INTRODUCTION

 

Stroke is the third leading cause of death and an important cause of disability worldwide¹. Thrombosis and atherosclerosis are major contributors to the development of ischemic stroke. Platelets play a critical role in triggering arterial thrombosis and in promoting atherogenesis.²

Furthermore, formation of platelet-leukocyte aggregates and leukocyte activation contribute to vascular repair and microcirculatory disturbances in ischemic tissue. Previously, in vitro, studies have demonstrated interaction of activated platelets with monocytes and neutrophils.³ Binding of platelets via P-selectin expressed on the surface of activated platelets to the leukocyte to the leukocyte counter receptor P-selectin GP ligand-1 may alter leukocyte recruitment and activation patterns⁴. 

Platelet activation is a crucial mechanism in arterial thrombogenesis and therefore in the pathophysiology of ischemic stroke.^5-8 Accordingly, antiplatelet therapy plays a central role in secondary prevention of ischemic stroke. Currently, decisions for antiplatelet drugs after stroke are based on clinical grounds but not on platelet activation studies in individual patients because of a lack of clinically applicable methods to monitor platelet function^5-8. 

Two articles recently showed that patients with acute cerebral ischemia have an excess of circulating platelets that express P-selectin (CD62p) and lysosome-associated membrane protein (CD63)^9,10.

P-selectin is a constituent of the membrane of -granules and serves as an adhesion receptor mediating the binding of platelets to leukocytes. CD63 is part of platelet lysosomes and may protect the plasma membrane from degradation by lysosomal enzymes. Both CD62P and CD63 are expressed exclusively on platelet activation^11,12.

The time course of these platelet activation markers after stroke has not yet been assessed. Whether their elevation during the subacute phase after stroke could predict recurrent ischemic events and whether they may be helpful in monitoring antiplatelet therapy are of interest⁹.

The aim of this study was to assess the degree of platelet activation in human cerebral ischemic stroke during acute stage and 90 days later. Furthermore, the current study also tested the correlation between platelet activation markers and clinical presentation of the studied patients.

 

SUBJECT AND METHODS

 

This study was undertaken on forty five patients (group I), recruited from the stroke unit of the Neurology Department of Tanta University Hospital within 24 hours after ischemic stroke and their clinical deficit lasted for at least 24 hours. Twenty healthy age and sex matched controls (Group II) and twenty risk factor control subjects (Group III) who were matched with patient in terms of age, sex, hypertension, diabetes mellitus, smoking and hyperlipidaemia were enrolled in the study.

Exclusion criteria for all subjects were trauma, surgery, or acute organ ischemia within the preceding 3 months (e.g. before the  stroke); severe liver disease; renal failure; cancer; chronic inflammatory diseases; and fever or acute inflammatory or infectious conditions at the study entry.

Patients with an infection after stroke or with a history of infection shortly before stroke were excluded to avoid including the occurrence of platelet activation attributable to parallel infection. However, we systematically assessed symptoms of infection and body temperature during the whole study period to recognize a possible influence of infection on the variables. Also, we excluded patients suffered from a new cerebral or cardiac ischemic event during the 90 days of follow-up.

For healthy control subjects, any vascular diseases or risk factors were additional exclusion criteria. Subjects with a history of myocardial infarction or stroke were excluded from the risk factor control group.

Vascular risk factors were defined as follows: arterial hypertension, on antihypertensive treatment or blood pressure 160/90 mm Hg at 2 readings before stroke or >5 days after stroke; diabetes mellitus, on antidiabetic treatment or elevated haemoglobin A1c or elevated blood glucose at 2 readings before stroke or >5 days after stroke; hyperlipidemia, on lipid-lowering medication or total cholesterol >220 mg/dL or triglycerides >180 mg/dL before stroke; and carotid stenosis, diameter reduction of 50% of the internal carotid artery on Duplex sonography¹³.

All stroke patients underwent a cranial CT excluding cerebral haemorrhage.

The origin of cerebral ischemia was classified according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria.¹⁴ Stroke severity was assessed with the National Institutes of Health Stroke Scale (NIHSS)¹⁵ on days 1, 14, and 90 after ischemia.

In stroke patients, blood samples were taken within 24 hours and on days 14, and 90 after ischemia. Measurements were performed once in both control groups. Serum and citrated plasma samples were collected at each venipunctue and were stored at -80^oC until measurements. Blood was obtained under minimal tourniquet pressure from the antecubital vein using a sterile 22-gauge needle and syringe in a single attempt. The first 3 mL blood was used to separate serum, and the following 3 mL was drawn into a Vacutainer tubes containing 3.8% buffered sodium citrate.  Mixtures of blood and sodium citrate were centrifuged for 15 min at 1.500 revolutions per minute at room temperature. The supernatant platelet-rich plasma was used to assess the platelet activity. The time between blood collection and antibody labelling was standardized to within 1 h.

Platelet activity was determined with respect to alpha granule degranulation (surface expression of P selectin or CD62p antigen). A Fluorescein isothiocyanate conjugated (FITC)–labeled antibodies to glycoprotein IIIa (CD61; Becton Dickinson) was used as an activation-independent marker of platelets. Phycoethrin (PE)–labeled antibodies (Immunotech) against CD62 or CD63 were used to assess platelet activation. The percentage of platelets expressing CD62p or CD63 was defined as the fraction exhibiting specific binding (i.e., CD62p or CD63) minus that exhibiting nonspecific binding (i.e. the percentage defined with the IgG-PE conjugate) from among the 10,000 platelets sorted.^16,17

 

Statistical Analysis

The data are presented as means and percentiles. Student-T test was used to compare different groups and to test significance. AOVA test also was used. P<0.05 was considered significant.                      

 

RESULTS

 

Forty five cerebral ischemic stroke patients (group I), twenty healthy controls (group II) and twenty risk controls (group III)  were included in this study. The mean age of group I  was 60.3 (SD±13.6) years , group II was 59.4(SD, ±14.2) years  and 61.5  years (SD, ±13.2) for group III .There was no significant difference between the three groups regarding age (P=0.51) and sex p=0.41) (Table 1).

The distribution of hypertension, diabetes mellitus, smoking, or cardiac disease did not vary among group I and group III subjects and the difference was non-significant (Table 2). There were no risk factors at all among group II.

The percentage of platelets expressing CD62p was significantly higher in  group I on day one after ischemia (mean, 3.1%) than that in both group II (mean, 1.4% ) and group III   (mean 1.6%;) subjects (P<0.001). The expression of CD62p on platelets was decreased over time after stroke. The difference between the three groups was no longer significant on days 14 and 90 after stroke  (Figure 1).

CD63 expression on platelets was significantly higher on day one among group I patients (mean, 3.1%) compared with group II and III subjects (mean1.5% and 1.4% respectively) (P<0.001). The percentage of platelets expressing CD63 decrease on  the 14 day and  90 day after stroke in group I patients but still higher than in group II and III. There was non significant difference when compared with group II and III subjects (Figure 1).

The most frequent etiology of ischemic stroke among patients  was large artery atherosclerosis 17 patients (37.7%), then 15 (33.3%) were due to undetermined aetiology, 8 (11.1%) from small artery disease and 5 patients (11.1%) had cardioembolic source of their ischemic stroke (Table 3).

The mean of  National Institutes of Health Stroke Scale (NIHSS) score for the  group I patients  at admission was 6 (1 to 27), after 14 days  was 3 (0 to 5 ) and after 90 days was 1 (0-18).

CD63 expression on day one was influenced by the stroke pathophysiological mechanism where cardioembolic stroke patients had a statistically significant higher difference [(median 3.25% ) with p value 0.012 ] compared to other stroke mechanisms. On the other hand, CD62p expression was not influenced by the stroke pathophysiology (Table 3) 

Stroke severity was not correlated with CD62P and CD63 expression at any time point  (Table 4).                  

Vascular risk factors did not influence the results for CD62p and CD63 among group I and group III subjects (Table 5).

On day 90 after stroke, CD62p and CD63 expression did not differ between patients treated with aspirin, clopidogrel, or anticoagulants (P>0.05 for all) (Table 6).

 

Table 1. Demographic data of the studied subjects.

 

Parameter	Group I (n=45)	Group II (n=20)	Group III (n= 20)		P
Age (y) Mean±SD	60.3±13.6	59.4±14.2	61.5±13.2	F=1.054	0.512
Male  n (%)	27 (60)	13(65)	12(60)	X2= 0.782	0.410

 

Table 2. Vascular risk factors among patients and risk factor control subjects.

 

Parameter	Group I n=45		Group III  n=20		Chi-square
	no	%	no	%	X2	P-value
Hypertension	22	48.8	9	45	0.08	0.77
Diabetes mellitus	14	31.1	7	35	0.09	0.75
Smoking	15	33.3	8	40	0.26	0.60
Cardiac disease	18	40	8	40	0.00	1.00
Hyperlipidemia	17	37.3	7	35	0.04	0.83

 

 

 

Figure 1. CD62P and CD63 in different groups.

Table 3. CD63 and CD62P in different Types of ischemic stroke in patients group

 

	N	%			Day 1	Day 14	Day 90
Large artery atherosclerosis	17	37.78	CD 62	Mean	3.135	1.453	1.376
				SD	0.889	0.265	0.249
			CD 63	Mean	3.112	2.971	2.765
				SD	0.877	0.796	0.687
Cardioembolism	5	11.11	CD 62	Mean	2.760	1.960	1.940
				SD	0.934	0.279	0.305
			CD 63	Mean	3.25	2.880	2.620
				SD	0.908	0.881	0.554
Small artery disease	8	17.78	CD 62	Mean	2.600	1.975	1.950
				SD	0.632	0.354	0.325
			CD 63	Mean	2.600	2.500	2.525
				SD	0.632	0.526	0.498
Undetermined etiology	15	33.33	CD 62	Mean	2.423	1.951	2.011
				SD	0.758	0.353	0.311
			CD 63	Mean	2.973	2.987	2.560
				SD	0.694	0.664	0.818
ANOVA			CD 62	F	0.897	1.554	2.075
				P-value	0.652	0.245	0.110
			CD 63	F	3.85	1.671	1.804
				P-value	0.012*	0.122	0.129

* significant at p<0.05

 

Table 4. CD63 and CD62p in different stroke severities in patients group.

 

	NIHSS and CD62P		NIHSS and CD 63
	r	P-value	r	P-value
Day 1	0.015	0.924	0.007	0.962
Day 14	0.176	0.566	0.020	0.897
Day 90	0.116	0.534	0.029	0.848

 

Table 5. Influence of vascular risk factors on serum levels of CD62p and CD63. 

 

Patient group		Positive		Negative		T-test
		N	Mean±SD	N	Mean±SD	t	P-value
Hypertension	CD 62 p	22	3.110±0.860	3	3.452±0.750	0.652	0.520
	CD 63	22	3.050±0.800	3	3.500±0.663	0.927	0.363
Diabetes mellitus	CD 62 p	14	3.000±0.770	11	3.343±0.946	1.001	0.327
	CD 63	14	3.110±0.860	11	3.096±0.686	0.043	0.966
Smoking	CD 62 p	15	2.990±0.800	10	3.393±0.925	1.159	0.259
	CD 63	15	2.890±0.650	10	3.425±0.966	1.661	0.110
Cardiac disease	CD 62 p	18	3.100±0.800	7	3.282±0.981	0.481	0.635
	CD 63	18	3.090±0.700	7	3.140±0.999	0.142	0.888
Hyperlipidemia	CD 62 p	17	3.010±0.810	8	3.451±0.938	1.208	0.239
	CD 63	17	2.990±0.711	8	3.346±0.943	1.053	0.303

Table 6. CD62p and CD63 expression in different drugs treated patients (day 90)

 

	Aspirin (n=24)	Clopidogrel (n=13)	Anticoagulants (n=8)	ANOVA
				F	P-value
CD62P	1.18±0.678	1.33±0.688	1.23±0.553	1.897	0.215
CD63	3.06±0.88	2.79±0.789	2.92±0.80	2.125	0.117

 

 

DISCUSSION

 

There is a well-recognized need for clinically applicable methods to monitor platelet function in the treatment and prevention of ischemic stroke. Platelet activation in the acute and chronic stages after stroke has been demonstrated with methods such as aggregometry, assessment of plasma levels of platelet release products, and urinary excretion of thromboxane metabolites. None of these methods proved to be a useful tool in daily clinical practice partly because of insufficient reproducibility of results and partly because of the expense of time and equipment required¹⁸.

The main methodical problem is artifactual in vitro activation of platelets during blood sampling and processing. Furthermore, measurements of release products may have a low sensitivity because of dilution effects in plasma. Flow cytometric analysis of platelet function has the advantage that it can use fixation procedures that block metabolic processes in cells within milliseconds but leave activation-dependent neoantigens on the cell surface preserved. Therefore, the possibility of artifactual cell activation is reduced to the venipuncture in this technique. We found good reproducibility of the assessment of both platelet activation markers with a low intratest variability and a low to moderate intertest variability in healthy subjects. Therefore, an important prerequisite for clinical application is fulfilled by this method¹⁹.

In accordance with previous reports^11,13, the current study found a significant increase in CD62p and CD63 expression within 24 hours after cerebral ischemia.

The differential expression pattern of CD62p and CD63 over time is an interesting finding that may be explained as follows. A recent report has shown that increased expression of CD62p or CD63 after stroke was not associated with an increased proportion of circulating platelets with shape change.⁹ The shape change is a very early and reversible event during platelet activation and a very sensitive marker of instantaneous platelet activation. This indicates that platelets after stroke are not particularly activated during their passage in peripheral venous blood. Thus, CD62p and CD63 are memory markers of platelet activation, and the increased expression of secretion-dependent antigens after stroke indicates that platelets had previously undergone activation. In a baboon model, degranulated platelets rapidly lost surface CD62p but continued to circulate and function.²⁰ A similar mechanism may be operative in humans, and the degranulated platelets may not lose CD63, which could explain the decline in CD62p expression parallel to continuously elevated CD63 expression after acute stroke. Platelets possess a lifespan of about 7 to 10 days. Therefore, increased CD63 expression in subacute stroke indicates continuously ongoing platelet activation. Altogether, in the subacute stage after ischemic stroke, CD63 is a more sensitive marker of platelet activation than CD62p, most likely because of shedding of CD62p.¹³

This study revealed that vascular risk factors and clinical stroke severity did not significantly modify CD62p and CD63 expression, and stroke patients had persistently higher CD63 expression than subjects with vascular risk factors. The simultaneous presence of multiple risk factors was not associated with higher platelet activation. This indicates that platelet activation markers may confer relevant information independently from and in addition to clinical risk factor assessment.

Regarding stroke pathophysiology, the findings of the current study did not support the previous observation that atherothrombotic stroke is associated with a particularly high platelet activation.²¹ However, our study was not designed to investigate the influence of different stroke origins on platelet activation, and results have to be viewed with caution because of the small numbers of subjects in subgroups. Moreover, we did not find a significant influence of medication for secondary prevention on platelet activation markers. The fact that medication with platelet inhibitors was not associated with lower antigen expression by platelets is in line with previous results showing that aspirin does not modify the excretion of a-granules.^22,23

Several studies have shown that fibrinogen, C-reactive protein (CRP), and leukocyte count are increased after ischemic stroke. Furthermore, all 3 parameters were shown to be independently associated with the risk of first-ever and recurrent vascular events^24,25, although the specificity of the link with vascular events was questioned recently.⁽²⁶⁾ The role of inflammatory parameters, mainly CRP, as an important predictive index for recurrent ischemic events after stroke was recently established.^24,25 An interesting question is whether the expression of platelet neoantigens, mainly of CD63, may provide additional information on future vascular events after stroke and thus may guide therapeutic decisions. This question needs to be answered in larger prospective studies.

 

[Disclosure: Authors report no conflict of interest]

 

REFERENCES

 

1.      Warlow CP. Epidemiology of stroke. Lancet. 1998; 352 (suppl 3): SIII1-4.

2.      Chamorro A. Role of inflammation in stroke and atherothrombosis. Cerebrovasc Dis. 2004; 17 (suppl 3): 15.

3.      Larsen E, Palabrica T, Sajer S, Gilbert GE, Wagner DD, Furie BC, Furie B. PADGEM-dependent adhesion of platelets to monocytes and neutrophils is mediated by a lineage-specific carbohydrate, LNF (CD15). Cell. 1990; 63: 467-474.

4.      Furie B, Furie BC, Flaumenhaft R. A journey with platelet P-selectin: the molecular basis of granule secretion, signalling and cell adhesion. Thromb Haemost. 2001; 86: 214-21.

5.      Toghi H, Suzuki H, Tamura K, Kimura B. Platelet volume, aggregation, and adenosine triphosphate release in cerebral thrombosis. Stroke. 1991; 22: 17–22.

6.      Grau A, Ruf A, Vogt A, Lichy C, Buggle F, Patscheke H, et al. Increased fraction of circulating activated platelets in acute and previous cerebrovascular ischemia. Thromb Haemost. 1998; 80: 298–301.

7.      Zeller JA, Tschoepe D, Kessler C. Circulating platelets show increased activation in patients with acute cerebral ischemia. Thromb Haemost. 1999; 81: 373–7.

8.      Fisher M, Francis R. Altered coagulation in cerebral ischemia: platelet, thrombin, and plasmin activity. Arch Neurol. 1990; 47: 1075-9.

9.      Grau A, Buggle F, Becher H, Werle E, Hacke W. The association of leucocyte count, fibrinogen and C-reactive protein with vascular risk factors and ischemic vascular diseases. Thromb Res. 2006; 82: 245–55.

10.    Beamer NB, Coull BM, Clark WM, Briley DP, Wynn M, Sexton G. Persistent inflammatory response in stroke survivors. Neurology. 1998; 50: 1722-8.

11.    Holmsen H. Platelet secretion. In: Colman RW, Hirsh I, Marder VJ editors. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 2^nd ed. Philadelphia: Lippincott Co; 1987: 606-17.

12.    Harrison P, Cramer EM. Platelet α-granules. Blood Rev. 1993; 7: 2-62

13.    Marquardt L, Ruf A, Mansman U, Winter R, Schuler M, Mayer H, Grau AJ. Course of platelet activation markers after ischemic stroke. Stroke. 2002; 33:2570-5.

14.    Adams HP, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE, for the TOAST Investigators. Classification of subtype of acute stroke: definitions for use in a multicenter clinical trial. Stroke. 1993; 24: 35-41.

15.    Goldstein LB, Bertels C, Davis JN. Inter rater reliability of the NIH stroke scale. Arch Neurol. 1989; 46: 660-2.

16.    Ruf A, Patscheke H. Flow cytometric detection of activated platelets: comparison of determining shape change, fibrinogen binding, and p-selectin expression. Semin Thromb Hemost. 1995; 21: 146-51.

17.    Beamer NB, Coull BM, Clark WM, Briley DP, Wynn M, Sexton G. Persistent inflammatory response in stroke survivors. Neurology. 1998; 50: 1722-8

18.    Koudstaal PJ, Ciabattoni G, van Gijn J, Nieuwenhuis HK, de Groot PG, Sixma JJ, Patrono C. Increased thromboxane biosynthesis in patients with acute cerebral ischemia. Stroke. 1993; 24: 219-23

19.    van Kooten F, Ciabattoni G, Patrono C, Schmitz PI, van Gijn J, Koudstaal PJ. Evidence for episodic platelet activation in acute ischemic stroke. Stroke. 1994; 25: 278-81.

20.    Michelson AD, Barnard MR, Hechtman HB, MacGregor H, Connolly RJ, Loscalzo J, et al. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface p-selectin but continue to circulate and function. Proc Natl Acad Sci U S A. 1996; 93: 11877-82

21.    Kannel WB, Anderson K, Wilson PWF. White blood cell count and cardiovascular disease: insights from the Framingham study. JAMA. 1992; 267: 1253–6.

22.    Gralnick HR, Vail M, Williams S, McKeown L, Shafer B, Hansmann K. Platelet alpha granule release is not inhibited by aspirin and is mediated by myosin light chain kinase and protein C kinase. Blood. 1990; 75: 128–38.

23.    Chronos NAF, Wilson DJ, Janes SL, Hutton RA, Büller NP, Goodall AH. Aspirin does not affect the flow cytometric detection of fibrinogen binding to, or release of -granules or lysosomes from, human platelets. Clin Sci. 1994; 87: 575-80.

24.    Di Napoli M, Papa F, Bocola V. Prognostic influence of increased C-reactive protein and fibrinogen levels in ischemic stroke. Stroke. 2001; 32: 133-8.

25.    Di Napoli M, Papa F, Bocola V. C-reactive protein in ischemic stroke: an independent prognostic factor. Stroke. 2001; 32: 917–24.

26.    Gussekloo J, Schaap MC, Frolich M, Blauw GJ, Westendorp RG. C-reactive protein is a strong but nonspecific risk factor of fatal stroke in elderly persons. Arterioscler Thromb Vasc Biol. 2000; 20: 1047–51.