Published online before print September 11, 2003, doi: 10.1161/01.STR.0000092400.93742.81
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(Stroke. 2003;34:e174.)
© 2003 American Heart Association, Inc.
Letters to the Editor |
In Vivo Platelet Activation in Ischemic Stroke Patients
Laboratory Hematology and Transfusion Medicine, University of Patras, Patras, Greece
Neurology Department, University of Patras, Patras, Greece
To the Editor:
Recently, Marquardt et al1 addressed the important issue of evaluating markers for monitoring patients after ischemic stroke. They evaluated the expression of the platelet activation markers CD62P and CD63 by flow cytometry for 90 days after ischemic stroke. They showed that although both markers are initially increased, the expression of CD62P decreases rapidly and by day 14 is no longer significantly increased, while that of CD63 remains elevated for the study period. The expression of both antigens was independent of the inflammation markers studied, indicating that expression of platelet neoantigens might be used as an additional marker for monitoring these patients. Expression was not influenced by the different medications for secondary stroke prevention, suggesting that they might reflect mechanisms that could predict future vascular events.
These results are very interesting because definition of reliable laboratory markers for diagnosing ischemic stroke, monitoring treatment, and predicting recurrence is a very important issue, particularly in the view of new and effective treatments that, however, are associated with significant complications.2,3
We performed a study investigating the expression of CD62P and CD63 antigens on circulating platelets in patients with acute ischemic disease on admission and after initiation of antiplatelet treatment. We studied 18 patients (14 men, 4 women; mean age, 63 years) with established stroke admitted to the Neurology Department of the University Hospital of Patras (Greece) within 24 hours after disease onset. All patients underwent a CT or MRI brain scan and carotid duplex scanning (ultrasound). The diagnosis of cerebral ischemia was based on the criteria of the third classification of cerebrovascular diseases by the National Institute of Neurological Disorders and Stroke and the Trial of Org 10172 in Acute Stroke Treatment classification.4,5 Exclusion criteria for patients were chronic systemic disease, febrile infection, and anticoagulant or antiplatelet treatment. All patients received ticlopidine on day 3 or 4 after the onset of the disease. Ten of the 18 patients with acute ischemia were also studied on days 7 and 30 after disease onset. As control subjects, we studied 25 normal volunteers (9 men, 16 women; mean age, 48 years). The study was approved by the ethics committee of the University Hospital. Informed consent was obtained from all subjects included in the study.
The blood for platelet activation studies was collected into tubes containing citrate and the platelet antiaggregants theophylline, adenosine, and dipyridamole (Diatube H, Diagnostica Stago). We used a whole-blood method for platelet immunophenotyping that is a modification of a previously described method.6 The samples were incubated with a combination of the monoclonal antibodies CD62-PE (clone CLBThromb/6) or CD63-PE (clone CLBGran12) with CD41-FITC (clone P2), all from Immunotech. All results are expressed as mean±SD. Multiple comparisons were made with Bonferroni’s correction. Values of P<0.05 between the 2 groups were considered significant.
Expression of CD62P and CD63 antigens on circulating platelets was significantly increased in patients with acute ischemic stroke at diagnosis (4.85±3.02 and 3.1±2.75, respectively) compared with control subjects (1.27±0.7 and 1.03±0.7, respectively). Expression of both markers declined gradually during follow-up. Our results, in agreement with those of Marquardt et al,1 showed that CD62P platelet expression decreased early after stroke; in contrast to their findings, we found that the expression of CD63 antigen also decreased and returned to that of normal control subjects during the study period. Our results are shown in the Figure. These differences in the time course of antigen expression might be due to differences in the study population and can be resolved only by large studies. In our study, the expression of CD62P antigen did not differ between the different subgroups of patients, whereas the expression of CD63 antigen was lower in patients with cardioembolic stroke, suggesting a different platelet activation profile in this subgroup of patients.
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Although previous studies have suggested that measurement of platelet secretion products or activation-dependent platelet antigens may define platelet activation profiles in patients with different subtypes of cerebral ischemia and can be used to monitor individual response to antiplatelet treatment,7–11 the lack of a reliable method for evaluation of platelet activation has for years hindered studies addressing questions regarding the diagnostic and prognostic value of markers of platelet activation.
The similar results in percentage of circulating platelets expressing activation markers obtained in our study and the others that used a whole-blood method for platelet immunophenotyping1,11 prove that, for the first time, there is a technique available that can be standarized and used in multicenter trials.12 Some of the questions that should be answered before these markers are introduced into clinical practice are the value of these antigens in discriminating the different subtypes of stroke, the way that different treatments affect their expression, and more importantly, whether persistence of platelet activation in these patients affects their clinical outcome.
References
1. Marquardt L, Ruf A, Mansmann U, Winter R, Schuler M, Buggle F, Mayer H, Grau AJ. Course of platelet activation markers after ischemic stroke. Stroke. 2002; 33: 2570–2574.
2. Bousser M-G. Antithrombotic strategy in stroke. Thromb Haemost. 2001; 86: 1–22.[Medline] [Order article via Infotrieve]
3. Mousa SA, Iqbal O, Fareed J. Antithrombotics and thrombolytics in stroke. Curr Opin Investig Drugs. 2002; 3: 878–885.[Medline] [Order article via Infotrieve]
4. Whisnant JP, Basford JR, Bernstein EF, Cooper ES, Dyken ML, Easton JD, Little JR, Marier JR, Millikan CH, Petito CK, et al. Special report from the National Institute of Neurological Disorders and Stroke: classification of cerebrovascular diseases III. Stroke. 1990; 21: 23–28.
5. Adams HP Jr., Bendixen BH, Kappelle LJ, Billes J, Love BB, Gordon DL, Marsh EE 3rd. Classification of subtype of acute ischemic stroke: definitions for use in a multicenter clinical trial TOAST: Trial of Org 10172 in Acute Stroke Treatment. Stroke. 1993; 24: 35–41.
6. Abrams CS, Ellison N, Budzynski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood. 1990; 75: 128–138.
7. Iwamoto T, Kubo H, Takasaki M. Platelet activation in the cerebral circulation in different subtypes of ischemic stroke and Dinswanger’s disease. Stroke. 1995; 26: 52–56.
8. Shah AB, Beamer N, Coull BM. Enhanced in vivo platelet activation in subtypes of ischemic stroke. Stroke. 1985; 16: 643–647.
9. Grau AJ, Ruf A, Vogt A, Lichy C, Buggle F, Patscheke H, Hacke W. Increased fraction of circulating activated platelets in acute and previous cerebrovascular ischemia. Thromb Haemost. 1998; 80: 298–301.[Medline] [Order article via Infotrieve]
10. Zeller JA, Tschoepe D, Kessler C. Circulating platelets show increased activation in patients with acute cerebral ischemia. Thromb Haemost. 1999; 81: 373–377.[Medline] [Order article via Infotrieve]
11. Yamazaki M, Uchiyama S, Iwata M. Measurement of platelet fibrinogen binding and P-selectin expression by flow cytometry in patients with cerebral infraction. Thromb Res. 2001; 104: 197–205.[CrossRef][Medline] [Order article via Infotrieve]
12. Schmitz G, Rothe G, Ruf A, Barlage S, Tschöpe D, Clemetson KJ, Goodall AH, Michelson AD, Nurden AT, Shankey TV. European Working Group on Clinical Cell Analysis: consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost. 1998; 97: 885–896.
Response
Neurologische Klink, Klinikum der Stadt Ludwigshafen a. Rh.
Center for Laboratory Medicine, Microbiology, and Transfusion Medicine, Klinikum Karlsruhe
We appreciate the comments of Dr Karakantza and coworkers on our recent publication and the presentation of their own data on platelet activation after acute ischemic stroke. Their results are in accordance with our own findings except for the data on CD63 expression in the subacute stage after ischemia, which we found to be persistently increased, whereas Karakantza et al did not detect any difference compared with the control group. As mentioned by Karakantza et al, the difference can be due to differences in study populations. Furthermore, the number of subjects was small in their study (n=18); therefore, a false-negative result cannot be excluded. In line with this, any subgroup analyses of their data should be viewed with great caution because of the small number of subjects.
We certainly agree with Karakantza and coworkers that analysis of platelet activation with flow cytometry is a rewarding topic in stroke research, given the potential of clinical application of this method.
Footnotes
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