Lack of uniform platelet activation in patients after ischemic stroke and choice of antiplatelet therapy

Thrombosis Research (2004) 113, 197 – 204

intl.elsevierhealth.com/journals/thre

Regular Article

Lack of uniform platelet activation in patients after ischemic stroke and choice of antiplatelet therapy Victor L. Serebruany a,*, Alex I. Malinin a, Benjamin R. Oshrine a, David C. Sane b, Aviv Takserman c, Dan Atar c, Charles H. Hennekens d a

Internal Medicine, Johns Hopkins University, 7600 Osler drive, ste. 307, Baltimore, MD 21204, USA Wake Forest University School of Medicine, Winston Salem, NC, USA c Aker University Hospital, Oslo, Norway d University of Miami School of Medicine, Miami, FL, USA b

Received 15 January 2004; received in revised form 26 February 2004; accepted 4 March 2004 Available online 12 April 2004

KEYWORDS Stroke; Platelets; Aspirin

ABSTRACT Introduction: Platelets play an important role in the natural history of ischemic stroke, and are known to be activated in the acute phase. Although aspirin reduces risks of myocardial infarction, stroke and cardiovascular death, the extent of platelet action and the effect of aspirin on platelet function in patients recovering from stroke remain unclear. Methods: We studied 120 individuals divided into three equal groups: aspirin-free patients after ischemic stroke, post-stroke patients receiving aspirin (81 – 650 mg/daily), and aspirin-free subjects with multiple risk factors for vascular disease. Conventional platelet aggregation induced by 5 AM ADP and 5 AM epinephrine, cartridge-based analyzers (UltegraR, and PFA-100k) readings, and expression of CD31, CD41a, CD42b, GPIIb/IIIa activity, CD51/CD61, CD62p, CD63, CD107a, CD154, CD165, formation of platelet – monocyte aggregates, intact (SPAN12), and cleaved (WEDE15) PAR-1 thrombin receptors by flow cytometry were analyzed. Results: There were no differences between aspirin-free poststroke patients and aspirin-free controls. Although aggregation was slightly higher, 12 out of the 14 receptor analyses, were surprisingly lower in the post-stroke cohort. Aspirin-treated patients exhibited highly significant inhibition of epinephrine-induced aggregation ( p = 0.0001), prolongation of the closure time ( p = 0.03), and reduction of the aspirin reactive units ( p = 0.02) measured by the UltegraR device. In addition, surface platelet expression of thrombospondin ( p = 0.001), GPIIb/IIIa activity ( p = 0.04), P-selectin ( p = 0.03), CD40-ligand ( p = 0.04), CD165

Abbreviations: ADP, adenosine diphosphate; ARU, aspirin response unit; ASA, aspirin; CD, cluster of differentiations; GP, platelet glycoprotein; LAMP, lyzosome associated membrane protein; NSAIDS, non-steroids anti-inflammatory drugs; PECAM, platelet/ endothelial cell adhesion molecule; PETA, platelet/endothelial tetraspan antigen; PFA, platelet function analyzer; RPFA, rapid platelet-function assay; TIA, transient ischemic attack. * Corresponding author. Tel.: +1-410-847-9490; fax: +1-443-583-0205. E-mail address: [email protected] (V.L. Serebruany).

0049-3848/$ - see front matter A 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2004.03.002

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V.L. Serebruany et al. / Thrombosis Research 113 (2004) 197 – 204 ( p = 0.02), the formation of the platelet – monocyte aggregates ( p = 0.01), and intact epitope of PAR-1 thrombin receptor ( p = 0.03) were significantly lower in the aspirin-treated group. Conclusions: Platelets are not activated in aspirin-free patients after ischemic stroke. Platelet function is significantly inhibited in those treated with aspirin when compared with healthy subjects with risk factors for vascular disease. Bleeding complications and hemorrhagic transformations after aggressive antiplatelet regimens could be related to the decreased or normal baseline platelet characteristics in such patients. Further analysis of platelet heterogeneity and its clinical significance remains to be determined in randomized trials. A 2004 Elsevier Ltd. All rights reserved.

Introduction Platelets play an important role in the natural history of acute ischemic stroke [1,2]. It has been established that aspirin, aggrenox (low-dose aspirin combined with the extended release dipyridamole), and ticlopidine reduce the frequency of secondary stroke events [3,4]. Clopidogrel has been shown to reduce the frequency of secondary vascular ischemic events when stroke, myocardial infarction, and peripheral arterial disease are considered together. These unquestionable clinical benefits of antiplatelet therapy, however, were never linked to the degree of platelet inhibition. There is substantial evidence of impaired platelet function in post-stroke patients, primarily based on in vitro [5,6], animal [7,8], and human observational studies [9,10]. There is agreement on the presence of elevated concentrations of plateletderived substances such as plasma P-selectin [11,12], or urinary thromboxane [13,14], which may serve as the indicators of enhanced platelet activity in patients with stroke. However, there is no consensus on the clinical meaningfulness of measuring platelet aggregation, and very limited knowledge regarding the assessment of major surface platelet receptor expression in such patients. Some discrepancies could be related in part to the limitation of the techniques used to assess platelet activity, and the absence of standard timing of blood sample collection. Indeed, the majority of the human studies lack a careful and precise description of the time course of blood drawing. Most studies have examined platelets during the first week after stroke when indirect confounding factors, including aggressive pharmacologic therapy affecting platelets, could not be excluded. It is not surprising that the determination of platelet function at random in patients after stroke has not led to helpful correlations regarding infarct volume or location, success of thrombolysis, or antiplatelet therapy, adequate secondary stroke prevention.

We still are clueless regarding the optimal magnitude of platelet inhibition which will derive the largest survival or/and secondary stroke prevention benefit. Most importantly, our knowledge of the platelet characteristics in the late post-reperfusion phase of ischemic stroke is very limited. However, it seems reasonable to expect that the platelet status of post-stroke victims may directly affect the chance for next stroke or transient ischemic attack (TIA). Thus, the purpose of the present study was to determine whether platelet activation occurs uniformly, and if antecedent aspirin therapy affects major platelet characteristics of the post-stroke patients, and to compare these data with those of controls with multiple risk factors for vascular disease. We employed a combined approach of assessing platelet aggregometry in response to different agonists, utilizing rapid cartridge-based platelet analyzers, together with the flow cytometric determination of 14 major platelet surface receptors.

Materials and methods Patient selection The study was conducted in two participating centers affiliated with the Johns Hopkins University with the core facility in Baltimore, MD, USA, and a satellite platelet laboratory at the Akers University Hospital, Oslo, Norway. The study was approved by local research ethics committees. Written informed consent was obtained from all participants. The enrollment phase lasted over 2 years (2001 – 2003). The study population consisted of three equal cohorts totaling 120 willing and eligible subjects. The baseline platelet data was collected from two prospective post-stroke studies, and from healthy volunteers.

V.L. Serebruany et al. / Thrombosis Research 113 (2004) 197 – 204 The first group was composed of patients aged z 40 years who had suffered an ischemic stroke

between 2 and 6 months earlier, and had not been treated with aspirin (ASA) at least 1 month, but had received acetylsalicylic acid (81 – 650 mg/day) previously and had discontinued its use for any reason including non-compliance. Patients receiving antecedent aspirin (81 – 650 mg/daily) for at least 1 month at the time of the enrollment, and who had stroke within the last 6 months were assigned to the second group. Eleven patients received 81 mg/ daily, 27 – 325 mg/daily, and two patients were treated with 650 mg/daily of aspirin. Large artery atherosclerosis was diagnosed by duplex studies or angiography showing stenosis of cerebral arteries of z 50% diameter reduction with a typical morphology for atherosclerotic lesions. Cerebral microangiopathy was diagnosed if neuroimaging showed ischemic lesions of < 1.5 cm and clinical symptoms in accordance with typical lacunar syndromes (pure motor stroke, pure sensory stroke, sensorimotor stroke, dysarthria clumsy hand syndrome, ataxic hemiparesis). Patients were excluded for a history of bleeding diathesis, drug or alcohol abuse, prothrombin time greater than 1.5 times control, platelet count < 100,000/mm3, hematocrit < 25%, or creatinine > 4.0 mg/dl, operation or angioplasty for symptomatic stenosis performed within 3 months or planned for the future, known allergy to ASA, history of gastrointestinal or other bleeding, history of drug-induced disorders, trauma or surgery within the last 3 months or any surgery planned for the next 3 months, cancer, rheumatic diseases, and epileptic seizures. Patients participating in other investigational drug trials within 1 month of completion were also excluded. No patients had received thienopyridines, non-steroid anti-inflammatory drugs (NSAIDs), or intravenous platelet glycoprotein IIb/IIIa inhibitors for the last 3 months.

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within the past 30 days; currently receiving aspirin therapy or any medications containing aspirin within the past month; have received any antiplatelet therapy including glycoprotein IIb/IIIa inhibitors, or thienopyridines within the past month; known history of platelet count < 100  109/l; or blood dyscrasia; gastrointestinal ulcer, and bronchial asthma.

Blood samples Baseline blood samples were obtained with a 19gauge needle by direct venipuncture and drawn into two 7-ml vacutainer tubes at room temperature containing 3.8% trisodium citrate. The vacutainer tube was filled to capacity and gently inverted three to five times to ensure complete mixing of the anticoagulant. The first 4 – 5 ml of blood were used for lipid profile analysis, or discharged. All samples were labeled with coded number and analyzed by blinded technicians.

Platelet aggregation The blood – citrate mixture was centrifuged at 1200  g for 2.5 min. The resulting platelet-rich plasma (PRP) was kept at room temperature for use within 1 h. The platelet count was determined in the PRP sample and adjusted to 3.5  108/ml with homologous platelet poor plasma. Platelets were stimulated with 5 Amol epinephrine, and 5 Amol adenosine diphosphate (ADP) (Chronolog, Havertown, PA) and aggregation was assessed as previously described using a Chronolog Lumi-Aggregometer (model 560-Ca) with the AggroLink software package. Aggregation was expressed as the maximal percent change in light transmittance from baseline, using platelet-poor plasma as a reference. Curves were analyzed according to international standards [15].

Control subjects Cartridge-based platelet analyzers Forty controls were eligible if they had met all of the following inclusion criteria: males and females 21 years and older; able to provide informed consent; and more than two out of the eight risk factors for vascular disease (i.e., family history of vascular disease; sedentary lifestyle; diabetes mellitus; hypertension; morbid obesity; known history of hypercholesterolemia; post-menopausal or surgically sterile females; current smokers or recent smokers). Subjects were ineligible for the study if they met any of the following criteria: participation in another clinical trial of an investigational drug

Platelet Function Analyzer (PFA-100k) Using the PFA-100 instrument (Dade Behring, Miami, FL), the blood – citrate mixture is aspirated under a constant negative pressure and contacts an adenosine diphosphate and collagen coated membrane. The blood then passes through an aperture that induces high shear stress and simulates primary hemostasis after injury to a small blood vessel under flow condition. The time to aperture occlusion (the closure time) is recorded in seconds and is inversely

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related to the degree of shear-induced platelet activation [16]. Rapid platelet-function assay cartridge test (RPFA-ASA, UltegraR) RPFA-ASA (UltegraR) is a rapid platelet-function assay aspirin cartridge test with propyl gallate used as agonist. Cationic propyl gallate is a novel platelet activator used to detect and monitor the amount of platelet inhibition induced by platelet prostanoid antagonists, including aspirin and other non-steroid anti-inflammatory drugs [17]. The Ultegra device (Accumetrics, San Diego, CA) is a turbidimetric-based optical detection system which measures platelet-induced aggregation as an increase in light transmittance. This particular test cartridge has been designed to specifically monitor antiplatelet effects of aspirin-containing medications. When the platelets are exposed to the fibrinogen-coated beads, activated with lyophilized and re-suspended propyl gallate, agglutination occurs in proportion to the number of available platelet receptors. The whole blood citrate mixture was added to the cartridge, and agglutination between platelets and coated beads was recorded [18]. Ultegra RPFA-ASA Assay results are reported as Aspirin Responsive Units (ARU). Both PFA-100 and Ultegra assays were performed in duplicate. An electronic quality control test was performed on each instrument daily prior to performing any patient samples.

Whole blood flow cytometry The surface expression of platelet receptors was determined by flow cytometry using the following monoclonal antibodies: CD41 antigen (GPIIb/IIIa); CD42b (GPIb), CD62p (P-selectin) (DAKO, Carpenteria, CA); PAC-1 (GPIIb/IIIa activity) CD31 (platelet/endothelial cell adhesion molecule [PECAM]-1), CD51/CD61 (vitronectin receptor), CD63 (lyzosome associated membrane protein or LAMP-3), CD107a (LAMP-1), CD151 (platelet endothelial tetraspan antigen, or PETA-3), CD154 (CD40-ligand), CD165 (GP37) (PharMingen, San Diego, CA); CD36 (thrombospondin, or GPIV), cleaved (WEDE15), and intact (SPAN12) platelet thrombin receptors (Beckman Coulter, Brea, CA). Formation of platelet – leukocyte aggregates was assessed by dual labeling with pan-platelet marker (CD151), and then with CD14, the macrophage receptor for endotoxin lipopolysaccharides. The blood – citrate mixture (50 Al) was diluted with 450 Al Tris buffered saline (TBS) (10 mmol/l Tris, 0.15 mol/l sodium chloride) and mixed by inverting an Eppendorf tube gently two times.

The appropriate primary antibody was then added (5 Al) and incubated at 37 jC for 30 min, and then a secondary antibody was applied if needed. After incubation, 400 Al of 2% buffered paraformaldehyde was added for fixation. The samples were analyzed on the FACScan flow cytometer (Becton Dickinson,

Table 1

Demographics, and clinical characteristics

Parameter

Demographics Age Male Ethnicity White Black Asian Multiple risk factors Previous history of stroke or TIA Family history of stroke/TIA Diabetes mellitus Hyperlipidemia Alcohol use Smoking Documented coronary artery disease Hypertension Hypotensive medications Statins Antiglycemic Antidepressants Time between acute event and platelet studies

ASA + ASA free ASA free post-stroke post-stroke controls (n = 40)a (n = 40)a (n = 40)a 64.8 F 7.7 26 (65%)

69.0 F 8.4 29 (72%)

41.6 F 9.2b 25 (62%)

14 24 2 22

17 22 1 29

27 (67%)b 13 (33%)b 0 40 (100%)b

(35%) (60%) (5%) (55%)

(42%) (55%) (2.5%) (72%)

6 (15%)

8 (20%)

16 (40%)

8 (40%)

6 (35%)

14 (35%)

17 (42%)

16 (40%)

28 14 18 18

26 16 16 20

20 (50%)b 19 (48%) 22 (55%) 0

(70%) (35%) (45%) (45%)

(65%) (40%) (40%) (50%)

0

26 (65%) 24 (60%)

24 (60%) 20 (50%)

22 (55%) 17 (42%)

22 (55%) 14 (35%) 16 (40%) 144 F 22 days

20 (50%) 12 (30%) 15 (37%) 108 F 31b days

10 (25%)b 0 8 (20%)b –

Stroke etiology Ischemic 40 (100%) 40 (100%) Stroke location Right 20 (50%) 18 (45%) hemisphere Left 12 (30%) 13 (32%) hemisphere Cerebellar 2 (5%) 2 (5%) Bilateral – – Brain stem 6 (15%) 7 (18%) a Data are presented in mean F SD. b p < 0.05.

– – – – – –

V.L. Serebruany et al. / Thrombosis Research 113 (2004) 197 – 204 San Diego, CA) calibrated to measure fluorescent light scatter as previously described [19]. All parameters were collected using four-decade logarithmic amplification. The data were collected in list mode files and then analyzed. P-selectin was expressed as percent positive cells as previously described [20]. Other antigens were expressed as log mean fluorescence intensity.

Statistical analysis Comparisons between and within treatment arms were made by two-tailed ANOVA with v2 and Fisher’s exact tests for discrete variables, and Wilcoxon rank-sum for continuous variables. Data were expressed as mean F SD, and p < 0.05 was considered significant. Differences between individual flow cytometric histograms were assessed using the Smirnov – Kolmogorov test incorporated in the CELLQuest’ (San Diego, CA) software. Statistical analyses were performed using SPSS/E11.5 (SPSS, Chicago, IL). Table 2

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Results Demographics The demographics, risk factors, and clinical characteristics of the post-stroke patients and control subjects with multiple risk factors are shown in Table 1. Age, gender, and race were distributed fairly even between post-stroke groups, while significantly more Caucasian ( p = 0.002), and younger subjects ( p = 0.0001) constituted the control group. Stroke patients experienced fewer risk factors ( p = 0.03), but higher incidence of previous stroke history ( p = 0.001), hyperlipidemia ( p =0.036), and coronary artery disease ( p = 0.0001). Frequency of diabetes, alcohol use, and smoking were similar among three groups. Statins ( p = 0.002), selective serotonin reuptake inhibitors ( p = 0.012), and antidiabetic agents ( p = 0.026) were more commonly used in the stroke groups when compared with the controls. The time interval between stroke occurrence and actual platelet studies was significantly

Platelet characteristics in patients after stroke, and subjects with multiple risk factors

Parameter Conventional plasma aggregometry Induced by 5 AM epinephrine (%) Induced by 5 AM adenosine diphosphate (%) Cartridge-based rapid analyzers Closure time by PFA-100 (s) Aspirin reaction units by Ultegra

Aspirin-free post-stroke (n = 40)a

Aspirin + post-stroke (n = 40)a

Aspirin-free controls (n = 40)a

77.4 F 8.8 73.5 F 9.0

39.2 F 5.5b 70.4 F 7.1

68.0 F 8.4 66.2 F 8.2

170 F 34 665 F 61

236 F 40 b 394 F 47 b

173 F 29 641 F 56

Whole blood flow cytometry c CD62p, p-selectin (+%) 10.2 F 3.0 7.3 F 3.1b 11.5 F 3.8 CD151 + CD14, platelet-monocyte 140 F 22 103 F 19b 157 F 24 aggregates (MFI) CD154, CD40-ligand (MFI) 8.9 F 3.0 6.2 F 3.1b 8.8 F 3.4 CD165, GP37 (MFI) 25.9 F 5.3 16.4 F 4.8b 28.1 F 5.5 PAC-1, GPIIb/IIIa activity (MFI) 13.8 F 3.5 9.2 F 3.0b 14.7 F 4.8 PAR-1 thrombin receptor, intact 34.8 F 8.0 19.0 F 4.4b 37.3 F 7.7 epitope (MFI) 13.9 F 3.0 Thrombospondin (MFI) 13.1 F 3.1 6.0 F 2.6b CD31, PECAM-1 (MFI) 56 F 16 52 F 19 60 F 11 CD41a, GpIIb antigen (MFI) 454 F 68 427 F 73 440 F 69 CD42b, GPIb (MFI) 253 F 38 271 F 51 284 F 44 CD63, LAMP-3 (MFI) 9.4 F 2.8 9.6 F 3.0 10.8 F 3.7 CD107a, LAMP-1 (MFI) 4.8 F 1.6 5.2 F 1.5 5.2 F 2.0 CD151, PETA-3 (MFI) 132 F 27 120 F 34 155 F 30 PAR-1 thrombin receptor, cleaved 21.4 F 4.2 16.5 F 5.0 25.9 F 7.8 epitope (MFI) a Data are presented as mean and standard deviation. b p values < 0.05 versus both aspirin-free groups. c All flow cytometry readings are expressed as log mean fluorescence intensity (MFI) except expression of Pselectin, which is (percent positive).

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shorter in patients treated with aspirin ( p = 0.03) than the aspirin-free cohort. In all patients stroke origin was ischemic, and right hemisphere was the most common stroke location.

Platelet data The platelet data dependent at presentation are presented in Table 2. Aspirin-free post-stroke patients versus aspirinfree controls The platelet data revealed no statistical differences between groups. Epinephrine-, and adenosine diphosphate-induced conventional platelet aggregation was slightly higher in the post-stroke patients. Rapid platelet analyzers revealed almost identical readings of the closure time by PFA-100 instrument, and aspirin reactive units measured by the UltegraR device. Moreover, 12 out of the 14 receptors measured were surprisingly lower, while only GPIIb antigen level, and CD40-ligand expression were higher in the post-stroke cohort.

Aspirin-free versus aspirin-treated poststroke patients Antecedent treatment with 81 – 650 mg of aspirin for at least 1 month in patients recovering after ischemic stroke resulted in a significant inhibition of various platelet characteristics as compared to the aspirin-free post-stroke group. Aspirin-treated patients exhibited highly significant inhibition of epinephrine-induced conventional platelet aggregation ( p = 0.0001). Cartridge-based rapid analyzers revealed prolongation of the closure time by the PFA-100 instrument ( p = 0.03), and reduction of the aspirin reactive units ( p = 0.02) measured by the UltegraR device suggestive of platelet inhibition in the aspirin-treated group. In addition, surface platelet expression of thrombospondin ( p = 0.001), GPIIb/IIIa activity ( p = 0.04), P-selectin ( p = 0.03), CD40-ligand ( p = 0.04), CD165 ( p = 0.02), and intact epitope of PAR-1 thrombin receptor ( p = 0.03) were significantly lower in the aspirin-treated group. Finally, the formation of the platelet – monocyte aggregates ( p = 0.01), suggestive of the degree of platelet – leukocyte interactions was significantly diminished in the aspirin group.

Discussion The data from the present study document a marked heterogeneity of platelet characteristics

in patients after ischemic stroke. The most important finding of this study is the fact that platelets are not uniformly activated during the post-stroke recovery phase even in the aspirin-free patients. Indeed, for the aggregation agonists, rapid analyzer reading, and surface antigens, there was a cohort of post-stroke patients with normal or even reduced range platelet status. Overall, platelet activity was remarkably heterogeneous, and was not overwhelmingly elevated after stroke. Indeed, there were no significant differences between aspirin-free groups of post-stroke patients and subjects with multiple risk factors. On the other hand, therapy with aspirin for at least 1 month resulted in substantial inhibition of platelet activity as reflected by conventional aggregometry, rapid cartridge-based platelet analyzers, and whole blood flow cytometry. Platelets play a pivotal role in acute cerebral artery thrombosis; however, the body of evidence is based mostly on experimental studies. Therefore, attempts to mimic and extrapolate clinical scenarios from the in vitro and animal studies have serious limitations, and should be considered with caution. Although there are some comprehensive clinical reports which have been performed mostly to assess platelet status in acute stroke after different reperfusion or/and antiplatelet strategies (e.g., Refs. [1,11,14,21]), the data are clearly underrepresented in the strokerecovering patients. The current study represents an attempt to simultaneously define platelet status in the chronic phase of stroke utilizing a variety of techniques including conventional aggregometry, platelet analyzers, and flow cytometry. We hypothesized that such a combined approach would permit detection of previously unrecognized signs of platelet activation in the post-stroke patients, and to define how aspirin may affect such activation. Therefore, inclusion of the aspirin-free groups was critical for these analyses. However, while aspirin did diminish platelet activation in post-stroke cohort, unexpectedly the degree of such activation was marginal, and not uniformly present in every patient, especially when measured by more sensitive flow cytometric techniques. Observations of the heterogeneity of certain platelet characteristics in patients with acute vascular events, and especially following myocardial infarction are not new. The first observation of the existence of ‘‘exhausted platelets’’ in patients after vascular occlusions was made over two decades ago [22]. Later, it was established that platelet secretion in response to thrombin, measured by flow cytometry varies dramatically [23], while platelet

V.L. Serebruany et al. / Thrombosis Research 113 (2004) 197 – 204 aggregation responses are markedly heterogeneous in both platelet-rich plasma and whole blood in myocardial infarction survivors [24]. The data in the current study are also concordant with our own report of the baseline platelet heterogeneity in patients with myocardial infarction enrolled in the GUSTO-III trial [25]. Another GUSTO-III-related observation was a case description which documented profound depression of platelets preceded the occurrence of hemorrhagic stroke in an elderly long-term aspirin user, treated with thrombolytic therapy [21]. Therefore, initial ‘‘exhausted’’ platelets may be responsible for the increased risk for hemorrhagic stroke following coronary thrombolysis, or hemorrhagic transformation of the ischemic attack. Our data are in full agreement with the report that the closure time, indicative of shear-induced platelet activation, and measured with the PFA-100 Analyzer is prolonged in the aspirin-treated patients after stroke [26]. On the other hand, our results did not match those from another observation that the escalating doses of aspirin inhibit adenosine phosphate aggregation, what most likely is related to the different concentrations of the agonists [27]. Obviously, the predictive value of platelet activation parameters requires investigation in prospective studies.

Study limitations This study represents a combined dataset of poststroke patients and volunteers, which has been collected at one time point (baseline) only, and has not been designed in the prospective fashion. Therefore, power calculations have not been made, and the study was underpowered to detect differences in platelet activation between the groups. Although the expression of multiple activation-dependent platelet receptors was studied, their individual roles in patients after stroke are unknown. Similarly, the relation of platelet receptor expression to established platelet function measurements is currently under investigation. The hemostatic response of platelets of post-stroke patients may be influenced by the genetic profile of the total population of receptors expressed on the platelet surface. Among the parameters potentially responsible for the observed heterogeneity could be the density of each receptor, and the presence of structural polymorphisms affecting function [28]. Moreover, there are other confounding factors such as shedding of receptors of the platelet surface [29], complex interactions between platelets and

203

leukocytes [30], sequestration of GPIIb/IIIa and Pselectin by lungs [31], and destruction of activated platelets by the liver and spleen [32]. Finally, the broad spectrum of concomitant medications may have affected the platelet characteristics. In conclusion, these findings suggest that the adequate and timely assessment of platelet activity may be critical when considering adjunctive antiplatelet regimens. Bleeding events after ‘‘one size fits all’’ aggressive antiplatelet therapy could be related to the decreased or normal baseline platelet characteristics in patients recovering after ischemic stroke. These data lead us to a clinical debate. Should we uniformly use combinations of antiplatelet agents, including platelet GPIIb/IIIa inhibitors without individual assessment of platelet status in post-stroke patients? Based on the present data, it is reasonable to speculate that bleeding complications, and hemorrhagic transformations after modern therapeutic strategies could be at least in part related to decreased or normal baseline platelet characteristics in some patients. Further analysis of platelet heterogeneity and its clinical significance remains to be determined in randomized trials.

Acknowledgements Patient enrollment was supported in part by Boehringer Ingelheim, (Ingelheim am Rhein, Germany), and Accumetrics (San Diego, CA, USA). The data analyses was supported by HeartDrugk Research (Wilmington, DE, USA). The authors thank all the nurses and laboratory personnel for their technical excellence and outstanding effort.

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