An inhibitory effect of beta-blockers on platelet function has been clearly demon strated in vitro at relatively high concentrations507'510 and also at therapeutic concen trations.510 However, conflicting data exists as to the effect of orally administered therapeutic doses of beta-blockers in normal individuals,509'515 and in those with CHD.483,491,516 In vitro inhibitory effects of calcium entry blockers on platelets have also been well documented,517'526 although controversy has also arisen regarding evidence for an effect in persons receiving oral calcium entry blockers.521,527'529 In vitro studies have demonstrated different modes of action for these two group of drugs on platelets. Thus, a combination of beta-blockers and calcium entry blockers may be sufficiently effective in suppressing platelet activity in vivo to be measurable. Furthermore, in vitro findings may not reflect the in vivo effects because of artifacts inherent in the preparation of PRP530 and metabolic changes in other blood compon ents which may influence platelet activation such as the release of ADP from red blood cells.349 Clearly, in vitro findings cannot be equated unquestionably to the in vivo situations, which must be evaluated specifically and independently. The inhibi tion of in vitro platelet function by nitrates has also been the subject of frequent reports.531
In the selection of subjects for a case-control study, it is of importance to determine whether or not it is necessary to stop cardiac medications used by the volunteers. This study therefore examines the in vivo effects of a combination of a beta-blocker (metoprolol) and calcium entry blocker (nifedipine) on platelet function at rest and after exercise. The effect of these anti-ischaemic drugs on platelet function before and after exertional ischaemia was also evaluated.
4.4.2 Methods
4.4.2.1 Patient population. The study population is the same group described in section 4.2.2.1, and the results constitute part of a single study.
4.4.2.2 Study Protocol. The methods have previously been described in full detail298 and in section 4.2.2.2. Importantly, all drugs were ceased for at least 5 days before the first exercise test. Aspirin and nonsteroidal antiinflammatory drugs were ceased for at least 2 weeks. Blood was sampled immediately before and after each maximal exercise test. After the first exercise test, 26 of the volunteers commenced metoprolol 50 mg twice daily and nifedipine 10mg three times daily for approximate ly 4 weeks. The patients were then exercised at the same time in the morning, 1.5 to 2 hours after taking the medications. Then 24 patients stopped the medications for a minimum of 5 days before undergoing a third treadmill test. Three patients withdrew for reasons unrelated to the study or drug side effects. All subjects performed a maximum treadmill exercise test.
4.4.2.3 Investigations. The laboratory investigations and angiographic scoring details are described in Chapters 3 and 8 respectively.
4A.2.4 Data Analysis. The data analysis methods are described in Chapter 2 and section 4.2.2.
4.4.3 Results
4.4.3.1 Plasma BTG and TXB2. The resting BTG level was slightly higher in the group with positive exercise tests whilst not on medication, but the difference was not significant (Fig. 4.4). Following exercise there was a small but significant increase in the BTG levels, which occurred in both the positive and negative stress test groups in the second and third treadmill test, although only in the negative test group for the first exercise test (Fig. 4.4). A significant increase in plasma BTG level in both groups following exercise was not altered by the study medications (Fig.4.4).
No difference was observed in resting plasma TXB2 levels between the two groups, nor were there detectable changes in plasma TXB2 levels following maximum exer cise. The medications had no measurable effect on the plasma TXB2 levels before or after exercise.
4.4.3.2 Serum TXB2. A modest but significant increase in TXB2 levels with exercise was observed in both tests without medications, and this increase was diminished in the presence of the study medications (Fig. 4.5). This small but signif icant increase in the formation of TXB2 in serum following exercise occurred in the ischaemic group, but not in non-ischaemic group (Fig. 4.5). There was also a signif icantly lower level of the resting serum TXB2 in the ischaemic group compared to the non-ischaemic subjects (Fig. 4.5).
4.4.3.3 Platelet count ratio. The PCR decreased following exercise in the initial exercise test without medications. This decrease was not reproducible in the third exercise test without the study medications. No significant change was demonstrated in the PCR whilst subjects were on medications (Fig. 4.3). The PCR was consistent ly lower in the non-ischaemic group, but this difference did not attain statistical significance. The mean PCR at rest was the same in subjects with or without medica tions, and when they were divided into positive or negative exercise groups (Fig. 4.3).
4.4.3.4 Blood Cell Indices. The medications had no affect on these variables at rest or after exercise (Table 4.4).
4.4.4 Discussion
Anti-platelet therapy has been clearly identified as having a protective effect follow ing acute ischaemic coronary events.97,98,104 An in vitro inhibitory effect on platelet fu n ctio n by both b eta-b lo ck ers and calcium entry b lo ck ers has been well established.507'509,510,517,519'526. The in vivo effects on platelets of these classes of drug are less well defined and controversial.509,511"515,521,527'529
In the previous section, evidence was provided that in patients with stable CHD exertional myocardial ischaemia does not activate circulating platelets.298 This result
confirm ed previous reports485 and has subsequently been verified by another study.532 Predictably, specific anti-platelet drugs have no significant effect on objec tive measures of exertional myocardial ischaemia in patients with chronic stable angina.533 However, our observations suggested that brief strenuous exercise with the induction of myocardial ischaemia creates a milieu in whole blood which leads to increased TXB2 release during in vitro thrombus formation.298 Unstable angina is asso ciated w ith disease p ro g re ssio n ,114,534 in traco ro n ary throm bus formation,23,26,115,535,536 and also the production of increased amounts of excreted thromboxane metabolites.537,538 Moreover, pathological evidence exists for the involvement of thrombus in disease progression.34,539
Given that thrombus formation may be involved in the formation of new stenoses and occlusions, a mechanism for drugs to prevent such lesion development is to contribute to the inhibition of thrombus formation. The combination of a beta-block er and a calcium entry blocker is more effective in the prophylactic treatment of symptomatic CHD than the use of single drugs.540 Beta-blockers reduce the in cidence of recurrent CHD events after an AMI, including the thrombotic event of recurrent MI.541,542 However, calcium entry blockers have not as yet been shown to be effective against further obstructive ischaemic events after an acute myocardial infarct,543 except possibly in specific subsets of patients.544 Yet, as discussed, they appear to lessen the developm ent of new occlusive lesions in patients with CHD.545,546 Some understanding of the mechanisms of the anti-ischaemic effect of these drugs has been provided following extensive investigation,547 although the m echanism (s) for the possible secondary p rev en tativ e effect has not been clarified.546,548 An inhibitory action on platelet reactivity and a reduction in a con tinuing thrombotic tendency, is one possible contributing mechanism that requires further investigation.
The results fail to demonstrate any effect on specific measures of in vivo platelet function by the combination of a beta-blocker and calcium entry blocker. Plasma TXB2 and BTG levels at rest did not change. A modest increase in plasma BTG levels with exercise was unaffected by the addition of the medications or the pres ence of exertional ischaemia. An initial significant difference between the PCR before and after exercise was not reproducible in the repeat test without treatment. It is possible that the repeat test without medication by chance failed to demonstrate a true difference. However, this is probably unlikely, the repeated measures of the PCR may represent the phenomenon of regression to the mean.549 In the absence of a control or placebo group, the average of two or more measures for classification of the initial variable for comparison can be used.550 Using this method to reduce the effect of regression in the PCR measures, that is averaging the values for the first and third measurement when not on treatment, then there is no difference before and after exercise, and with and without treatment.
Following exercise, the serum TXB2 levels increased significantly in the group with a positive exercise test but not in the group with a negative exercise test.298 Although the increase in serum TXB2 level was small, the change within the group was clear ly attenuated by the drug combination. Serum TXB2 measures prostaglandin activity of blood cells during thrombus formation ex vivo. The production of prostaglandin metabolites will be influenced by platelet agonists present in the blood, or formed
during thrombus production. Suppression of TXB2 formation may arise from a number of factors, including a reduction in agonist concentration, reduced platelet prostaglandin metabolism, and/or reduced platelet response to endogenous agonists. The beta-blocker propranolol in vitro appears to have inhibitory activity on BTG release and TXB2 production by whole blood and on spontaneous clotting at concen trations near the therapeutic range.510 This may be due to an inhibition of collagen and thrombin-induced platelet aggregation and TXB2 synthesis.510 The present find ings are consistent with these in vitro observations, although the mechanism of the effect of the drug combination on platelet prostaglandin activity measured ex vivo
cannot be deduced from this study. Inhibition of platelet function in vivo following the infusion of isosorbide mononitrates at increasing does has been reported and includes a reduction in TXB2 production in PRP following agonist induced aggrega tion.531 The degree of inhibition was related to the rate of the infusion and it was concluded that the variability of the functional platelet response was related to the haemodynamic response.531 Eight of 11 patients in the study population had stable CHD with exercise ECG changes of myocardial ischaemia but characterisation according to the presence of inducible ischaemia was not reported. However, the observations suggest a relationship to the decrease in BP. Therefore it is possible that the abolition in the increase in serum TXB2 is related to the decrease in BP response to exercise whilst on the study medications rather than a direct effect on the platelet.
The explanations for the increased serum TXB2 production after exercise in ischae mic patients cannot be determined from the current study. However, it would appear from the lack of response seen with the measures of in vivo platelet activity, that this increase is unlikely to be due to an increase in circulating platelet activation. It is when platelets are activated during thrombus formation ex vivo, following exertional myocardial ischaemia, that a capacity for increasing thromboxane production occurs. Should this increase in prostaglandin production occur during thrombus formation in vivo, then a greater stimulus would be available for propagation of thrombus. Whether or not the combination of drugs contributes to a reduction in platelet pros taglandin activity in vivo and diminished thrombus propagation during endothelial desquamation, or atherosclerotic plaque rupture, can only be speculative.
The results of the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT) study confirmed previous experimental studies demonstrating a signifi cant reduction in newly formed coronary artery lesions (stenoses and occlusions) in patients on nifedipine compared to placebo, especially in the presence of early CAD.546 A proposed explanation, which will be briefly stated and elaborated on subsequently, ensues from the proposal that disruption of small atherosclerotic plaques with subsequent mural thrombosis and fibrotic organisation of thrombus seems to contribute to the progression of atherosclerosis.32 Firstly, nifedipine reduc es the development of new lesions.546 Our results show that the combination of nifidepine and metoprolol reduces the amount of thromboxane produced during whole blood clotting ex vivo. Progression of CAD involves new lesion formation that is not entirely related to the underlying atherosclerosis, implicating additional mechanisms such as thrombosis. Pathological evidence exists for the involvement of thrombus in disease progression.34,539 and clinical syndromes (unstable angina and AMI) are associated with new lesion development, in association with a fissured
plaque, and increased thromboxane production. M oreover, increased TXB2 produc tion occurs in whole blood clotting in the presence o f provoked ischaem ia.298 There fo re, a reduction in throm boxane p ro d u ctio n m ight red u ce the lik e lih o o d o f the production of those new CAD lesions that involve thrombosis.
Such a scenario im plies that disease progression, particularly the form ation of dis crete lesions, has a m ajor non-atherosclerotic com ponent, namely throm bus form a tion. The following brief review o f disease progression, as interpreted from angio graphic studies, supports this concept. D espite being able to say which areas of the vessel have a com m on site o f p ro g ressio n ,551 the use o f previous angiogram s to predict which vessels in an individual would be involved in progression has not been reliable.552 In fact, not only does knowledge o f the initial pathology not provide the ability to accurately predict progression, but the frequency o f progression does not appear to be related to the initial num ber o f vessels with greater than 50% lum inal o b stru c tio n s or lu m in al o c c lu sio n s.552 F u rth erm o re , alth o u g h a n g io g ra p h ica lly norm al coronary arteries have a low rate o f progression,553 those with some intralu m inal disease w ithout a significant lum inal obstruction have a rate o f progression almost equivalent to those with established CAD.554 Undoubtedly progression occurs in old existing atherom atous lesions. However, new areas o f significant narrow ing frequently occur in previously apparently norm al segm ents555 and the progressive nature o f the disease is quite ep iso d ic.556 Indeed, progression o f o cclu siv e CAD occurs commonly in areas that have previously been m inim ally diseased in patients w ith o u t i n i t i a l l y c l in ic a l d is e a s e r e q u ir in g a P T C A o r c o r o n a ry b y p a s s grafting.555,557,558. A natom ical studies have shown that a substantial percentage o f lesions associated with coronary artery thrombosis and AMI are moderate in severi- t y 12,559
In summ ary, a high incidence o f progression o f CAD in vessels that are norm ally m inim ally diseased has been well docum ented.20,555,557,558,560 although the progres s io n to to ta l o c c lu s io n a ls o o c c u r s f r e q u e n tly in v e ry s e v e r e ly n a rro w e d lesions.21,43,555 The progression o f coronary atherosclerosis is a highly unpredictable process that follows a non-linear course and information about its dynamics derived from sequential coronary arteriograms hardly improves prediction o f future progres sio n .561 K now ledge o f the in itial pathology does not allow accurate pred ictio n o f w here new lesions will occur.551,562,563 In addition, there appear to be tim e depend ent and time independent factors.551,564 The time dependence does not appear to be present for those developing unstable angina and progression com pared with individ uals having chronic stable angina and disease progression.
4.4.5 Summary
1. There is no evidence o f activated circulating platelets follow ing exer tional ischaemia and no measurable effect of the drug combination o f m etoprolol and nifedipine on in vivo measures of platelet activity.
2. An increase in prostaglandin biosynthesis ex vivo in whole blood asso ciated with exercise induced myocardial ischaemia is attenuated by a combination of a beta-blocker and a calcium entry blocker. The drugs may produce their effect by reducing m yocardial ischaem ia, BP or, alternatively, by directly decreasing blood
cell prostaglandin formation.
3. In addition, it may be possible that a reduction of whole blood prosta glandin activity during thrombus formation in individuals with myocardial ischaemia may contribute to the protective effect of these drugs against acute, and transient obstructive thrombotic coronary episodes.
4. For evaluation of platelet function in a cross-sectional study, such a potentially confounding factor will not be a major bias. For patient safety, all car diovascular medications were not ceased beforehand. This allowed the serial selec tion of patients with stable premature CHD, rather than waiting and only selecting patients without medications, or who are sufficiently stable to ethically stop medica tions.
4.5 VARIABILITY OF SERUM TXB2 LEVELS AND INTERACTION