Platelet Activation Is Not Involved in Acceleration of the Coagulation System in Acute Cardioembolic Stroke With Nonvalvular Atrial Fibrillation
Background and Purpose It is generally accepted that the coagulation system is activated in ischemic stroke and that platelet activation is involved in the pathogenesis of this disease. However, little is known about how and to what extent platelet activity participates in coagulation system enhancement. We evaluated the hemostatic condition, especially with regard to platelet function and the coagulation system, within 3 days of onset of acute stroke. The study participants were limited to elderly patients with cardioembolic stroke due to nonvalvular atrial fibrillation.
Methods Seventeen elderly patients with acute cardioembolic stroke due to nonvalvular atrial fibrillation were investigated. Within 3 days of stroke onset, β-thromboglobulin (BTG), platelet factor 4 (PF4), thrombin-antithrombin III complex (TAT), and d-dimer from arterial blood were carefully evaluated in these patients. Blood samples from 19 healthy age- and sex-matched control subjects were also examined.
Results The two studied markers of platelet activity did not change in the patients or the control subjects, and the between-group differences between the stroke and control groups were not statistically significant (BTG, 43.8 versus 31.9 ng/mL; PF4, 9.06 versus 5.78 ng/mL; respectively). In contrast, the two studied coagulation-system indicators were markedly elevated in the patients compared with the control subjects (TAT, 13.8 versus 3.5 ng/mL, P<.01; d-dimer, 366.3 versus 147.2 ng/mL, P<.01; respectively).
Conclusions Platelet function was not enhanced in the acute stage of cardioembolic stroke with nonvalvular atrial fibrillation. This result indicates that enhancement of the coagulation system in cardioembolic stroke is not the result of platelet hyperfunction, ie, “platelet-fibrin” thrombi, but rather of “stasis-related” thrombi formation.
Recent ischemic stroke research has indicated that acceleration of coagulation-system activity plays an important role in the development of ischemic stroke. Takano et al1 2 and Yamazaki et al3 reported a marked activation of the coagulation system and enhancement of the secondary fibrinolytic system in cardioembolic stroke. This coagulation-system enhancement is considered to be not the result of stroke but a reflection of its mechanisms of thrombi formation and vessel occlusion.
Enhancement of the coagulation system may be induced through either activation of the platelet pathway or direct stimulation due to abnormal blood stasis. Controversy still exists as to whether activation of platelets contributes to this hemostatic condition. Fisher and Francis,4 Shah et al,5 and Woo et al6 reported elevation of plasma BTG in cardioembolic stroke, whereas we7 8 and Waki et al9 found that platelet function in cardioembolic stroke was modestly activated. Shah et al5 also reported that in about one fourth of patients with cardioembolic stroke platelet function was normal. One reason for this difference in results may be that these previous investigators did not exclude valvular and other kinds of heart disease, which may activate platelet function.
Thus, we evaluated four hemostatic molecular markers: BTG and PF4, which reflect platelet α-granule release, and TAT and d-dimer, which are coagulation-system and secondary fibrinolytic-system indicators, in patients with acute cardioembolic stroke and NVAF to clarify the involvement of platelet activation in the pathogenesis of acute cardioembolic stroke.
Subjects and Methods
This investigation included 17 consecutive and eligible patients with acute cardioembolic stroke and NVAF who were admitted to Tokyo Metropolitan Tama Geriatric Hospital from January 1990 through December 1993. All of the patients were evaluated within 3 days of stroke onset, and all had a history of chronic atrial fibrillation that had been previously diagnosed in our hospital before stroke onset. The diagnosis of cardioembolic stroke was made using the criteria of the National Institute of Neurological Disorders and Stroke.10 Following previously reported11 12 13 14 15 methodology, electrocardiography and echocardiography were performed immediately after admission for the diagnosis of NVAF. We ruled out patients with atrial fibrillation and valvular heart disease, a prosthetic heart valve, or other organic heart diseases (eg, myocardial infarction and dilated cardiomyopathy). Patients with complications that may affect systemic hemostasis, such as hematologic diseases, infectious diseases, malignancies, ongoing large and peripheral vessel diseases (especially deep vein thrombosis), and pulmonary embolism were also excluded from our study. Patients with severe carotid artery lesions were not included. None of our patients were receiving antiplatelet therapy or had been previously treated with anticoagulants or anti-inflammatory drugs. Patient profiles are summarized in Table 1⇓.
Special care was taken not to affect coagulation status during blood sampling. Thus, blood examination was performed before any other procedure on admission. To avoid alteration of coagulation status, we restricted blood sampling to a single arterial puncture using a 22-gauge “butterfly” needle and collected less than 10 mL of blood. Specimens drawn after two or more venipunctures were excluded. Blood sampling was performed only by the doctors in our department. The investigators who performed the laboratory measurements were blinded to the status of the patients.
Blood was collected immediately after sampling into a precooled, siliconized glass tube containing (in mg/mL) 5.78 anhydrous theophylline, 1 adenosine, 0.1 dipyridamole, 5.78 citrate monohydrate, and 24.26 trisodium citrate dehydrate (Diatube-H; Terumo Europa Ltd). Plasma was separated by centrifugation at 2000g for 30 minutes at +2°C and was stored at −70°C until assayed. Plasma levels of BTG and PF4 were determined using Asserchem β-TG and PF4 kit-EIA (Diagnostica Stago). Because of the possibility of in vitro platelet activation after blood sampling,16 we excluded samples with a ratio of BTG to PF4 of less than 2.0. Urinary analysis of BTG and PF4 levels were not performed.
Simultaneously, we also measured TAT and d-dimer in the same specimens. Blood was added to a siliconized glass tube that contained 3.8% trisodium citrate. Plasma was separated by centrifugation at 2000g for 30 minutes at room temperature. Plasma levels of TAT and d-dimer were determined using the EIA Enzygnost-TAT (Behringwerke AG) and the Dimertest (AGEN Ltd), respectively.
As a control, blood specimens from 19 healthy age- and sex-matched elderly individuals without cerebrovascular disease or hematologic disorders were also examined following the same method and precautions.
Neither the patient nor the control group included individuals with arteriosclerotic diseases such as arteriosclerosis obliterans, myocardial infarction, renal sclerosis, and aortic aneurysm. Both groups were also matched for “degree of atherosclerotic disease” to avoid the effect of arteriosclerosis on blood coagulation.
Statistical analyses were performed using ANOVA followed by Scheffé’s F test method. Significance was set at P<.05.
There was no significant difference between the patients and control subjects in age and sex. Platelet function indexes are depicted in Table 2⇓ and the Figure⇓. In cardioembolic stroke, the levels of BTG and PF4 (mean±1 SD) were 43.8±23.2 and 9.06±7.04 ng/mL, respectively. Corresponding levels of these two markers in the control subjects were 31.9±12.7 and 5.68±3.53 ng/mL, respectively. For both markers, there was no statistically significant difference between the two groups.
However, plasma values of TAT and d-dimer from the same specimens were significantly elevated in the patients compared with the control subjects.8 9 This finding was consistent with the results of many previous reports.1 2 3 4
Many recent studies have suggested that marked hemocoagulation abnormalities, such as accelerated coagulation or platelet activation,1 2 3 4 5 6 7 exist not only in patients with acute ischemic stroke but also in those at risk of stroke.17 18
Enhancement of the coagulation system in acute ischemic stroke is considered to be not the result of stroke but a reflection of its mechanisms of thrombi formation and vessel occlusion due to various stroke risk factors. It may not be the result of the size of the stroke lesion, the influence of medical procedures, or deep vein thrombosis due to long-term bed rest.
In addition, we19 and Gustafsson et al20 previously reported the existence of the same coagulation abnormalities seen in patients with NVAF without stroke history. The coagulation abnormalities were not a complication for all of the patients with NVAF, occurring in only part of the arrhythmia group.19 We think that the hemostatic condition in NVAF is very heterogeneous. Therefore, we used healthy elderly subjects for our control group in this study.
In the control group, the mean levels of the two studied platelet markers were slightly higher than those seen in some previous investigations.4 We believe that this difference was caused by the slightly higher mean age of our control group compared with that used in many other reports. The effects of aging must be taken into account. However, the degree of platelet activation, which was represented by these two molecular markers, remained within the normal range.21 Indeed, some recent studies5 6 22 23 have demonstrated levels similar to ours.
The present study revealed that platelet function was not significantly enhanced in patients with cardioembolic stroke and NVAF within 3 days of onset compared with the value in healthy control subjects. However, the coagulation system of the patients was markedly stimulated. Although their mean levels of BTG and PF4 were slightly but not significantly higher than those of the control subjects, most of the patient values remained within ±2 SD of those of the control group (Figure⇑). The sample size was very small in this study; however, our results indicate that greater statistical significance could be obtained if the sample size were increased.
We investigated the activity levels of BTG and PF4, substances derived from platelet α-granules. The release of these markers mainly occurs on the arterial vessel surface when atherosclerosis exists. The absence of an increase in these two molecular markers does not indicate inactivity of the entire platelet pathway. However, we speculated that such a small degree of platelet activation, which has no effect on the alteration of BTG and PF4, could not induce the observed activation of coagulation factors. Several previous investigations represented by that of Doyle et al21 support our speculation. The activation levels of our two molecular markers reflecting platelet function were within the normal range in both stroke and control groups.
Vingerhoets et al24 recently demonstrated that atrial fibrillation can be a symptom of stroke. In our study, all of the patients had chronic atrial fibrillation that had been diagnosed before stroke onset. Therefore, atrial fibrillation cannot be categorized as a symptom of stroke in our patients.
In patients with artificial heart valves or rheumatic valvular disease, Stein et al25 and Chesebro et al26 proposed that platelet activation participates in the enhancement of hemostasis. Cardioembolic stroke due to these heart conditions may involve acceleration of both the coagulation system and platelet activity. Previous investigators4 5 6 have reported enhanced platelet function in cardioembolic stroke. Their studies included patients with many other heterogeneous cardiac diseases (a potential cardiac source of emboli) and ignored the possibility that there may be a difference in platelet activation between these heart diseases. Thus, it is very important to identify underlying heart disease as a potential cardiac source of cardiogenic embolism.
Our results indicated the absence of platelet α-granule release (indicating platelet activation) in patients with cardioembolic stroke due to NVAF. Therefore, we hypothesized that the common pathophysiological mechanism of the hemostatic condition in cardioembolic stroke may be through the pathway of so-called “stasis-related thrombi” or “fibrin-rich thrombi” formation, which may be directly induced by abnormal blood stasis.25 26
On the other hand, the thrombi induced by platelet activation chiefly develop on the atherosclerotic arterial wall. Stein and Chesebro termed this process “platelet-fibrin thrombi”25 26 formation. Indeed, some of our patients showed slightly enhanced platelet activation (Figure⇑). These changes may reflect platelet activation due to advanced systemic atherosclerosis rather than to intracardiac thrombus formation.
Recently, some investigators have mentioned the possibility that thrombin promotes platelet activation. In this study, there was no correlation between levels of TAT and the platelet markers. Because other researchers have proposed that platelet activation contributes to enhancement of the coagulation system in patients with cardioembolic stroke and various cardiac disorders, we speculate that our results cannot be explained by some other mechanism such as intermittent thrombin activation or differences in clearance between molecular markers.
Atrial fibrillation is an independent risk factor for stroke27 28 29 in the elderly. There is no doubt that the most common and important arrhythmic cause of cardioembolic stroke in the elderly is NVAF.27
This hypothesis of stasis-related thrombi in the development of stroke has major implications in establishing methods to prevent and treat the disease. If our hypothesis holds true, antiplatelet therapy for the secondary prevention of cardioembolic stroke in elderly patients may have little if any effect.15 30 31
This study suggests the necessity of anticoagulant therapy for the secondary prevention of cardioembolic stroke, especially that due to NVAF, in the elderly.
Selected Abbreviations and Acronyms
|NVAF||=||nonvalvular atrial fibrillation|
|PF4||=||platelet factor 4|
|TAT||=||thrombin-antithrombin III complex|
We thank Professor Tamotsu Matsuda (Third Department of Internal Medicine, Kanazawa University), Dr Kentaro Takano (Second Department of Internal Medicine, Kyushu University), and Dr Kenji Inamura (our department, Nippon Medical School) for reviewing our article and providing critical suggestions.
- Received June 28, 1994.
- Revision received May 1, 1995.
- Accepted May 1, 1995.
- Copyright © 1995 by American Heart Association
Takano K, Yamaguchi T, Kato H, Omae T. Activation of coagulation in acute cardioembolic stroke. Stroke. 1991;22:12-16.
Takano K, Yamaguchi T, Uchida K. Markers of a hypercoagulable state following acute ischemic stroke. Stroke. 1992;23:194-198.
Shah A, Beamer N, Coull B. Enhanced in vivo activation in subtypes of ischemic stroke. Stroke. 1985;16:643-647.
Woo E, Huang C, Chan V, Chan Y, Yu Y, Chan T. Beta-thromboglobulin in cerebral infarction. J Neurol Neurosurg Psychiatry. 1988;51:557-562.
Nagao T, Hamamoto M, Kanda A, Ichiseki H, Miyazaki T, Yamazaki M, Terashi A. Altered Hemostatic Molecular Markers in Acute Ischemic Stroke. In: Program of the Second World Congress of Stroke; September 8-12, 1992; Washington, DC. Abstract S72.
Nagao T, Hamamoto M, Kanda A, Tsuganesawa T, Ueda M, Miyazaki T, Terashi A. New strategy for secondary stroke prevention using hemostatic molecular markers. Can J Neurol Sci. 1993;20(suppl 4):S204. Abstract.
Waki R, Okada Y, Tashiro M, Miyashita T, Yamaguchi T. The plasma levels of β-thromboglobulin in the acute phase of cerebral infarction. Jpn J Stroke. 1987;13:433-439.
National Institute of Neurological Disorders and Stroke. Classification of cerebrovascular diseases III. Stroke. 1990;21:637-676.
Stroke Prevention in Atrial Fibrillation investigators. Design of a multicenter randomized trial for the Stroke Prevention in Atrial Fibrillation study. Stroke. 1990;21:538-545.
Ezekowitz MD, Bridgers SL, James KE, Carliner NH, Colling CL, Gornick CC, Krause-Steinrauf H, Kurtzke JF, Nazarian SM, Radford MJ, Rickles FR, Shabetai R, Deykin D. Warfarin in the prevention of stroke associated with nonrheumatic atrial fibrillation. N Engl J Med. 1992;327:1406-1412.
Kaplan K, Owen J. Plasma levels of β-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood. 1981;57:199-202.
el Khawand C, Jamart J, Donckier J, Chatelain B, Lavenne E, Moriau M, Buysschaert M. Hemostasis variables in type I diabetic patients without demonstrable vascular complications. Diabetes Care. 1993;16:1137-1145.
Kanda A, Hamamoto M, Nagao T, Ichiseki H, Miyazaki T, Kaku T, Terashi A. Alterations of hemostatic markers in elderly patients with atrial fibrillation without a history of stroke. Jpn J Geriatr. 1993;30:382-386.
Gustafsson C, Blomback M, Britton M, Hamsten A, Svensson J. Coagulation factors and the increased risk of stroke in nonvalvular atrial fibrillation. Stroke. 1990;21:47-51.
Doyle DJ, Chesterman CN, Cade JF, McGready JR, Rennie GC, Morgan FJ. Plasma concentration of platelet-specific proteins correlated with platelet survival. Blood. 1980;55:82-84.
Landi G, D’Angelo A, Boccardi E, Candelise L, Mannucci PM, Nobile Orazio E, Morabito A. Hypercoagulability in acute stroke: prognostic significance. Neurology. 1987;37:1667-1671.
De Caterina R, Gazzetti P, Mazzone A, Marzilli M, L’Abbate A. Platelet activation in angina at rest: evidence of paired measurement of plasma beta-thromboglobulin and platelet factor 4. Eur Heart J. 1988;9:913-922.
Vingerhoets F, Bogousslavsky J, Regli F, Van Melle G. Atrial fibrillation after acute stroke. Stroke. 1993;24:26-30.
Stein B, Fuster V, Halperin J, Chesebro J. Antithrombotic therapy in cardiac disease: an emerging approach based on pathogenesis and risk. Circulation. 1989;80:1501-1513.
Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation: a major contributor to stroke in the elderly. The Framingham study. Arch Neurol. 1987;147:1561-1564.
Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation as an independent risk factor for stroke: the Framingham study. Stroke. 1991;22:983-988.
Britton M, Gustafsson C. Non-rheumatic atrial fibrillation as a risk factor for stroke. Stroke. 1985;16:182-188.
Miller VT, Rothrock JF, Pearce LA, Feinberg WM, Hart RG, Anderson DC. Ischemic stroke in patients with atrial fibrillation: effect of aspirin according to stroke mechanism. Neurology. 1993;43:32-36.
Kistler JP, Singer DE, Millenson MM, Baier KA, Gress DR, Barzegar S, Hughes RA, Sheehan MA, Maraventano SW, Oertel LB, Rosner B, Rosenberg RD. Effect of low-intensity warfarin anticoagulation on level of activity of the hemostatic system in patients with atrial fibrillation. Stroke. 1993;24:1360-1365.