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J Am Coll Cardiol, 2002; 40:484-490 © 2002 by the American College of Cardiology Foundation |
a Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, United Kingdom
Manuscript received March 1, 2002; revised manuscript received April 16, 2002, accepted May 7, 2002.
* Reprint requests and correspondence: Prof. Gregory Y. H. Lip, Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham B18 7QH, United Kingdom.
g.y.h.lip{at}bham.ac.uk
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Abstract |
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BACKGROUND: Dose-adjusted warfarin is beneficial as thromboprophylaxis in AF, but potentially serious side effects and regular monitoring leave room for alternative therapies.
METHODS: We randomized 70 patients with nonvalvular AF who were not on any antithrombotic therapy to either dose-adjusted warfarin (international normalized ratio 2 to 3) (Group I) or combination therapy with aspirin 75 mg and clopidogrel 75 mg (Group II). Plasma indices of thrombogenesis (fibrin D-dimer, prothrombin fragment 1+2) and platelet activation (beta-thromboglobulin [TG] and soluble P-selectin) were quantified, along with platelet aggregation responses to standard agonists, at baseline (pretreatment) and at six weeks posttreatment.
RESULTS: Pretreatment levels of fibrin D-dimer (p = 0.001), beta-TG (p = 0.01) and soluble P-selectin (p = 0.03) were raised in patients with AF, whereas plasma prothrombin fragment 1+2 levels and platelet aggregation were not significantly different compared with controls. Dose-adjusted warfarin reduced plasma levels of fibrin D-dimer, prothrombin fragment 1+2 and beta-thromboglobulin levels at six weeks (all p < 0.001), enhanced plasma levels of soluble P-selectin (p < 0.001) and had no significant effect on platelet aggregation. Aspirin-clopidogrel combination therapy made no difference to the plasma markers of thrombogenesis or platelet activation (all p = NS), but the platelet aggregation responses to adenosine diphosphate (p < 0.001) and epinephrine (p = 0.02) were decreased.
CONCLUSIONS: Aspirin-clopidogrel combination therapy failed to reduce plasma indices of thrombogenesis and platelet activation in AF, although some aspects of ex vivo platelet aggregation were altered. Anticoagulation with warfarin may be superior to combination aspirin-clopidogrel therapy as thromboprophylaxis in AF.
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Clinical trials have established the role for adjusted-dose warfarin (target international normalized ratio [INR] 2 to 3) as thromboprophylaxis in AF, whereas the benefits of antiplatelet therapy such as aspirin are inconsistent (2). However, warfarin poses a significant threat of bleeding and warrants regular anticoagulation monitoring, contributing to a general reluctance to commence anticoagulation therapy and to noncompliance among patients (3,4). Efforts have thus been made to find a viable alternative to warfarin for thromboprophylaxis in AF. Nevertheless, trials with low intensity or fixed-minidose warfarin, or a combination of fixed-dose warfarin with aspirin, have been disappointing (5).
The utility of measuring various markers of hypercoagulability to indicate the effectiveness of antithrombotic therapy has been demonstrated by randomized trials where fixed low-dose (1 mg) warfarin, warfarin-aspirin combination therapy or aspirin 300 mg did not significantly reduce elevated indices of thrombogenesis and platelet activation in nonvalvular AF, although conventional warfarin therapy did normalize levels of these markers (6,7). These observations were consistent with the beneficial effect of full-dose warfarin in preventing stroke and thromboembolism in patients with AF in clinical trials, and suggested that fixed low-dose (1 mg) warfarin, warfarin-aspirin combination therapy or aspirin 300 mg may not exert similar beneficial effects in reducing thrombogenesis, in keeping with the subsequent publication of the results from their respective clinical trials (5).
The role of platelet activation in AF needs to be better defined (8) and an investigation into the influence of combination antiplatelet therapy in AF is warranted. Aspirin exerts its antithrombotic effect by inhibiting the synthesis of thromboxane A2, which potentiates platelet aggregation and the release of granule contents (9–11). However, aspirin is not a universally effective antithrombotic agent, as platelet activation could still occur through cyclooxygenase-independent pathways (12–15). The thienopyridine antiplatelet agents ticlopidine and clopidogrel specifically interfere with the adenosine diphosphate (ADP)-induced activation pathway to inhibit ADP-induced platelet activation (14,15). In clinical studies, a combination of aspirin and antiplatelet therapy (such as dipyridamole or clopidogrel) has proven to be of higher benefit than either drug alone in reducing platelet-induced thrombus formation in a variety of atherosclerotic vascular disorders, such as ischemic heart disease and unstable angina (16). Indeed, combination antiplatelet therapy appears to be superior to warfarin regimens in reducing stent thrombosis and hemorrhagic complications (17).
We hypothesized that introducing combination therapy with aspirin (75 mg) and clopidogrel (75 mg) could reduce indices of thrombogenesis when compared to dose-adjusted anticoagulation with warfarin (achieving an INR of 2.0 to 3.0). To test this, we performed a prospective randomized trial of aspirin-clopidogrel combination therapy versus adjusted-dose warfarin in nonvalvular AF.
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Subjects and methods |
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4 weeks apart). We excluded any patients with hematological, renal (creatinine >200 mg/dl), hepatic (liver enzymes > twice the upper limit of normal), inflammatory or neoplastic disorders and those who suffered recent (<3 months) myocardial infarction or stroke, as these disorders might influence platelet activation and the levels of the markers measured. Patients who consumed regular nonsteroidal anti-inflammatory drugs, corticosteroids or hormone replacement therapy were also excluded. Furthermore, patients with rheumatic mitral valvular disease, prosthetic cardiac valves and acute AF precipitated by thyrotoxicosis or any acute infection were excluded from the study. The study was approved by the City Hospital ethics committee and informed consent was obtained from patients. We randomized the 70 patients with chronic nonvalvular AF to either dose-adjusted warfarin (INR 2-3) (Group I, n = 35; 23 men; mean age 69 years, SD 11) or combination therapy with aspirin 75 mg and clopidogrel 75 mg (Group II, n =35; 21 men; mean age 71 years, SD 8). Patients were seen at baseline (visit 1) and then at six weeks (visit 2) after randomization. The patients randomized to warfarin were referred to and regularly seen in the hospital anticoagulation clinic to achieve and maintain an INR of 2.0 to 3.0. Because of patient withdrawals, complete samples for haemostatic markers were taken at six-week follow-up visit in only 33 patients in Group I and only 32 patients in Group II; paired comparisons between baseline and posttreatment samples were performed on these 33 and 32 patients, respectively.
Baseline blood results in patients with AF were compared with "healthy controls," which consisted of 50 normal subjects (23 men; 70 years, SD 13) recruited from healthy hospital staff, relatives of the patients and those attending the hospital for routine senile cataract surgery. The subjects were nonsmokers with no clinical evidence of vascular, metabolic, neoplastic or inflammatory disease on careful history, examination and routine laboratory tests. Clinically, these subjects were normotensive and in sinus rhythm. The reason for including this healthy control group was not to emphasize a case/control comparison (as previously addressed by many other authors [1]), but to indicate approximate "normal" levels of the hemostasis markers for comparisons with the AF patient group.
Laboratory. Blood samples were drawn from an antecubital vein with atraumatic venipuncture into plastic tubes with 3.2% sodium citrate for platelet aggregation study, and also for the quantification of plasma indices of thrombogenesis (fibrin D-dimer, prothrombin fragment 1+2) and platelet activation (beta-thromboglobulin [beta-TG] and soluble P-selectin). Blood for plasma beta-TG levels were collected in vacutainers containing citrate, theophylline, adenosine and dipyridamole.
Blood samples for the quantification of plasma markers were separated within 1 h of collection by centrifugation at 3,000 rpm for 10 min. Aliquots were stored at –70°C to allow batch analysis. Soluble P-selectin (R and D Systems, Abington, United Kingdom), beta-TG levels (Asserachrom beta-TG, Diagnostica Stago, Asnieres Sur Seine, France), fibrin D-dimer (Technoclone, Wien, Austria) and prothrombin fragment 1+2 (Enzygnost F 1+2 micro, Dade Behring Marburg GmbH, Marburg, Germany) were all determined by enzyme-linked immunosorbent assay. Intraassay and interassay coefficients of variation for all enzyme-linked immunosorbent assays were <5% and <10% respectively.
Citrated blood for platelet aggregation was maintained at room temperature (22°C) until sample preparation to maintain platelet activity. Citrated blood tubes were centrifuged within 1 h of collection, at room temperature at 1,000 rpm for 10 min. The supernatant platelet-rich plasma was separated. The residual sample was then centrifuged at 2,000 rpm for 20 min to obtain a clear citrated supernatant, platelet-free plasma. Ex vivo platelet aggregation was measured in a plasma platelet aggregometer (Platelet aggregation profiler, model PAP-4, Bio/Data Corporation, Horsham, Pennsylvania). Aggregation was induced by the addition of the following reagents: 1) ADP (final concentration of 0.1 µmol/ml), 2) collagen (final concentration of 2 mg/ml), epinephrine (final concentration of 0.1 µmol/ml) (Sigma Diagnostics, St. Louis, Missouri) and 3) thrombin (final concentration of 10 NIH U/ml) (Pacific Hemostasis, Huntersville, North Carolina). Platelet aggregation curves were recorded and the extent of platelet aggregation was evaluated by measuring the percentage platelet aggregation at the end of 3 min.
Power calculations. We have previously demonstrated that six weeks of dose-adjusted warfarin reduced median levels of fibrin D-dimer by 50% among 51 patients with AF (6) and by 40% in 61 different patients (7). However, in the current study, because we intended to measure additional indices, we more conservatively hypothesized a reduction in median D-dimer levels (due to either intervention) of one-third of its pretreatment (baseline) level. In order to achieve this difference at p < 0.05 and a 1-beta of 0.8 we needed good data from 25 subjects per intervention group. However, as stated, we recruited in excess of this figure to improve our chances of minimizing Types I and II errors. We compared our 70 patients at baseline to 50 healthy controls. The reason for including this latter group is not to provide hard case/control data but to indicate approximate levels of the hemostasis markers in the patient group. However, these numbers provide sufficient power to detect a difference of 0.4 of a standard deviation, under the same probability conditions, should one be present.
Statistical analysis.
Results are expressed as mean, SD or as median (interquartile range) for the normally distributed data and nonparametrically distributed data respectively. Data between patients with AF and healthy controls were analyzed using the unpaired t test or Mann-Whitney U tests as appropriate. Paired data, which were comparisons of baseline levels with those for six weeks posttreatment, were performed using the paired t test or paired Wilcoxon test, as appropriate. Correlations were performed using the Spearman rank correlation method, and categorical data were compared with use of the 
2 test. All statistical calculations were performed on a microcomputer using a commercially available statistical package (Minitab release 12, Minitab Inc., State College, Pennsylvania). A p value <0.05 was considered as statistically significant.
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Results |
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Treatment with warfarin (Group I) or aspirin-clopidogrel combination therapy (Group II). Patients with AF in two treatment groups were of similar age and gender ratio (Table 3). The majority of clinical cardiovascular risk factors or underlying diseases did not differ significantly between the subgroups of patients with AF, although the AF patients subsequently randomized to combination antiplatelet therapy had a higher prevalence of ischemic heart disease (p = 0.03), whereas a significantly higher proportion of patients randomized to warfarin had a previous history of stroke/transient ischemic attacks (p = 0.01).
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Discussion |
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In the present study, dose-adjusted warfarin significantly reduced plasma levels of fibrin D-dimer and prothrombin fragments 1+2 at six weeks, in keeping with a reduction in thrombogenesis and fibrin turnover. This confirms the previous similar findings (6,7,19) and is consistent with the beneficial effects of warfarin in the clinical setting. Warfarin also reduces plasma beta-TG levels, perhaps reflecting warfarin’s inhibitory action on the production of thrombin, which is a potent platelet agonist (6). Therefore, warfarin possibly exerts its beneficial effect in the reduction of stroke and thromboembolism by two mechanisms: 1) a reduction in hypercoagulability and thrombogenesis, related to the fibrin-rich atrial thrombi and 2) (possibly) a reduction in platelet activation, resulting in a decrease in platelet-rich thrombi, associated with atherothrombotic vascular disease commonly present in patients with AF.
Interestingly, warfarin significantly enhanced plasma levels of soluble P-selectin in patients with AF. However, this finding needs to be interpreted very cautiously. Soluble P-selectin is thought to originate from platelets (22,23), and has been hypothesized to be a new marker of platelet activation (24). However, whether soluble P-selectin levels represent platelet activation or simply a destruction of platelets by the reticulo-endothelial system is not entirely clear. Except for occasional studies investigating plasma levels of soluble P-selectin in AF (21), other work examining the role of platelet activation and its correlation to stroke in AF have predominantly concentrated on platelet surface expression of P-selectin, rather than plasma soluble P-selectin levels per se (25–27). Furthermore, plasma soluble P-selectin levels did not correlate with plasma beta-TG levels in patients with AF, in keeping with our previous observations (21) and the lack of correlation between plasma beta-TG and P-selectin expression on platelets (28). However, given the complexity of platelet activation in AF (8), it is still possible that warfarin enhances some aspects of platelet activation while suppressing the others. Furthermore, there is some evidence of warfarin causing platelets to be hyperaggregable in AF (29).
Clopidogrel selectively and irreversibly inactivates platelet-ADP receptors and reduces vascular events by 30% to 35% (30). Previous experimental studies in both animals and humans and large-scale clinical studies have demonstrated that a combination of aspirin and clopidogrel results in a combined inhibition of cyclooxygenase and ADP effects, and provides marked enhancement of antithrombotic efficacy in experimental and clinical studies of patients with atherosclerotic vascular disorders (15,16,31–33). Aspirin selectively and irreversibly interrupts thromboxane A2 production and decreases thromboembolic events by 20% to 25%; interestingly, aspirin therapy results in a nonsignificant 25% reduction in the markers of thrombogenesis (6,18).
However, platelet aggregation was no different in our patients with AF when compared with healthy controls in sinus rhythm, and the effect of combination antiplatelet therapy on platelet aggregation in our patients with AF would thus seem to be of limited clinical interest. Furthermore, previous studies have failed to relate platelet aggregation to surrogate markers of thromboembolism, such as spontaneous echo contrast in AF (34). Although there was reduction in platelet aggregation response to ADP and epinephrine with combination antiplatelet therapy, the latter failed to reduce plasma indices of thrombogenesis and platelet activation in AF. Our observations confirm previous observations of only partial inhibition of platelet aggregation by aspirin (alone) in AF (29) and the inhibition of platelet aggregation in response to ADP and epinephrine, but not in response to collagen and thrombin (35). It is believed that epinephrine results in platelet aggregation through a thromboxane-mediated pathway, whereas collagen and thrombin mediate their platelet aggregatory effects through thromboxane-independent pathways (35). Furthermore, clopidogrel is an ADP antagonist (36) and it is therefore not surprising that in the present study, platelet aggregation to ADP was reduced by aspirin-clopidogrel combination therapy. Thus, it appears that although various aspects of platelet activation may often be intimately linked, each process can occur independently of the other, depending upon the stimulus used.
The findings from our study raise two important issues. First, the consistent clinical benefit of antiplatelet therapy in atherosclerotic vascular disorders, but not in AF, raises the question whether the nature of platelet activation in AF differs from that seen in atherothrombotic vascular disorders (8,11). Second, the available clinical data and the present study suggest that even though platelet activation exists in AF, it is probably the enhanced coagulation rather than platelet activation that plays an important role in the stroke and thromboembolic risk of AF.
Study limitations. The main objective of our study was to study the effects of combination aspirin-clopidogrel antiplatelet therapy and dose-adjusted warfarin on plasma indices of thrombogenesis and platelet activation, and platelet aggregation. Therefore, we have not made any detailed attempt at correlating these parameters with the patient’s clinical, demographic or echocardiographic findings at baseline, in view of many previous analyses on this topic (1). Thus, there may be the potential for unmeasured differences (e.g., comorbidity) between groups that may have affected the results. However, available evidence already points towards a lack of a significant relationship between markers of thrombogenesis and patients’ cardiovascular risk profile or structural cardiac abnormalities on echocardiography (1,18,20). The study methods also do not allow inference regarding the clinical or prognostic value of combination antiplatelet therapy compared to dose-adjusted warfarin, which would necessitate a large clinical trial. Furthermore, whether in vitro or ex vivo platelet activation (as measured by platelet aggregometry) reflects true in vivo activity (as reflected by plasma beta-TG and soluble P-selectin levels) is not exactly known (8,11). Although many workers (including our group) have published extensively on soluble P-selectin as an index of platelet activation, we also recognize that many plasma markers of platelet activation do exist (11), with some debate over which is the best one. The possibility also arises that the increase in soluble P-selectin with warfarin could merely reflect a regression to the mean or some putative seasonal variation in soluble P-selectin.
In conclusion, we have confirmed previous observations that AF is associated with enhanced thrombogenesis and platelet activation. Dose-adjusted warfarin significantly reduced thrombogenesis and (at least one aspect of) platelet activation in AF, consistent with its observed clinical benefit. However, aspirin-clopidogrel combination therapy failed to reduce plasma indices of thrombogenesis and platelet activation in AF, although some aspects of ex vivo platelet aggregation were altered. Anticoagulation with warfarin may be superior to combination aspirin-clopidogrel therapy as thromboprophylaxis in AF.
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