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Circulation. 1997;95:125-132

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(Circulation. 1997;95:125-132.)
© 1997 American Heart Association, Inc.

Articles

Different Effects of Calcium Antagonists, Nitrates, and ß-Blockers on Platelet Function

Possible Importance for the Treatment of Unstable Angina

Charles J. Knight, MRCP; Manvinder Panesar, BSc; Darren J. Wilson, MSc; Nicolas A.F. Chronos, MRCP; Deven Patel, MRCP; Kim Fox, MD; Alison H. Goodall, PhD

the Department of Cardiology, Royal Brompton Hospital, (C.J.K., N.A.F.C., D.P., K.F.) and the Vascular Cell Biology Laboratory, Royal Free Hospital School of Medicine (M.P., D.J.W., A.H.G.), London, UK.

Correspondence to Dr Alison Goodall, Vascular Cell Biology Laboratory, Royal Free Hospital School of Medicine, Rowland Hill St, London NW3 2PF, UK.


*    Abstract
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 *Abstract
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Background The three major classes of antianginal drug all inhibit platelet aggregation at high concentrations in vitro, but detecting clinically relevant effects has proved to be more difficult. We used whole-blood flow cytometry, a sensitive method that allows direct measurement of activation antigens on the surface of individual platelets in whole unfixed blood, to evaluate the effect of representative antianginal drugs on platelet function in vivo in healthy volunteers.

Methods and Results The effects of glyceryl trinitrate (GTN), amlodipine, and atenolol were studied in nine normal volunteers. Fibrinogen binding to activated GP IIb/IIIa and expression of P-selectin, GP Ib, and GP IIb/IIIa on the platelet surface were measured. In addition, fibrinogen binding and P-selectin expression were measured in response to ex vivo stimulation with the agonists ADP and thrombin. The three drugs had very different effects on platelets. GTN inhibited platelet fibrinogen binding and expression of P-selectin at rest and in response to agonist stimulation, whereas amlodipine enhanced P-selectin expression and atenolol increased fibrinogen binding in response to agonists. Atenolol did not block the stimulatory effects of epinephrine on ADP-induced platelet activation. GTN neutralized the proactivatory effects of amlodipine, whereas the effects of atenolol and amlodipine were not additive.

Conclusions The three main classes of antianginal medication have different and possible clinically relevant effects on platelet behavior in vivo, nitrates causing inhibition of aggregation (fibrinogen binding) and degranulation (P-selectin expression), calcium antagonists enhancing degranulation, and ß-blockers enhancing aggregation.


Key Words: platelets • drugs • angina


*    Introduction
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 *Introduction
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There is increasing evidence that platelet activation is involved in atherogenesis and the development of the thrombotic complications of atheroma.1 2 Furthermore, antiplatelet therapy with aspirin is effective in limiting complications in patients with unstable angina.3 Clearly, it is desirable that drugs administered to patients with atherosclerosis should not have adverse effects on platelets, particularly in the context of the treatment of acute coronary syndromes. Nitrates, calcium antagonists, and ß-blockers are frequently administered to such patients, and these agents, via release of nitric oxide, modification of calcium flux, and alteration of catecholamine balance, respectively, might be expected to alter platelet function. In view of reports of an unfavorable outcome in patients with acute coronary syndromes treated with some classes of calcium antagonist,4 5 it is important to establish whether the adverse prognostic effects of such drugs could be mediated, at least in part, through platelet activation.

Although all three of these classes of drug have been shown to inhibit platelet aggregation in vitro at high concentrations, demonstration of therapeutically relevant effects has proved to be more difficult, and different methods of investigation have produced contradictory results for each drug studied.

Nitrates may exert an antiplatelet effect by breakdown and liberation of NO, causing both inhibition and reversal of platelet aggregation6 via elevation of intracellular cGMP. Nitrates also attenuate oxidation of arachidonate and its conversion to proaggregatory endoperoxides and TxA2.7 GTN and other nitrates inhibit platelet aggregation in vitro (eg, see Reference 7) but not at doses achievable in vivo. Contradictory results have been observed with both intravenous and oral nitrates in vivo, some reports showing no effect8 9 10 and others (eg, see References 11 and 12) reporting inhibition.

Changes in intracellular calcium concentration, as a result of either calcium influx or mobilization of intracellular stores, are fundamental to the platelet response to activation.13 Although human platelets probably do not possess voltage-dependent calcium channels,14 calcium antagonists might affect platelet function by a variety of other mechanisms.15 16 17 18 19 In general, the doses of calcium channel blockers required for in vitro platelet effects are high, and their in vivo relevance is questionable. In vivo studies to date have not produced a consensus: both inhibitory effects20 and no effects21 of verapamil treatment have been shown, whereas in a small uncontrolled study, amlodipine showed diminished platelet aggregation in 10 hypertensive patients.22

An inhibitory effect of ß-blockade on platelet function might be expected, because physiological doses of epinephrine have been shown to enhance, and ß-blockers to inhibit, agonist-induced platelet aggregation in vitro. Moreover, intravenous infusion of epinephrine to high physiological levels has been shown to increase platelet aggregation as assessed by filtragometry23 and to sensitize single platelets to agonist stimulation as assessed by flow cytometry.24 However, the majority of in vivo studies of platelet function after ß-blockade have not produced a clear picture.23

The conflicting results obtained with each of the main classes of antianginal compound reflect both the inadequacy of isolated in vitro testing of drugs that have multiple cardiovascular effects and the methodological problems of measuring platelet activation in vivo.

Earlier studies of the effects of these drugs have relied on indirect measurement of the consequences of platelet activation in vivo by aggregometry or the measurement of platelet release products. Direct assessment of the activation status of individual platelets can be measured by fluorescence-activated flow cytometry with fluorescently labeled antibodies to detect the expression of activation antigens on the platelet surface.25 Activation causes a conformational change in the platelet membrane GP IIb/IIIa complex, exposing the receptor site for fibrinogen and leading to binding of plasma fibrinogen, which can be recognized with specific antibodies.26 27 28 Platelet degranulation is accompanied by translocation of granule membranes to the platelet surface, bringing with them specific membrane glycoproteins, such as {alpha}-granule P-selectin,29 that appear as neoantigens. Activation of platelets in vivo can also lead to increased numbers of GP IIb/IIIa complexes and reduced numbers of GP Ib/IX complexes on the cell surface.30 Whole-blood flow cytometry of unfixed blood also allows the responsiveness of the platelets to ex vivo stimulation with platelet agonists such as thrombin and ADP to be studied in the presence of other blood components. Thus, in addition to detecting changes in the basal activation state of platelets, the technique can also detect more subtle alterations in platelet behavior.

Here, the effects of therapeutic levels of a representative nitrate (GTN), calcium antagonist (amlodipine), and ß-blocker (atenolol), alone and in combination, were investigated in a group of healthy volunteers and found to produce effects on platelet function in vivo that could have consequences for their use in patients.


*    Methods
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Subjects
Nine healthy male volunteers 21 to 29 years old participated in the studies. All subjects underwent a pretrial examination, including ECG. No subject had ingested any platelet-active drug during the 2 weeks preceding the study period, platelet counts were all within the normal range, and none of the subjects smoked. All subjects gave written consent to participate in the study, which was approved by the Ethical Practices Committee of the Royal Free Hospital.

Procedures and Blood Sampling
The study was divided into two parts: the first to assess the effects of GTN and the calcium antagonist alone and in combination and the second to assess the effects of the ß-blocker and the calcium antagonist alone and in combination. Seven subjects took part in the first study and six (four from the first study and two new subjects) in the second. All experiments were performed at the same time of day to avoid any effects of diurnal variation. The subjects were asked to avoid caffeine on the day of the test, and all subjects rested supine for 30 minutes before sampling on each occasion.

On the first occasion, blood was drawn from the subject, and an infusion of GTN (50 mg in 50 mL 0.9% wt/vol saline) was commenced via an indwelling venous cannula in the contralateral arm with an IVAC 770 syringe pump at a rate of 17 µg/min for 10 minutes before increasing to 33, 67, and 133 µg/min at 10-minute intervals. The dose of 133 µg/min was maintained for 20 minutes before repeat blood sampling, after which the infusion was stopped. The total dose of GTN infused was 4.4 mg. The subjects were then instructed to take amlodipine 5 mg/d for 7 days. After 1 week they returned, and the experiment as outlined above was repeated. Heart rate and blood pressure were monitored at 10-minute intervals throughout the GTN infusions.

After a minimum washout period of 6 weeks, baseline samples were taken for the second part of the study, and subjects were instructed to take atenolol 50 mg/d for 1 week, after which time sampling was repeated. They were then issued a further week's supply of atenolol in addition to a week's supply of amlodipine 5 mg/d. After 7 days of combination therapy, the last samples were obtained.

Samples were also collected from five patients with unstable angina who were receiving treatment with atenolol 50 mg/d. These subjects gave verbal consent, and sample collection had been approved by the Ethical Practices Committee of the Royal Brompton Hospital.

All blood samples were obtained from the noninfused arm by clean venipuncture under minimal stasis, and any repeated venipunctures were made distal to the initial site, conditions designed to avoid artifactual activation of platelets during phlebotomy. Blood was taken via a 21-gauge butterfly needle into three Monovette tubes (Sarstedt, Beaumont Leys); the first 2.7 mL into EDTA, used to obtain blood counts; the second into 0.105 mol/L trisodium citrate, used for flow cytometric analysis; and the third into an ice-cold citrate tube that was centrifuged within 5 minutes for 30 minutes at 3000g and 4°C. Plasma was taken from the center of the tube and frozen in aliquots at -70°C until analyzed for drug levels, plasma fibrinogen, and vWF.

Flow Cytometric Analysis of Platelet Activation
Platelets were analyzed for expression of GP Ib, GP IIb/IIIa, P-selectin, and bound fibrinogen as described previously.28 31 Briefly, 5 µL of citrated blood was added within 10 minutes of collection to 50 µL HEPES-buffered saline containing 5 µL of appropriate concentrations of antibodies. Platelet agonist responsiveness was investigated as follows: fibrinogen binding was measured in response to ADP (0.1 to 10 µmol/L) and thrombin (0.02 to 0.32 U/mL). When atenolol was studied, ADP-stimulated samples were also costimulated with epinephrine at 50 nmol/L, and the buffer was supplemented with L-ascorbic acid at 0.1 mmol/L to prevent oxidation of the epinephrine. P-selectin expression was measured in response to maximal ADP stimulation (10 µmol/L) and to thrombin (0.02 to 0.32 U/mL). All samples incubated with thrombin contained 0.125 mmol/L GPRP peptide to inhibit fibrin cross-linking and consequent clot formation.32 After gentle mixing, the samples were incubated for 20 minutes at 20°C to 22°C, then diluted with 0.5 mL of 0.2% (vol/vol) formyl saline and analyzed within 1 hour in a Coulter EPICS Profile II flow cytometer (Coulter Electronics Ltd). Five thousand platelets were analyzed, and the results represent the means of duplicate samples. The percentage of platelets positive for the marker and the mean fluorescence intensity (MFI) for each sample were used to calculate the binding index (BI) for the marker from the following equation: BI=(Percent PositivexMFI)/100.

Plasma Assays
Amlodipine levels were measured by gas-liquid chromatographic assay after 7 days' dosing. Plasma fibrinogen was determined by the Clauss method in a KC10 coagulometer, and vWF was measured by ELISA (Shield Diagnostics Ltd).

Statistical Analysis
All data are shown as mean±SD. Comparison of variables was performed with nonparametric (Wilcoxon) tests to investigate the relationship between independent variables. For analysis of heart rate and blood pressure changes, paired t tests were used.


*    Results
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 *Results
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Physiological Changes
No side effects of the drugs were experienced apart from mild headache in two subjects during their first infusion of GTN. Changes in heart rate and blood pressure on therapy are shown in Table 1Down. Subject compliance was monitored by tablet count. Plasma levels of amlodipine at the end of treatment and physiological parameters after atenolol were consistent with full compliance in all subjects. Plasma levels of fibrinogen and vWF were all within the normal range and did not change with any treatment.


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  		Table 1. Physiological Changes in Response to GTN+Amlodipine and Atenolol+Amlodipine

Flow Cytometry
Glyceryl trinitrate
There was a significant fall in fibrinogen binding (P=.03) and P-selectin expression (P<.05) in unstimulated samples after GTN infusion (Table 2ADown). All subjects had reduced fibrinogen binding and P-selectin expression in response to agonist stimulation. Fibrinogen binding in response to ADP (Fig 1aDown) and thrombin (not shown) was reduced, but only the former reached statistical significance. There was also a significant reduction in P-selectin expression in response to stimulation with thrombin (Fig 1bDown) and with maximum ADP (Fig 1cDown).


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  		Table 2. Levels of Platelet Antigens in Unstimulated Samples Before and After Drug Administration



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  		Figure 1. Dose-response curves of platelet fibrinogen binding in response to stimulation by ADP (a) and P-selectin expression in response to thrombin (b) before and after intravenous GTN. The drug caused a significant inhibition of the response (*P<.05). The inhibitory effect of GTN on P-selectin expression with maximum ADP (10 µmol/L) is shown in inset (c).

Amlodipine
Fibrinogen binding was unchanged after amlodipine in unstimulated samples (Table 2BUp) or on stimulation with ADP (Fig 2aDown) or thrombin (not shown). There was a rise in P-selectin expression on unstimulated samples, but this was not significant (P=.11, Table 2BUp). There was, however, increased P-selectin expression in response to stimulation with thrombin and ADP in all but one of the subjects, the increase for the group as a whole reaching statistical significance (Fig 2b and 2cDownDown). GP IIb/IIIa levels were not affected by amlodipine, but GP Ib expression was significantly lower (P=.04, Table 2BUp).



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  		Figure 2. Dose-response curves of platelet fibrinogen binding in response to stimulation by ADP (a) and P-selectin expression in response to thrombin (b) before and after amlodipine. Fibrinogen binding was unaffected by the drug, but there was significant enhancement of P-selectin expression. The stimulatory effect of amlodipine on P-selectin expression with maximum ADP (10 µmol/L) is shown in inset (c) (*P<.05).

Amlodipine and GTN
Although compared with baseline, platelet activation (Table 2BUp) and responsiveness were no different on the combination of amlodipine and GTN, the effect of GTN administration to subjects on amlodipine was to cause a significant fall in fibrinogen and P-selectin expression (Table 2BUp) in unstimulated samples and to reverse the increase in P-selectin expression in response to ADP and thrombin (Fig 3Down).



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  		Figure 3. Dose-response curves for P-selectin expression in response to stimulation with thrombin at baseline ({bullet}), after amlodipine ({circ}), and after the amlodipine/GTN combination ({blacksquare}).

Atenolol
Atenolol therapy did not alter platelet activation significantly in unstimulated samples (Table 2CUp). However, the drug enhanced fibrinogen binding in response to agonist stimulation in all subjects, reaching statistical significance for the response to thrombin (Fig 4bDown). Although mean P-selectin expression in response to agonists was higher after atenolol, the effect was neither consistent nor significant (Fig 4c and 4dDownDown). GP Ib levels were not significantly altered by atenolol, but GP IIb/IIIa expression increased (Table 2CUp). Blood samples from subjects before and after atenolol therapy were also stimulated ex vivo with ADP (1 to 10 µmol/L) in the presence of low-dose epinephrine (50 nmol/L). Epinephrine significantly enhanced fibrinogen binding. This effect was seen both before (Fig 5aDown) and after (Fig 5bDown) atenolol, suggesting that the drug did not block the effects of epinephrine on platelet fibrinogen binding. Enhanced ADP responsiveness by epinephrine was also observed in a group of patients with unstable angina receiving ß-blockers (Fig 5cDown).



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  		Figure 4. Dose-response curves of platelet fibrinogen binding in response to stimulation by ADP (a) and thrombin (b) and P-selectin expression in response to thrombin (c) and ADP (inset, d) before and after atenolol (*P<.05).



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  		Figure 5. Dose-response curves of platelet fibrinogen binding in response to stimulation by ADP (0.1 to 10 µmol/L) with and without epinephrine (adrenaline) (50 nmol/L) before (a) and after (b) atenolol and in patients with unstable angina on atenolol (c).

Atenolol and amlodipine
There was no additive effect on platelet activation of the two drugs. Fibrinogen binding and P-selectin expression both in unstimulated samples (Table 2CUp) and in response to agonist stimulation (not shown) was unchanged after combination therapy compared with atenolol alone.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Our study, using the sensitive technique of whole-blood flow cytometry for the first time to investigate the platelet effects of antianginal medication in standard therapeutic doses in vivo, shows that it is possible to detect changes in platelet behavior after administration of these compounds to normal volunteers and furthermore, that the three classes of antianginal drugs alter platelet behavior in different ways.

Whereas intravenous GTN caused a fall both in the basal state of activation of platelets and in their secretory and proaggregatory responses to stimulation by agonists, the calcium antagonist amlodipine enhanced degranulation in response to agonist stimulation. The effect of GTN in subjects taking amlodipine was to reduce both activation state and responsiveness significantly, suggesting that the effects of GTN (inhibitory) and amlodipine (stimulatory) are to some extent antagonistic.

Atenolol did not alter platelet activation significantly. However, it did enhance platelet agonist responsiveness, suggesting a proactivatory effect, although a significant increase was seen only for fibrinogen binding in response to thrombin. Atenolol did not inhibit the stimulatory effects of ex vivo epinephrine on platelet ADP responses, either in normal volunteers or in patients with unstable angina. The effects of amlodipine and atenolol were not additive, with no difference in activation or responsiveness observed after combination therapy compared with atenolol alone.

The effects of intravenous GTN were not unexpected, because nitrates are thought to exert an antiplatelet effect by liberation of NO, with a consequent increase in intracellular cGMP, and increased intraplatelet cGMP in response to nitrate-like compounds has been shown to inhibit platelet aggregation6 and fibrinogen binding33 in experimental models. However, previous in vivo investigations of the effects of nitrates on platelets have produced contradictory results, primarily as a result of methodological differences.

Intravenous GTN failed to inhibit ex vivo aggregation in platelet-rich plasma in four of five normal volunteers8 and had no effect on in vivo aggregates and ex vivo aggregation in seven patients with congestive cardiac failure.9 However, using the more sensitive technique of ex vivo filtragometry, Karlberg et al34 showed that GTN significantly increased aggregation in eight healthy volunteers. Intravenous GTN has been shown to inhibit aggregation in patients with unstable angina and myocardial infarction,11 stable angina,35 and cardiomyopathy.36 Intravenous isosorbide dinitrate37 and isosorbide mononitrate38 reduced the number of circulating platelet aggregates and inhibited ADP- and epinephrine-induced aggregation ex vivo in patients with stable angina.

Our results would therefore concur with the majority of previous investigations. The pharmacological and pathophysiological relevance of such an effect, however, is still open to question. Oral administration may not produce the same effects on platelets as an intravenous infusion. Previous studies with oral nitrates have failed to show effects on conventional assays: one double-blind study of oral isosorbide mononitrate in patients with stable angina39 failed to show any effect on ex vivo aggregation. In addition, ß-TG levels and ex vivo filtragometry were unchanged by oral isosorbide dinitrate in a double-blind placebo-controlled crossover study in normal volunteers40 and in patients with stable angina.10

Patients with coronary artery disease have diseased, functionally incompetent endothelium, which may result in altered platelet responsiveness. Their response to nitrates may therefore not correspond to that observed in healthy, young male volunteers. Furthermore, tolerance to nitrates may limit the antiplatelet as well as the hemodynamic effects of nitrates in the longer term. It is not clear whether any antiplatelet effect of GTN will persist in the presence of increased plasma catecholamines, such as might occur during myocardial infarction. It has also been suggested41 that higher doses of nitrates may produce a fall in blood pressure sufficient to cause a rise in plasma catecholamines that might have an antagonistic and deleterious effect on platelet activation.

Changes in intracellular calcium concentration, either as a result of calcium entry from the exterior or from mobilization of intracellular stores, are fundamental to the platelet response to activation.13 Calcium antagonists, which selectively inhibit the inward calcium current in myocytes, might therefore be expected to have an effect on platelet activation, and it has been shown that in vitro, verapamil inhibits epinephrine but not ADP-induced aggregation.42 To produce a similar effect, however, much larger doses of the dihydropyridine calcium antagonist nifedipine are needed,43 indicating that different classes of calcium antagonists may differ in their precise mechanisms of action.

It has been suggested, however, that human platelets do not possess voltage-dependent calcium channels14 44 and that calcium antagonists might therefore affect transmembrane calcium transport in the platelet by other mechanisms. It has been proposed that the GP IIb/IIIa complex may function as a calcium channel,15 although its inhibition requires much higher concentrations of antagonist than those required to inhibit the classic voltage-gated calcium channel.45 Calcium antagonists can also affect mobilization of calcium from intracellular stores,16 they are capable of inducing an "anesthetic-like" effect on cell membranes,17 and they have been shown to increase intracellular cAMP by inhibiting cAMP phosphodiesterase.18 TxA2 formation may also be inhibited by calcium channel blockers.19 Verapamil, but possibly not other calcium antagonists,19 46 inhibits epinephrine-induced platelet aggregation by interaction with the platelet serotonin47 and {alpha}-adrenergic receptors.48 There are therefore several potential mechanisms by which calcium antagonists might alter platelet function, and different classes of calcium antagonist may have different effects on platelets.

In vivo studies to date have not produced a clear picture. With Born aggregometry, both inhibitory effects49 50 and no effects21 51 of verapamil treatment have been reported in healthy volunteers and patients with coronary artery disease. Nifedipine has also been reported to decrease aggregation52 but had no effect on aggregation in a double-blind randomized trial by Rostagno et al,21 whereas with the same methods in a small uncontrolled study, amlodipine was shown to diminish aggregation in 10 hypertensive patients.22 Using the more sensitive technique of ex vivo filtragometry, Wallen et al20 detected inhibition of aggregation with verapamil in 48 patients with stable angina, and Lacoste et al53 demonstrated a significant inhibition of platelet thrombus formation on porcine thrombogenic arterial wall media exposed to circulating blood of patients with coronary artery disease after verapamil treatment.

Our results do not support an antiplatelet effect of the dihydropyridine calcium antagonist amlodipine. Indeed, there was evidence of in vivo platelet activation after the drug, as evidenced by an increase in the degranulation response at rest and in response to agonist stimulation. Infusion of GTN into amlodipine-treated subjects produced a significant fall in the expression of both P-selectin and fibrinogen, suggesting that the two drugs had an antagonistic effect on platelet function.

The effects of dihydropyridines may differ from that of verapamil, the calcium blocker for which an in vivo antiaggregatory effect is perhaps best established. The differing effects of these two classes of compound on secondary prevention after myocardial infarction5 54 could be explained in part by differences in their antiplatelet effects, and comparative studies are indicated.

Intravenous infusion of epinephrine at high physiological levels increases platelet aggregation, as assessed by filtragometry,23 and sensitizes single platelets to stimulation with ADP24 and thrombin,55 as assessed by flow cytometry. It might therefore be expected that the administration of a ß-blocker would oppose some of these catecholamine-mediated effects. However, we have shown that the response to ex vivo stimulation with epinephrine is unaffected by in vivo treatment with a ß-blocker. This concurs with previous work showing that the proaggregatory effects of physiological levels of catecholamines are mediated by the {alpha}- rather than the ß-receptor.56 Norepinephrine has also been shown to increase platelet aggregation and plasma ß-TG levels, the proaggregatory effect persisting despite aspirin pretreatment,57 whereas the ß-agonist isoprenaline decreases aggregation as assessed by ex vivo filtragometry.58 It therefore seems likely that the proaggregatory effects of catecholamines are mediated by {alpha}2-receptors and that the platelet ß-receptors are of minor importance in vivo.58

Furthermore, our results suggest that although ß-blockers have little effect on basal platelet activation, they do enhance the aggregatory response to agonists. The mechanism for this may be an unmasking of {alpha}-mediated epinephrine proaggregation, because ß-blockade has been shown to potentiate rather than inhibit the effects of epinephrine at low concentrations in vitro.58

The majority of previous in vivo studies of platelet function after ß-blockade have not demonstrated consistent effects.23 Although propranolol has been shown to inhibit ADP-induced aggregation in two studies in healthy volunteers,59 60 Larsson et al61 showed in a double-blind placebo-controlled crossover trial that metoprolol had no effect on platelet function assessed by ex vivo filtragometry; in vitro aggregometry; and urinary excretion of ß-TG, thromboxane, and prostacyclin metabolites in healthy volunteers. Similar results have been found in patients with coronary artery disease.20 Interestingly, in view of our findings, the in vivo platelet aggregatory response to sympathoadrenal stimulation after metoprolol was enhanced, rather than blocked, in the study by Larsson et al.61

In conclusion, small, consistent, and potentially significant changes in platelet behavior were produced by the three representative antianginal drugs. By use of whole-blood flow cytometry, which allows direct assessment of platelet activation status in vivo and measurement of agonist responsiveness in vitro, it has been possible to detect small but significant changes in platelets in response to drug treatment.

These effects were produced in young, healthy subjects in whom vascular endothelial function should be relatively normal. In patients with established atherosclerotic disease, the resultant vascular endothelial cell dysfunction might add to the platelet-activatory effects of these drugs. The potential importance of these findings is indicated by recent reports suggesting adverse prognosis in some groups of patients taking short-acting calcium antagonists.62 If adverse prognosis with these drugs is confirmed, platelet effects are a possible mechanism for the phenomenon, and further studies of different classes of calcium antagonists, alone and in combination with other antianginal drugs, are indicated in patients with coronary artery disease.


*    Selected Abbreviations and Acronyms
 
GTN = glyceryl trinitrate
ß-TG = ß-thromboglobulin
TxA2 = thromboxane A2
vWF = von Willebrand factor


*    Acknowledgments
 
Dr Knight is a British Heart Foundation Junior Research Fellow, Dr Patel was supported as a fellow by the Augustus Newman Foundation, and Ms Panesar is funded by a grant from the Royal Brompton Hospital Clinical Research Committee. We thank Anita Jagroop and Dale Owens (Royal Free Hospital School of Medicine) for measurement of plasma fibrinogen and Dr Peter Meredith (Department of Medicine and Therapeutics, Gardiner Institute, Western Infirmary, Glasgow, UK) for assays of plasma amlodipine levels. ELISA kits for vWF analysis were kindly supplied by Dr Rob Ford, Shield Diagnostics Ltd, Dundee, UK.

Received April 29, 1996; revision received May 13, 1996; accepted July 11, 1996.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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