Abstract
Clinical and experimental data suggest that ergotamine compounds and triptans may contribute to vascular events such as myocardial infarction and stroke. The role of blood cell aggregation in this context is, however, not clarified. We aimed to evaluate the impact of different acute antimigraine compounds on platelet and erythrocyte aggregation in a human ex vivo experimental design. In 20 healthy subjects without migraine and in 20 healthy subjects with migraine without aura, platelet and erythrocyte aggregation were measured before and after intake of placebo, acetylsalicylic acid, ergotamine tartrate, zolmitriptan and sumatriptan. Platelet aggregation was measured by the so-called platelet reactivity index. Erythrocyte aggregation was measured by photometric assessment in an aggregometer. Ergotamine tartrate induced a significant increase of platelet aggregation, whereas acetylsalicylic acid induced a significant decrease in both subject groups. After placebo, after sumatriptan and after zolmitriptan, no significant changes of platelet aggregation were noted. Erythrocyte aggregation was affected by neither compound. We can conclude that platelet aggregation, but not erythrocyte aggregation, is increased after intake of ergotamine tartrate. This may in part contribute to vascular side-effects of this compound. Acetylsalicylic acid and the triptans appeared to be safe with respect to platelet and erythrocyte aggregation.
Introduction
Since the introduction of ergotamine-containing compounds and, again, of the triptans into acute migraine therapy, it has been debated whether these drugs can induce an acute vascular event such as stroke or myocardial infarction. For ergotamine compounds, risk for such a vascular event has been suggested in case series and in studies on their impact on vessel properties (1, 2). For the triptans, similar case reports have been published which proposed an increased incidence of stroke or myocardial infarction after intake of these drugs (3). However, this has not been confirmed in controlled epidemiological studies which show no increased risk of vascular events in patients regularly taking triptans (4). Such epidemiological studies have, however, not been performed for ergotamine compounds.
Beside the receptor-specific effects of ergotamine compounds and the triptans on vessel wall properties (5, 6), other mechanisms might contribute to the ischaemic events which have been discussed to be associated with the intake of these drugs. One of these mechanisms could be an increased risk of blood cell aggregation. Little is known about the direct influence of the specific antimigraine compounds on platelet and erythrocyte aggregation. For sumatriptan, one study has been published, showing no effect of this drug on platelet responsiveness in different in vitro models (7). Ergotamine has long been suggested to induce platelet aggregation (8). However, modern experimental studies on this issue are lacking.
Previous studies on the impact of drugs on platelet functions have examined in vitro models of platelet aggregation. We wanted to study the impact of antimigraine compounds on the complex mechanisms of platelet and erythrocyte aggregation in an ex vivo model which has been shown to be useful and valid in other conditions with disturbances of platelet function (9, 10). Such a model is of particular importance, since migraine patients show disturbances of platelet function with increased aggregation (11).
We therefore designed a study in order to evaluate changes of platelet and erythrocyte aggregation caused by different antimigraine drugs in an ex vivo model of platelet and erythrocyte aggregation. We choose acetylsalicylic acid, ergotamine tartrate, sumatriptan and zolmitriptan in their (for Germany) recommended oral standard dose. Placebo was included to control for the impact of the study procedure on platelet and erythrocyte aggregation. The study was performed in a crossover blinded design.
Methods
Subjects and study procedure
We enrolled 20 healthy subjects without any neurological or psychiatric disease and without any drug intake. In addition, 20 otherwise healthy subjects with migraine without aura according to the criteria of the International Headache Society were enrolled. They were matched for age and sex. These subjects were non-smokers, without any drug intake, and did not take any hormones. All migraine subjects were studied outside an attack. Data were considered for statistical analysis only if the last migraine attack had finished at least 3 days previously and if the next migraine attack did not start the day after the investigation. The study was approved by the Ethics Committee of the University of Münster.
After giving written informed consent, the subjects were asked to return to the clinic on five different days with at least 2 days between two visits and to come always at the same time of day. On every visit, after a rest of 30 min, the subjects received one of the following oral drugs with 100 ml water: placebo (gelatine capsule with glucose), zolmitriptan 2.5 mg, sumatriptan 100 mg, acetylsalicylic acid 1000 mg or ergotamine tartarte 2 mg. The order of the drugs was randomized for all subjects, who were blinded to the drug they received. Directly before and 2 h after drug intake, a venous blood sample was taken from the left anticubital vein. The subjects had to rest between the blood samples. The blood was analysed immediately after intake of the drugs by an investigator who was blinded to the respective drug taken by the subject.
Aggregation analysis
Platelet aggregation was determined ex vivo according to a previously described procedure (12). In brief, a platelet reactivity index was calculated which quantifies the ratio of the number of non-aggregated platelets and the total number of platelets measured immediately after blood sampling. Blood was collected from the left anticubital vein by using an R21 butterfly in 3-ml syringes with the first 200 µl being discarded. Then, 300 µl blood was collected directly into two syringes containing either 1000 µl EDTA buffer (11.4 mmol Na-EDTA in 1 : 15 mmol NaK hydrogenphosphate, pH 7.4) or EDTA-formaldehyde buffer (EDTA buffer containing 1% formaldehyde). Erythrocytes were counted in both samples which were then centrifuged for 20 min at 52 g. Supernatants were collected and platelets were counted by means of a commercial platelet counter. The platelet reactivity index (PR) was calculated by the following formula:
PR = (platelets in EDTA × erythrocyte number in EDTA-formaldehyde)/(platelets in EDTA-formaldehyde × erythrocytes number in EDTA)
Erythrocyte aggregation was determined by a commercial aggregometer (device MA1, Myrenne, Germany) according to a method published previously (13). For this procedure, EDTA blood was taken and centrifuged. Then, a standardized blood sample was produced consisting of 60% plasma and 40% cell sediment. Twenty microlitres of this sample was given in a glass chamber of the aggregometer. The measurement of erythrocyte aggregation is based on the photometric assessment of the changes in light transmission that accompany the spontaneous formation of the erythrocyte rouleaux and aggregates of rouleaux that occurs when a sample of erythrocytes is first sheared and then suddenly brought to a stop. The numerical output of the MA1 device is equivalent to the percent of the maximum possible extent of erythrocyte aggregate formation during the first 5 s after shear stop. The laboratory normal value of this system is 30.0 ± 6.6 (arithmetic mean with twofold SD; no dimension); higher values denote more aggregates.
Statistics
All values are presented as arithmetic mean with 1 SD. In larger series, both erythrocyte and platelet aggregation measured as described above follow a parametric distribution. However, since we had a small sample size, we could not be sure that our data were parametric. Therefore, we used non-parametric tests (Wilcoxon test with Bonferroni correction for the comparisons between the time before and after intake of the study drug; Kruskal–Wallis analysis for the comparison between the five study drugs at baseline). The significance level was set at P = 0.05.
Results
Demographic data of the healthy and migraine subjects and the time pattern of the migraine subjects are shown in Table 1.
Demographic data of the healthy and migraine without aura subjects and time pattern of the migraine subjects
Since both subject groups were matched for age and sex, there were no significant differences.
In Table 2, data of platelet aggregation are presented for both the healthy and the migraine subjects. There were no significant differences between the five drugs in the baseline values. After intake of placebo, no significant changes in platelet aggregation could be observed. After intake of acetylsalicylic acid a significant decrease and after intake of ergotamine tartrate a significant increase of platelet aggregation could be noted in both subject groups. Sumatriptan and zolmitriptan induced no significant changes of platelet aggregation.
Platelet aggregation directly before and 2 h after intake of the different study drugs given as arithmetic mean and simple standard deviation
Comparison between the time before and after drug intake by Wilcoxon test with Bonferroni correction. The data are presented separately for healthy subjects without and with migraine. NS, Not significant.
In Table 3, data of erythrocyte aggregation are presented for both the healthy and the migraine subjects. There were no significant differences between the five drugs in the baseline values. We could detect no significant changes of this aggregation by any of the study drugs. However, a trend (P = 0.093) towards decreased erythrocyte aggregation was noted after intake of acetylsalicylic acid in healthy subjects without migraine.
Erythrocyte aggregation directly before and 2 h after intake of the different study drugs given as arithmetic mean and simple standard deviation
Comparison between the time before and after drug intake by Wilcoxon test with Bonferroni correction. The data are presented separately for healthy subjects without and with migraine. NS, Not significant.
There were no significant differences between healthy subjects without and healthy subjects with migraine at baseline. Furthermore, we could detect no significant differences in platelet or erythrocyte aggregation with respect to sex or age (data not shown).
Discussion
Our most important finding is that ergotamine tartrate, but not sumatriptan or zolmitriptan, induced increased platelet aggregation 2 h after intake of the standard dose for treating migraine attacks. As expected, acetylsalicylic acid induced a decrease in platelet aggregation. Erythrocyte aggregation was not affected in any way beside a non-significant trend for a decrease after intake of acetylsalicylic acid in healthy subjects without migraine. For sumatriptan, a previous in vitro study had also shown that this drug has no direct effect on platelet aggregation (7). In a recent study, it has even been shown that triptans can normalize the increased platelet activation of patients with migraine without aura (14).
The observed difference between ergotamine and the triptans may be due to the different receptor affinity of these drugs. 5-HT1 receptors are rarely found on the surface of platelets (15, 16). Therefore, the triptans probably do not have any activity on platelet receptors and, thus, not on platelet metabolism. The same is true for erythrocytes. Ergotamine, however, has several biological agonistic properties on serotonergic and other (e.g. adrenergic) receptors (17). It has been shown that 5-HT and adrenaline are able to induce platelet aggregation in vitro (18), although the exact receptors mediating this effect are, as yet, not identified. Vice versa, 5-HT receptor antagonists are able to suppress the activation of platelets by 5-HT (19). Probably, ergotamine compounds with their known activity on several serotonergic and adrenergic receptors are able to induce platelet aggregation through this mechanism.
Furthermore, ergotamine can cause marked vasoconstriction (6) and decreased arterial vessel wall distensibility (5) in extracerebral vessels by adrenergic mechanisms and by direct action on vascular smooth muscle cells. These mechanisms lead to modification of specific arterial vessel wall properties which can activate platelets and indirectly induce platelet aggregation. Since triptans have no adrenergic activity (3), it is unlikely that these drugs induce similar changes of arterial vessel walls to those of ergotamine.
Acetylsalicylic acid has long been known as an agent with anti-aggregating properties. Several studies have shown this effect in vivo, ex vivo and in vitro (9, 20–22). We can confirm the inhibiting effect of acetylsalicylic acid on platelet aggregation. Furthermore, there was a trend for a decrease of erythrocyte aggregation by acetylsalicylic acid, which has also been shown in a recent study (23).
Interestingly, there were no significant differences between subjects with and those without migraine. Activation of platelets by ergotamine and the lack of effect of triptans on platelet and erythrocyte aggregation were similar for both subject groups. The well-known differences in platelet properties between healthy subjects and subjects with migraine do not seem to play a major role in platelet aggregation induced by ergotamine or inhibited by acetylsalicylic acid.
There are two limitations of our study. First, we did not examine the situation in a migraine attack. It might be that activation of platelets in the attack is affected by antimigraine compounds in a different way. Second, we did not examine patients with migraine with aura. This was due to the difficulty in finding enough healthy aura patients able to make five visits. The major advantage of the method used in this study to determine platelet and erythrocyte aggregation is that the influence of the different drugs could be measured ex vivo. On the other hand, the method evaluated platelet and erythrocyte aggregation as a whole process and could not distinguish between the different components of the complex physiological mechanism of aggregation. However, it was not the aim of this study to analyse the influence of the drugs on pharmacological mechanisms of aggregation, but to analyse influence on the amount of aggregation from a clinical perspective.
The increase of platelet aggregation induced by ergotamine tartrate is within 1 SD of the values before ergotamine intake and of the values after placebo intake. Therefore, in our interpretation, the increase in platelet aggregation induced by ergotamine tartrate is clinically of minor relevance and cannot be a major cause of vascular events caused by ergotamine. However, this mild increase may contribute to other biological effects of ergotamine such as vasoconstriction and decreased vessel wall distensibility and may, thus, be one factor among others leading to vascular incidents which have been widely associated with ergotamine in the literature.
In summary, we have demonstrated that the 5-HT1B/D receptor agonists sumatriptan and zolmitriptan do not induce significant changes of platelet or erythrocyte aggregation in an ex vivo examination. Ergotamine tartrate induced a mild but significant increase of platelet aggregation, whereas acetylsalicylic acid induced a significant decrease of platelet aggregation.