the 2-fluoroadenine nucleotides, but with significant increases in the concentrations of F-AMP and F-ADP. This suggests that F-ATP can replace ATP as the energy donor for the aggregation and release phenomena.As reported elsewhere, adenosine strongly inhibits platelet aggregation induced by ADP. However, this inhibitory effect disappears after preincubation for about 30 min. If the preincubation is carried out in the presence of coformycin, a tight-binding inhibitor of adenosine deaminase (K_i ? 1 × 10^?10M), the inhibition of aggregation by adenosine is markedly prolonged, indicating that the loss of inhibition results from conversion of adenosine to inosine by adenosine deaminase. ADP-induced aggregation is powerfully inhibited by F-Ado, and the inhibition becomes more pronounced on prolonged incubation. This is consistent with the observation that F-Ado has very weak substrate activity with adenosine deaminase. The analog, N⁶-phenyladenosine, an inhibitor of adenosine kinase that does not form analog nucleotides in platelets, inhibits aggregation strongly, and the inhibition is maintained during incubation of 1 hr. Several other adenosine analogs only weakly inhibit ADP-induced aggregation even in the presence of coformycin. These include 2'-deoxyadenosine, 3'-deoxyadenosine (cordycepin), arabinosyladenine, and formycin A, a C-nucleoside. However, significant quantities of nucleotides of formycin A are formed in platelets in the presence of coformycin.     

Effects of the calmodulin antagonists fendiline and calmidazolium on aggregation,secretion of ATP,and internal calcium in washed human platelets

Summary Ca²⁺-calmodulin dependent phosphorylation of myosin is essential for the induction of platelet shape change and subsequent reactions. Therefore, we studied the effects of the calmodulin antagonists fendiline and calmidazolium on the thrombin-induced aggregation, secretion of ATP, and increases in the intracellular free calcium concentration ([Ca²⁺]_i) in washed human platelets in the absence and presence of extracellular Ca²⁺. In Ca²⁺ free medium, fendiline (10–100 M) and calmidazolium (3–30 M) concentration-dependently inhibited aggregation. The effect of fendiline could be partly reversed by extracellular Ca²⁺ and higher thrombin concentrations. Furthermore, aggregations induced by the calcium ionophore ionomycin and by the protein kinase C-activator 4--phorbol 12-myristate 13-acetate were inhibited by fendiline, although to a smaller degree than the thrombin-induced aggregation. Thrombin-induced secretion of ATP was attenuated by low concentrations of fendiline (1–30 M) and calmidazolium (1 M) but enhanced by higher concentrations (10–30 and 3–10 M, respectively), independently of extracellular Ca²⁺. Fendiline (1–10 M) did not affect [Ca²⁺]_i in resting and thrombin-stimulated platelets. At higher concentrations (30–100 M), it induced increases in [Ca²⁺]_i in unstimulated platelets and attenuated the response to thrombin in Ca²⁺ free medium, whereas thrombin-induced Ca²⁺ influx was markedly enhanced. Similar results were obtained with calmidazolium (1–3 M). These stimulating effects on ATP secretion and on [Ca²⁺]_i of fendiline and calmidazolium may be attributed to interactions with platelet membranes by which the permeability of small cations is increased. In contrast to these actions that would be normally considered platelet-activating, our study demonstrates that the two calmodulin antagonists effectively antagonize [Ca²⁺]_i ^– mediated induction of platelet aggregation. Send offprint requests to A. Lückhoff at the above address     

Differential inhibition of human platelet aggregation and thromboxane A2 formation by L-arginine in vivo and in vitro

We compared the effects of L-arginine (L-ARG), the precursor of endogenous NO, on platelet aggregation and thromboxane A₂ formation in vivo and in vitro. Human platelet-rich plasma (PRP) was anticoagulated with citrate (which decreases extracellular Ca²⁺) or with recombinant hirudin (which does not affect extracellular Ca²⁺). Two groups of 10 healthy male volunteers received intravenous infusions of L-ARG (30 g or 6 g, 30 min) or placebo. Blood was collected immediately before and at the end of the infusions for aggregation by ADP or collagen. Infusion of L-ARG inhibited ADP-induced aggregation in PRP anticoagulated with citrate by 37.5 ± 6.3% (P < 0.05). In PRP anticoagulated with hirudin, aggregation was inhibited by 33.6 ± 16.0% (P < 0.05). L-ARG infusion also inhibited platelet TXB₂ formation and slightly, but not significantly decreased the urinary excretion rate of 2,3-dinor-TXB₂; cGMP concentrations in PRP were significantly elevated during L-arginine infusion. In vitro preincubation with L-ARG (10 μM–2.5 mM) inhibited platelet aggregation in PRP anticoagulated with r-hirudin, but not citrate. This effect was stereospecific for L-arginine, as D-arginine had no effect. It was dependent upon NO synthase activity, as indicated by increased cGMP levels in PRP. Moreover, both the NOS inhibitor L-NMMA and the inhibitor of soluble guanylyl cyclase ODQ antagonized the effects of L-ARG. Haemoglobin, an extracellular scavenger of NO, partly antagonized the antiplatelet effects of L-ARG. 8-Br-cyclic GMP and the exogenous NO donor linsidomine inhibited aggregation in PRP anticoagulated with citrate or r-hirudin. The inhibitory effects of L-ARG on platelet aggregation in vitro were paralleled by increased cyclic GMP levels; L-ARG also inhibited platelet TXB₂ formation in PRP anticoagulated with r-hirudin, but not citrate. We conclude that the L-arginine/NO pathway is present in human platelets as a Ca²⁺-dependent anti-aggregatory pathway. In vivo the formation of NO from L-ARG by endothelial cells may contribute to the platelet-inhibitory effects of L-ARG. NO-releasing compounds like linsidomine inhibit platelet aggregation in vitro independent of extracellular Ca²⁺. Received: 17 April 1997 / Accepted: 17 October 1997