Inhibitory mechanism of dipyridamole on platelet aggregation ex vivo

The effects of dipyridamole on platelet aggregation ex vivo and in vitro and on platelet cyclic AMP phosphodiesterase (PDE) were studied, and the mechanism of the ex vivo effects was assessed. Both the ADP- and collagen-induced aggregations ex vivo were inhibited dose-responsively by oral administration of dipyridamole. Maximum dipyridamole levels in the plasma were reached at 30 min after the administration. The inhibitory effects of dipyridamole on platelet aggregation ex vivo reached a maximum at between 1 and 2 hrs. On the other hand, the ADP-induced aggregation in vitro and cyclic AMP PDE activity were not inhibited until after 10 min of incubation at a low concentration of dipyridamole. This mode of inhibition of platelet aggregation in vitro and of cyclic AMP PDE activity agreed with the mode of inhibition in the case of platelet aggregation ex vivo. It is suggested therefore that the ex vivo effects, observed with only a low dipyridamole concentration in the plasma, may be due primarily to inhibition by dipyridamole of the cyclic AMP PDE in platelets.     

Increased dipyridamole plasma concentrations associated with salicylate administration. Relationship to effects on platelet aggregation in vivo

Dipyridamole, in relatively low doses, when used in combination with aspirin was shown to be effective in normalizing reduced platelet survival seen in patients with prosthetic heart valves, whereas either drug, when tested alone, was ineffective. To determine whether this combined drug effect was due to a synergism of the two drugs or due to an alteration in the pharmacokinetics of one drug in the presence of the other, we examined the effects of aspirin and sodium salicylate on 1) the effects of dipyridamole on platelet function and 2) the concentration of dipyridamole in the plasma. Both aspirin and salicylate altered the plasma concentration of dipyridamole and prolonged the effect of dipyridamole on collagen-induced platelet aggregation in the rabbit. This effect appeared to be due to saturation of the glucuronide pathway(s) which is the major route of clearance of dipyridamole from the circulation.     

Inhibition of platelet aggregation in whole blood by dipyridamole and aspirin

We have examined the effects of dipyridamole on platelet aggregation in whole blood both in vitro and after administration to man. The effects of dipyridamole ex vivo were compared with those of aspirin and a combination of dipyridamole and aspirin. In vitro dipyridamole was most effective as an inhibitor of platelet aggregation induced by platelet activating factor (PAF) and low concentrations of arachidonic acid (AA). Its inhibitory effect was always potentiated by adenosine suggesting that its effect on aggregation may be via inhibition of adenosine uptake into blood cells. Ex vivo, dipyridamole, aspirin and the combination of these drugs inhibited the platelet aggregation induced by PAF and AA. Again, adenosine increased the degree of inhibition. These results stress the importance of measuring platelet aggregation in the natural whole blood environment for detection of the inhibitory effects of dipyridamole and suggest a mode of action for the drug.     

Dipyridamole inhibits platelet aggregation in whole blood

Dipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine-5'-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p less than 0.001). A statistically significant inhibition of both collagen (p less than 0.0025) and ADP-induced (p less than 0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37 degrees C with dipyridamole (3.9 microM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood. Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.     

Comparison of the inhibitory effects of the TXA2 receptor antagonist, vapiprost, and other antiplatelet drugs on arterial thrombosis in rats: possible role of TXA2.

The antithrombotic effect of the thromboxane A2 receptor antagonist, vapiprost, was compared with those of other antiplatelet drugs using an arterial thrombosis model which utilized photochemical reaction in the rat femoral artery. Vapiprost prolonged the time required to occlude the artery with thrombus and inhibited collagen-induced rat platelet aggregation in whole blood ex vivo, in a dose-dependent manner. The potency ranking of antithrombotic effect was vapiprost > ketanserin (serotonin 5-HT2 receptor antagonist) > ticlopidine (inhibitor of ADP-induced platelet aggregation) = dipyridamole (adenosine uptake inhibitor) > aspirin (cyclooxygenase inhibitor). On the other hand, the ranking of antiplatelet effect was ticlopidine > or = vapiprost > or = aspirin. Ketanserin and dipyridamole were ineffective. Relative to their antiplatelet effect, vapiprost and ketanserin had powerful antithrombotic effects. It is possible that the potent antithrombotic effects of vapiprost and ketanserin in vivo reflect the ability of these drugs to inhibit mediator-induced vascular contractions in addition to platelet aggregation. The results of the present study also suggest that TXA2 may play an important role in thrombogenesis in rats.     

Enhanced antiplatelet effects of clopidogrel plus acetylsalicylic acid compared with acetylsalicylic acid alone or combined with extended-release dipyridamole in healthy volunteers

BACKGROUND: Previous studies have shown the potential benefit of using antiplatelet agents with complementary modes of action. METHODS: Using a crossover design, the ex vivo antiplatelet effects of 10 days' treatment with clopidogrel 75 mg + acetylsalicylic acid (ASA) 75 mg daily, ASA 75 mg/day, or extended-release dipyridamole 200 mg/low-dose ASA 25 mg twice daily were compared, using various platelet agonists. RESULTS: Clopidogrel + ASA was significantly more effective than dipyridamole + ASA in inhibiting collagen-induced platelet aggregation in whole blood (mean 44.9 +/- 5.6% inhibition vs. 16.5 +/- 6.7%; p = 0.0009). Clopidogrel + ASA was significantly more effective than ASA or dipyridamole + ASA in inhibiting ADP-induced platelet aggregation in whole blood (p < or = 0.0001) and platelet-rich plasma (PRP) (p < or = 0.0001), and in inhibiting collagen-induced aggregation in PRP (p < or = 0.0001). ASA alone and clopidogrel + ASA were significantly more effective than dipyridamole + ASA in inhibiting arachidonic acid-induced platelet aggregation in whole blood (p < or = 0.0001). CONCLUSIONS: Based on ex vivo platelet aggregometry, clopidogrel + ASA is a more potent antiplatelet regimen than either ASA alone or the marketed combination of dipyridamole + ASA. However, the clinical significance of this finding remains to be confirmed.     

Effect of the combination of antiplatelet agents in man : Combination of aspirin, trapidil, ticlopidine and dipyridamole

An study of how platelet aggregation would be inhibited by the combination of aspirin or ticlopidine irreversibly inhibitory to platelet aggregation and trapidil or dipyridamole reversibly inhibitory, was carried out. The measured 50% inhibition concentrations indicated that aspirin was most inhibitory to collagen-induced platelet aggregation, followed by trapidil, ticlopidine and dipyridamole in decreasing sequence of inhibition. The combination of either aspirin or ticlopidine with trapidil inhibited platelet aggregation more intensely than the combination of either agent with dipyridamole. Thus, in clinical use of aspirin or ticlopidine, it may be expected that the lower dosage of aspirin or ticlopidine with lower frequencies of side effects inhibits platelet aggregation effectively with the combination of trapidil rather than dipyridamole.     

Effect of therapy on platelet activating factor-induced aggregation in acute stroke

Platelet activating factor, a potent inducer of in vivo platelet activation and thrombosis, has been shown to be excessively active in acute ischemic stroke patients. Therefore, we studied the effect of aspirin/dipyridamole therapy in inhibiting platelet activating factor-induced platelet activation in acute ischemic stroke patients, 23 taking aspirin/dipyridamole and 21 untreated. Aspirin/dipyridamole-treated patients failed to show suppression of platelet activating factor-induced platelet aggregation even though collagen-induced activation was inhibited, suggesting that platelet activating factor acts by cyclooxygenase-independent mechanisms. Failure to suppress cyclooxygenase-independent mechanisms of platelet activation may explain the limited usefulness of current antiplatelet therapy, aspirin in particular, in stroke prevention. The role of selective platelet activating factor antagonists both in isolation and combined with aspirin needs to be investigated for their usefulness in the treatment and prevention of ischemic stroke.     

The inhibitory effect of aspirin on human endothelial cells.

Human endothelial cell monolayers prepared from umbilical veins have been incubated with aspirin (1--2 mM) dissolved in Hepes modified solution and in platelet-rich plasma. They have also been incubated with plasma prepared from subjects before and after intake of aspirin giving a mean plasma concentration of 0.5 mM. The effects of the endothelial cells on ADP and collagen-induced platelet aggregation and malondialdehyde production in platelet-rich plasma have been tested. The endothelial cells had a spontaneous inhibitory effect on all three parameters. This effect was abolished when the cells were incubated with aspirin dissolved in MHS for 20 min and the increase in effect observed when platelet-rich plasma was incubated with endothelial cells for a period of 30 min was similarly inhibited when aspirin was dissolved in plasma or when plasma prepared from subjects who had taken aspirin were used. Aspirin had no inhibitory effect on prostacyclin (PGI2) with regard to the effect of PGI2 on platelets. On the contrary, the two compounds had an additive inhibitory effect on platelet aggregation induced by ADP and collagen. These findings should be considered with regard to the use of aspirin as an antithrombotic agent.     

Effect of 1-methyl-2-mercapto-5-(3-pyridyl)-imidazole (KC-6141) on rabbit platelet aggregation in vitro and rat platelet retention

Effect of 1-methyl-2-mercapto-5-(3-pyridyl)-imidazole (KC-6141) on rabbit platelet aggregation in vitro and rat platelet retention investigated. In the in vitro study, KC-6141 inhibited ADP-induced aggregation by 27% at 5 X 10(-4)M, being more active than dipyridamole but much less than adenosine. Inhibition of arachidonic acid- and collagen-induced aggregation by KC-6141 was more effective than that of ADP-induced one and its ED50 was 2.1 X 10(-5) and 8 X 10(-5)M, respectively. KC-6141 was 10 and 4 times more potent than aspirin in arachiodonic acid- and collagen-induced aggregation, respectively. The dose-response curve of KC-6141 was parallel to that of aspirin, suggesting it is an aspirin-like compound. In the platelet retenion study, a method for determining platelet retintion in rats was devised so that platelet retention can be measured with a volume of blood as small as possible. By use of the method, effects of KC-6141, aspirin and dipyridamole were compared. When deministered intraperitoneally at 100 mg/kg, KC-6141 indicated 54.8% inhibition of platelet retention, whereas aspirin and dipyridamole showed only 23.5 and 5.2% inhibition, respectively. On the oral administration at 200 mg/kg KC-6141 inhibited by 60.8% and its ED50 was 125 mg/kg. The activity lasted over 32 hr. The above results demonstrated that KC-6141 is a compound with more potent action on the platelet aggregation, as well as on the platelet retention than aspirin and dipyridamole-a known antithrombotic drug.     

Ex vivo evaluation of anti-GPVI antibody in cynomolgus monkeys: dissociation between anti-platelet aggregatory effect and bleeding time

Recent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (> 80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0.2 mg/kg with a slight prolongation of bleeding time (1.3 times baseline value). Furthermore, at 18.8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1.9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5.0 times at 0.35 mg/kg, the lowest effective dose on platelet aggregation. In a pharmacodynamic study, a bolus injection of OM2 at 0.4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exert a potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.     

Effect of ticlopidine on human platelet responsiveness ex vivo: Comparison with aspirin

Platelet responsiveness in a variety of tests was measured in seven volunteers twice prior to and once after treatment with ticlopidine (250mg bds for seven days). The drug caused moderate but significant inhibition of both collagen-induced aggregation and the release of [¹⁴C]5-HT associated with aggregation induced by collagen, ADP and adrenaline. Platelet responses to ADP after ticlopidine treatment were unusual in that a marked tendency to disaggregate was observed. First-phase aggregation in response to ADP and adrenaline was not affected by ticlopidine, nor was platelet retention in glass bead columns or adhesion to collagen. A single low dose of aspirin (300mg) had a significantly greater effect than seven days treatment with ticlopidine on aggregation and [¹⁴C]5-HT release, and also reduced adhesion to collagen. However, aspirin did not induce disaggregation. Although ticlopidine seems relatively weak as an anti-aggregant drug when tested in humans $\text{ex vivo}$, its capacity to induce disaggregation distinguishes it from aspirin and could be important in a potential antithrombotic drug.     

Effects in mice of testosterone and dihydrotestosterone on platelet aggregation in injured arterioles and ex vivo

Mice were implanted subcutaneously with placebo pellets, or with pellets containing either testosterone (T) or dihydrotestosterone (DHT). Eight to 19 days later platelet aggregation was produced in pial or mesenteric arterioles by injuring their endothelium with a noxious light/dye stimulus. The onset of platelet aggregation was significantly shortened in the mesenteric arterioles of male mice following implantation of 1.0 mg T or 0.1 mg DHT. However no effect was observed in females, nor did T or DHT alter aggregation in pial arterioles of either sex. Ex vivo studies showed that sodium arachidonate produced greater aggregation of platelets in plasma from testosterone treated males compared with controls. No effect of testosterone was observed ex vivo in platelets from females. These ex vivo results paralleled in vivo data from mesenteric vessels, but not from pial vessels. Moreover DHT failed to influence aggregation ex vivo in either sex. Thus enhanced aggregation observed in vivo in mesenteric arterioles of androgen treated males may not reflect direct action of androgen on the platelet. Rather enhanced aggregation may reflect hormonal action on endothelium or adjacent tissue. In addition to the preceding studies we tested the hypothesis that testosterone's action was due to its conversion to estradiol. This was considered because our data with T and mesenteric vessels resembled that previously reported by us with estradiol. However only minimal elevations in plasma estradiol levels resulted from testosterone treatment.     

Inhibition of arterial thrombosis and platelet function by nafazatrom

Nafazatrom (1-[2-(naphthyloxy) ethyl]3-methyl-2-pyrayolin-5-one) has been shown to be a potent antithrombotic agent in experimental animals, but its mode of action is unknown. In this study, we examined its antithrombotic effect on arterial and venous thrombosis, vessel wall prostacyclin synthesis, and platelet aggregation in vivo and ex vivo in the rabbit. We found that arterial but not venous thrombus formation was significantly inhibited with low doses of nafazatrom. This antithrombotic effect was associated with a normalization of reduced platelet survival. Nafazatrom had no effect on basal or stimulated prostacyclin production but inhibited ADP- and collagen-induced platelet aggregation in vivo and ex vivo. This platelet effect was associated with a reduction in platelet thromboxane B₂ release and an inhibition of irreversible ADP-induced platelet aggregation.     

Effect of picotamide on platelet aggregation and on thromboxane A2 production in vivo

Effects of picotamide (900 mg in 3 oral administrations for 7 days) on ex vivo and in vivo platelet TxA2 production and on platelet aggregation were evaluated in 8 patients with peripheral arteriopathy and in 8 normal subjects. Picotamide significantly reduced ADP-induced platelet aggregation, but had no effect on that induced by arachidonic acid or the thromboxane analogue U46619. Though ex vivo platelet TxA2 production (TxB2 concentration after arachidonic-acid-induced aggregation) was reduced from 946 +/- 141 (mean +/- SD) to 285 +/- 91 ng/ml in controls and from 1515 +/- 673 to 732 +/- 420 ng/ml in patients with arteriopathy, there was no effect on urinary excretion of 2,3-dinor-TXB2 (in vivo indicator of platelet TxA2 production), or on in vivo PGI2 production (urinary excretion of 6-keto-PGF1 alpha and 2,3-dinor-6-keto-PGF1 alpha). In the same subjects, single-dose aspirin reduced ex vivo TxB2 production by at least 98% and 2,3-dinor-TxB2 excretion from 116.7 +/- 61.4 to 32.6 +/- 17.0 ng/g creatinine in control subjects, and from 156.3 +/- 66.1 to 59.1 +/- 19.2 ng/g creatinine in patients with peripheral arteriopathy. Our data suggest that inhibition of platelet TxA2 production in vivo may not be picotamide's main mechanism of action.     

Effects of estradiol on platelet aggregation in mouse mesenteric arterioles and ex vivo

Mice were implanted subcutaneously with a pellet containing 0.5 mg estradiol or with a placebo. Eight to 12 days later platelet aggregation was produced in mesenteric arterioles by injuring their endothelium in vivo with a noxious light/dye stimulus. The time between the onset of the noxious stimulus and the appearance of platelet aggregates was significantly shortened (p less than .01) in the estradiol treated mice. In contrast to this enhancement of aggregation, when platelets were tested ex vivo in platelet rich plasma (PRP), aggregation to sodium arachidonate was inhibited in estradiol treated mice. Aggregation of PRP by ADP was not affected by estradiol treatment of the mice. Thus the enhanced aggregation observed in injured mesenteric arterioles of estradiol treated mice may not reflect direct effects of estradiol on the platelet itself. Rather enhanced aggregation may reflect an effect of estradiol on endothelium or adjacent tissue. The data are discussed in light of other literature concerning estradiol effects in rodents, and in light of literature suggesting that an increased number of thromboembolic events occurs in humans receiving high doses of estrogens.     

Effects of antiepileptic drugs on rat platelet aggregation: ex vivo and in vitro study

The influence of conventional antiepileptic drugs (valproate, phenobarbital, diazepam, clonazepam, carbamazepine and diphenylhydantoin) on rat platelet activation induced by arachidonic acid (AA) or adenosine-5'-diphosphate (ADP) was investigated both ex vivo and in vitro on platelet-rich plasma (PRP). It was found that only diazepam, and to a smaller extent clonazepam, impaired rat platelet function. These benzodiazepines did not affect ex vivo platelet aggregation induced by ADP but dose-dependent inhibition of platelet aggregation and malondialdehyde (MDA) synthesis were observed, when the platelets were stimulated with AA (ED(50) of diazepam for aggregation was 2.7 mg/kg and that for MDA synthesis - 3.9 mg/kg). In in vitro study, diazepam was found to be a potent inhibitor of AA-induced platelet aggregation (IC(50) 1.2 microg/ml) and MDA synthesis (IC(50) 4.0 microg/ml). Higher concentrations of diazepam were required to inhibit ADP-induced aggregation (IC(50) 29.0 microg/ml). Clonazepam also exhibited a concentration-dependent inhibitory effect on AA-induced platelet aggregation and MDA synthesis but this effect was weaker when compared to diazepam. The present data demonstrate that diazepam possesed a strong inhibitory effect on rat platelet activation. The correlation between the reduction of platelet aggregation and the synthesis of MDA may suggest that the observed effect of diazepam is due to the inhibition of the cyclooxygenase pathway of the AA metabolism in platelet.     

Effects of low dose aspirin on platelet function in patients with recent cerebral ischemia

We tested the antiplatelet effects of low-dose aspirin in patients with occlusive cerebrovascular disease, because conventional dosage aspirin inhibits vascular synthesis of prostacyclin at the same time that it inhibits platelets. The effects on platelet function and thromboxane A2 synthesis of 40 mg of aspirin daily or 40 mg aspirin plus dipyridamole were measured in 23 patients starting within a week after the onset of cerebral ischemia. All patients had normal baseline platelet aggregation responses to four stimuli: arachidonate, epinephrine, adenosine diphosphate and collagen. The generation of thromboxane A2 by platelets, measured as serum thromboxane B2, was also normal. After 3 to 7 days of low dose aspirin therapy, platelet aggregation responses were suppressed to the extent observed with higher dosage aspirin. Serotonin release during platelet aggregation was inhibited by more than 95% and thromboxane B2 levels in clotted blood fell by more than 95%. Responses to aspirin treatment were similar in patients with transient ischemic attacks and in those with stroke and were also similar in both sexes. No differences in platelet responses were observed between patients receiving aspirin alone and aspirin plus dipyridamole. Thus 40 mg aspirin daily inhibited platelet responses as effectively as higher doses of aspirin in patients who had recent cerebral ischemia and showed a cumulative antiplatelet effect.     

Role of plasma adenosine in the antiplatelet action of HL 725, a potent inhibitor of cAMP phosphodiesterase: species differences

The potent inhibitor of platelet cAMP phosphodiesterase (PDE) HL 725 (9,10-Dimethoxy-2-mesitylimino-3-methyl-3, 4,6,7-tetrahydro-2H-pyrimido(6,1-A)-isoquinoline-4-one-hydrochloride), was examined for its effects on human and rat platelet aggregation. Strong inhibitory effects are seen on collagen-induced platelet aggregation both in rat platelet-rich plasma (PRP) (IC50, 54 +/- 12 nM) and whole blood (IC50, 57 +/- 25 nM). Compared to the effects on rat platelets, HL 725 is about two-fold less inhibitory in human PRP (IC50, 94 +/- 29 nM) and whole blood (IC50, 126 +/- 50 nM). The inhibitory action of HL 725 can be reversed by washing and resuspension of the platelets, suggesting that HL 725 does not bind tightly to cAMP PDE. If human or rat PRP is pretreated with adenosine deaminase, an enzyme that degrades adenosine or 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, the inhibitory effect of HL 725 is reversed. Similar blockade of the inhibitory actions of several other inhibitors of cAMP PDE such as RA 233, RX-RA 69 (analogs of dipyridamole) and oxagrelate is seen by adenosine deaminase pretreatment. The nucleoside transport inhibitors, dilazep and dipyridamole which are non-inhibitory alone to platelet aggregation, strongly potentiate (about 10-fold) the inhibitory action of HL 725 on collagen-induced platelet aggregation in human whole blood. However, if the whole blood is pretreated with adenosine deaminase, no inhibitory effect of dipyridamole plus HL 725 is seen on platelet aggregation. These studies demonstrate that plasma adenosine plays a crucial role in the antiaggregatory actions of HL 725 and several other inhibitors of cAMP PDE both in human and rat blood.     

Antithrombotic activities of green tea catechins and (-)-epigallocatechin gallate

The antithrombotic activities and mode of action of green tea catechins (GTC) and (-)-epigallocatechin gallate (EGCG), a major compound of GTC, were investigated. Effects of GTC and EGCG on the murine pulmonary thrombosis in vivo, human platelet aggregation in vitro, and ex vivo, and coagulation parameters were examined. GTC and EGCG prevented death caused by pulmonary thrombosis in mice in vivo in a dose-dependent manner. They significantly prolonged the mouse tail bleeding time of conscious mice. They inhibited adenosine diphosphate- and collagen-induced rat platelet aggregation ex vivo in a dose-dependent manner. GTC and EGCG inhibited ADP-, collagen-, epinephrine-, and calcium ionophore A23187-induced human platelet aggregation in vitro dose dependently. However, they did not change the coagulation parameters such as activated partial thromboplastin time, prothrombin time, and thrombin time using human citrated plasma. These results suggest that GTC and EGCG have the antithrombotic activities and the modes of antithrombotic action may be due to the antiplatelet activities, but not to anticoagulation activities.