Journal of Thrombosis and Haemostasis, 8: 1185–1190
DOI: 10.1111/j.1538-7836.2010.03851.x
ORIGINAL ARTICLE
Lower coated-platelet levels are associated with early hemorrhagic transformation in patients with non-lacunar brain infarction C . I . P R O D A N , * J . A . S T O N E R , à L . D . C O W A N à and G . L . D A L E § *Department of Neurology, University of Oklahoma Health Sciences Center; Veterans Aairs Medical Center; àDepartment of Biostatistics and Epidemiology, University of Oklahoma Health Sciences Center; §Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
To cite this article: Prodan CI, Stoner JA, Cowan LD, Dale GL. Lower coated-platelet levels are associated with early hemorrhagic transformation in patients with non-lacunar brain infarction. J Thromb Haemost 2010; 8: 1185–90.
Introduction Summary. Background: Coated-platelets, representing a subset of platelets with procoagulant potential, are elevated in patients with non-lacunar ischemic stroke and decreased in patients with spontaneous intracerebral hemorrhage. However, within the non-lacunar patient population there are individuals with lower levels of coated-platelets, which raises the possibility that these individuals would be susceptible to early hemorrhagic transformation (HT) of ischemic stroke. Objective: Because extremes in coated-platelet potential may be associated with either thrombotic or hemorrhagic events, we undertook a pilot study to investigate whether there is an association between coatedplatelet production and the presence of early HT in patients with non-lacunar ischemic stroke. Patients and methods: Coated-platelet levels were determined in 115 consecutive eligible patients with a diagnosis of non-lacunar ischemic stroke. Early HT was determined on CT scan examination and confirmed by MRI studies. The distribution of coated-platelet levels was summarized using the median and interquartile range (25th–75th percentiles) and compared statistically between patients with and without early HT using the non-parametric Wilcoxon rank sum test. Results: The median coated-platelet level in all non-lacunar stroke patients was 38.0% (interquartile range 30.5–48.3%). Early HT was detected in 11 patients (9.6%), and these patients had significantly lower coatedplatelet levels compared with those without early HT [median 25.1% (interquartile range 20.4–35.5%) vs. 39.2% (31.6– 49.5%), P = 0.003]. Conclusions: Lower levels of coatedplatelets are associated with the presence of early HT in patients with non-lacunar ischemic stroke. Keywords: hemorrhage, ischemia, platelets, thrombosis. Correspondence: Calin I. Prodan, 711 S.L. Young Blvd, PPOB Suite 215, Oklahoma City, OK 73104, USA. Tel.: +1 405 271 4113; fax: +1 405 271 5723. E-mail:
[email protected] Received 5 January 2010, accepted 3 March 2010 2010 International Society on Thrombosis and Haemostasis
Coated-platelets are a subpopulation of platelets observed upon dual stimulation with collagen and thrombin. First described in 2000 and previously referred to as COAT-platelets (collagen and thrombin activated platelets), this subset of activated platelets supports a robust prothrombinase activity, releases microparticles and retains high levels of several procoagulant proteins on the cell surface, including factor V, fibrinogen, thrombospondin and von Willebrand factor [1–3]. These unusual properties suggested the possibility that extremes in coated-platelet potential among individuals may be associated with either thrombotic or hemorrhagic events [1]. We previously demonstrated that mean coated-platelet levels are elevated in patients with non-lacunar ischemic stroke compared with either controls or patients with lacunar ischemic stroke [4]. Despite the increase in the mean levels of coatedplatelets in non-lacunar ischemic stroke patients, there was still a remarkably wide range (5.2–66.2%) for these procoagulant platelets [4]. In contrast to the findings in ischemic stroke, we noted that patients with spontaneous intracerebral hemorrhage had significantly lower coated-platelet levels shortly after the hemorrhagic event compared with normal controls [5]. In addition, an absence of coated-platelet production is associated with a potentially life threatening bleeding diathesis in dogs [6]. Early hemorrhagic transformation (HT) of brain infarction consists of secondary bleeding into the ischemic brain tissue, ranging from petechial hemorrhage to parenchymal hematoma with variable clinical presentations [7–13]. Prompted by the procoagulant potential of coated-platelets and the abnormalities in coated-platelet production noted in non-lacunar ischemic stroke and spontaneous intracerebral hemorrhage [1,4,5], we conducted a pilot study to investigate whether coatedplatelet levels in patients with non-lacunar stroke measured shortly after the ischemic event are associated with the presence of early hemorrhagic transformation (HT). We hypothesized that non-lacunar ischemic stroke patients with early HT would have lower coated-platelet levels compared with non-lacunar ischemic stroke patients without early HT.
1186 C. I. Prodan et al
Materials and methods Patients
The study was approved by the Institutional Review Board (IRB) of the University of Oklahoma Health Sciences Center. Individual informed consent was obtained for all study subjects in accordance with the University of Oklahoma Health Sciences Center Institutional Review Board and the Oklahoma City Veterans Administration Hospital rules and regulations. The research was carried out according to the principles of the Declaration of Helsinki. One hundred and fifteen consecutive, eligible patients with a diagnosis of non-lacunar ischemic stroke based on TOAST criteria [14] were recruited from the Neurology service at the University of Oklahoma Health Sciences Center and the Veterans Affairs Medical Center. All subjects were recruited between 30 September 2007 and 31 October 2009. During this time interval a total of 491 patients were admitted with an initial diagnosis of ischemic stroke. Of these patients, 112 were subsequently diagnosed with small-vessel (lacunar) ischemic stroke, 74 with transient ischemic attack, 47 with a diagnosis other than stroke and 16 declined to participate. Of the remaining 242 patients with a diagnosis of ischemic stroke, 115 (48%) were recruited for our study. The main factors precluding recruitment were initiation of anticoagulants for the current stroke prior to collection of blood for determining coated-platelet levels and > 96 h between the actual onset of the symptoms and enrollment [1]. Exclusion criteria included initiation of anticoagulants or administration of thrombolysis for the current stroke prior to enrollment, prolonged coagulation tests (PT, PTT or INR), > 96 h between the onset of the symptoms and enrollment, imaging evidence of small-vessel (lacunar) stroke as defined by TOAST criteria [14], prior dementia or evidence of primary intracerebral hemorrhage or tumor on imaging studies. These exclusion criteria were prompted by the potential confounding effects of heparin and thrombolytics on coated-platelet measurements, the observation that changes in coated-platelet production in animals after physiological manipulation require a minimum of 4 days to be manifest [1], and previously published coated-platelet abnormalities in Alzheimer disease, respectively [15]. All patients were diagnosed with non-lacunar ischemic stroke by a board certified neurologist and underwent initial brain CT scan followed by brain MRI studies, or repeat brain CT (if MRI was contraindicated) within 12–24 h to confirm the presence of cerebral ischemia [16]. The diagnosis of nonlacunar ischemic stroke was based on clinical and imaging data and published criteria [14]. Additional investigations included carotid ultrasound studies and echocardiogram/ECG, along with routine laboratory studies including coagulation studies and measurement of blood count, serum chemistry, troponin and creatine kinase levels. A neuroradiologist provided the definitive reading for every CT and MRI scan obtained. Early HT was defined as small
petechiae along the margins of the infarct, confluent petechiae within the infarcted area without space-occupying effect, or parenchymal hematoma occupying £ 30% or > 30% of the infarcted brain area [11,17,18] on the initial CT scan and/or the repeat MRI or CT scan, as determined by the neuroradiologist. For analysis purposes, we considered all patients with early HT transformation as a single group. Of note, the neuroradiology staff was not aware of the study protocol or the results of coated-platelet measurements. Smoking status [19], gender, race, age and use of medications that may influence coated-platelet levels, such as selective serotonin reuptake inhibitors (SSRI), HMG-CoA reductase inhibitors (statins) or antiplatelet medication [19,20], were recorded at the time of enrollment for each patient and reflected prehospitalization status and medication use. Additional information recorded included a history of diabetes or end-stage renal disease as previous reports noted increased coated-platelet potential in these conditions [21,22]. The presence of high blood glucose, platelet count based on a blood draw taken the same day as the blood draw for the coated-platelet measure, or cardioembolism as the source of stroke, was also noted as these conditions have been reported as predictors of early HT [10,12,13,23,24]. Coated-platelet assay
After obtaining informed consent, 5 mL of blood was drawn into a plastic syringe containing 0.5 mL of acid citrate dextrose (ACD), and platelet-rich plasma (PRP) was prepared as described [4]. Coated-platelets were assayed as previously described [4] with 1 lL of PRP in a 100 lL assay with the following reagents (final concentrations): 1.0 lg mL)1 biotinfibrinogen, 0.4 mM gly-pro-arg-pro-amide, 500 ng mL)1 convulxin, 0.5 U mL)1 bovine thrombin, 2 mM CaCl2, 1 mM MgCl2, 150 mM NaCl and 10 mM N-(2-hydroxyethyl)-piperazine-NÕ-(4-butanesulfonic acid) (HEPES), pH 7.5. After 5 min at 37 C, 0.8 lg of phycoerythrin-streptavidin and 0.5 lg of FITC-abciximab were added. After an additional 5 min at 37 C, the reaction was stopped with 0.2 mL of 1.5% (w/v) formalin in 150 mM NaCl, 10 mM HEPES, pH 7.5. The percentage of abciximab-positive events (platelets) with bound biotin-fibrinogen was quantitated by flow cytometry. Results are reported as percentage of cells converted to coated-platelets. Additional details concerning this assay are provided in Data S1. The individuals performing the coated-platelet assay were not aware of the clinical diagnosis or the results of imaging studies corresponding to the blood sample analyzed. Statistical analyses
Data were analyzed using SAS (SAS System for Windows, ver. 9.1; SAS Institute Inc., Cary, NC, USA) and SPSS (SPSS for Windows, rel. 15.0; SPSS, Chicago, IL, USA). Descriptive statistics, including the median and interquartile range (25th– 75th percentile values), were used to summarize the distribution 2010 International Society on Thrombosis and Haemostasis
Coated-platelets and early hemorrhagic transformation 1187
of patient characteristics. The distributions of coated-platelet levels and clinical characteristics were compared between groups of patients with and without early HT using the nonparametric Wilcoxon rank sum test for continuous measures and a chi-square or FisherÕs exact test when expected cell counts were small, for categorical measures. The correlation between continuous measures was summarized using a PearsonÕs correlation coefficient. Significance for all tests was set at P £ 0.05. The current sample size was calculated to achieve > 90% power to detect a difference of 15% points in mean coatedplatelet levels between subjects with and without early HT assuming a standard deviation of 11% points, an approximate 10% incidence of early HT in patients with ischemic stroke [12,13], and a two-sided 0.05 alpha level. The effect size and standard deviation estimates were based on our previously published research data on coated-platelet levels in ischemic stroke and spontaneous intracerebral hemorrhage [4,5]. Sample size calculations were performed using PASS software with a non-parametric adjustment to the comparison of independent means [25]. Results Table 1 lists demographic variables, coated-platelet levels and relevant comorbidities/medications for all non-lacunar ischemic stroke patients (n = 115). The median coated-platelet level for the entire group was 38.0% (interquartile range 30.5–
Table 1 Summary of baseline demographic variables, coated-platelet levels, and relevant comorbidities and medications for all patients All subjects (n = 115) Baseline values*
Min
Max
Median
Mean
SD
Age (years) Coated-platelets (%) Platelet count (140–400 · 103 mm)3) Time to initial CT (h) Time to blood collection (h)
35 13.3 87
95 67.3 486
63 38.0 242
64.6 39.4 248.6
11.2 12.8 70.3
18 24.6 44 47.6 Count (%)
19.9 22.6
Male Race/ethnicity Caucasian African American Hispanic American Indian Smokers Anti-platelets SSRIs Statins Diabetes mellitus End stage renal disease Cardioembolism High blood sugar
3 14
80 92
82 (71) 76 (66) 37 (32) 1 (1) 1 (1) 59 (51) 53 (46) 10 (9) 42 (37) 39 (34) 5 (4) 34 (30) 57 (50)
*Data summarized using the minimum (Min), maximum (Max), median, mean and standard deviation (SD) or count (%). 2010 International Society on Thrombosis and Haemostasis
48.3%) with a minimum of 13.3% and maximum of 67.3%, consistent with our previous data regarding coated-platelet synthesis in patients with non-lacunar ischemic stroke [4]. Stroke patients consisted of 33 women and 82 men, age 64.6 ± 11.2 years (mean ± SD), with the following racial distribution: 37 African American, one Hispanic, one Native American and 76 white (see Table 1 for additional details). Of these patients, 60 were veterans of the United States armed forces, resulting in an over-representation of men in our patient population, a consequence of the military composition during the time these veterans served. Early HT was present in 11 patients, representing 9.6% of the study group. The changes observed with early HT consisted of small petechiae along the margins of the infarct (one case), more confluent petechiae within the infarcted area (eight cases) or parenchymal hematoma that occupied < 30% of the infarcted area with slight space-occupying effect (two cases). Early HT changes were noted by the neuroradiologist on the initial CT scan and confirmed by repeat MRI imaging performed within 12–24 h in 10 patients. One patient (coated-platelet level 25.1%) had no evidence of early HT on the initial CT scan, but deteriorated clinically within 6 h. Emergent repeat CT scan showed a parenchymal hematoma of < 30% of the infarcted area with space-occupying effect and MRI scan obtained within 12 h confirmed the presence and size of HT within the ischemic stroke lesion. Table 2 lists demographic variables, coated-platelet levels and relevant comorbidities/medications for stroke patients subdivided as patients with early HT (n = 11) and patients without early HT (n = 104). Stroke patients with early HT had significantly lower median coated-platelet levels compared with stroke patients without early HT [median 25.1% (interquartile range 20.4–35.5%) vs. 39.2% (31.6–49.5%), P = 0.003]. The association between coated-platelet levels and the presence of early HT is summarized in Fig. 1. Based on a descriptive comparison, stroke patients with early HT tended to have a shorter time to coated-platelet blood collection after the onset of stroke symptoms and were more likely to have high blood sugar levels. They were also less likely to be African American, to have a cardioembolic source of stroke, or to be on statins compared with stroke patients without early HT. However, none of these differences were statistically significant (Table 2). Other factors, including smoking status, gender, age, use of SSRIs or antiplatelets, history of diabetes, end-stage renal disease, platelet count, and the time period between the onset of symptoms and the initial CT scan, had similar distributions and also did not differ statistically between patients with early HT and patients without early HT (Table 2). To investigate the potential modifying and confounding effects of the recorded subject characteristics and clinical measures, differences in coated-platelet levels between subjects with and without early HT were plotted after stratifying by smoking status, gender, race/ethnicity, age, use of medications that may influence coated-platelet levels (SSRIs, statins, or antiplatelets) [19], history of diabetes [21], end-stage renal
1188 C. I. Prodan et al Table 2 Summary of baseline demographic variables, coated-platelet levels, and relevant comorbidities and medications according to early hemorrhagic transformation (HT) status Patients without (n = 104) Baseline values*
Median
Age (years) Coated-platelets (%) Platelet count (140–400 · 103 mm)3) Time to initial CT (h) Time to blood collection (h)
62 39.2 244 18 44
Male Race/ethnicity (white/minorities) Caucasian African American Hispanic American Indian Smokers Anti-platelets SSRIs Statins Diabetes mellitus End stage renal disease Cardioembolism High blood sugar
early
Mean 64.3 40.4 279.1 24.4 48.0 Count (%)
HT
Patients with early HT (n = 11)
SD
Median
11.2 12.4 71.3 20.0 22.7
69 25.1 232 18 34
Mean 67.4 29.1 243.5 25.9 43.8 Count (%)
SD 10.8 12.4 62.1 20.1 22.2
P-value 0.3 0.003 0.7 0.7 0.6
74 (71)
8 (73)
> 0.9
67 36 1 0 54 49 10 40 34 4 32 49
9 (82) 1 (9) 0 1 (9) 5 (45) 4 (36) 0 2 (18) 5 (45) 1 (9) 2 (18) 8 (73)
0.3
(64) (35) (1) (52) (47) (10) (38) (33) (4) (31) (47)
0.7 0.5 0.6 0.3 0.5 0.4 0.5 0.1
*Data summarized using the median, mean and standard deviation (SD) or count (%).
regardless of the subgroup under consideration. In patients with cardioembolism as the source of stroke (n = 34), coatedplatelets appear to be higher among subjects with (n = 2) vs. those without early HT (n = 32). Table S1 (Supporting information) summarizes the association between coatedplatelet levels and early HT status for subgroups defined by the above characteristics. Table S2 presents coated-platelet levels in all patients with or without relevant chronic use medications. No significant linear correlation was found in stroke patients between coated-platelet levels and the time elapsed from the onset of symptoms to blood drawing (r = 0.1, P = 0.2) or the patientsÕ age (r = )0.08, P = 0.4).
Coated-platelet level (%)
60.00
40.00
20.00
0.00 Absent Present Early hemorrhagic transformation Fig. 1. The association between coated-platelet levels and the presence of early HT. The upper and lower edges of the box correspond to the 25th and 75th percentiles, respectively. The middle bar corresponds to the median. The whiskers are drawn to the minimum and maximum values within 1.5 times the interquartile range (distance from the 25th to the 75th percentile) of the box edge. Outlying points (falling between 1.5 and 3 times the interquartile range beyond the box edge) are denoted with a * symbol and extreme points (falling more than three times the interquartile range beyond the box edge) are denoted with a d symbol.
disease [22], the presence of high blood glucose, platelet count or cardioembolism as the source of stroke [10,12,13,23,24]. With the exception of patients with cardioembolism as the source of stroke, subjects with early HT tended to have lower coated-platelet levels as compared with those without early HT
Discussion As previously described, the combination of bound procoagulant proteins and exposed phosphatidylserine present on coated-platelets defines their single most important characteristic: the ability to support a robust prothrombinase activity [1]. Because thrombin generation is the central event in coagulation, coated-platelets are considered to be pro-thrombotic [1]. This is supported by the genetically-determined absence of coated-platelets in dogs with a bleeding diathesis, significantly lower coated-platelet levels in patients with SICH, and elevated levels of coated-platelets in patients with non-lacunar ischemic stroke [4–6]. It should be noted that the level of coated-platelets observed is a function of the agonist concentrations utilized (i.e. higher agonist concentrations result in higher levels of coatedplatelets and lower levels of agonists in lower coated-platelet 2010 International Society on Thrombosis and Haemostasis
Coated-platelets and early hemorrhagic transformation 1189
levels). However, in a clinical study, such as that detailed here, the rank order of patient coated-platelet values established with our standard assay would not change if agonist concentrations were varied. The patients with the highest and lowest coatedplatelet levels retain that order independent of the actual agonist concentrations utilized. Most importantly, the relative levels of coated-platelets among patients still reflect the comparative thrombotic propensity of each patient regardless of the absolute coated-platelet value. The current study indicates that coated-platelet formation is lower in patients with non-lacunar ischemic stroke with early HT compared with patients with non-lacunar ischemic stroke without early HT. This difference in coated-platelet formation is consistent not only with the pro-thrombotic potential of these activated platelets [1], but also with previous observations indicating that extremes in coated-platelet potential may be associated with either thrombotic or hemorrhagic events [4–6]. The rationale for focusing our current investigation on nonlacunar stroke patients instead of all ischemic stroke patients (lacunar and non-lacunar) was based on previous findings of significant differences in coated-platelet potential between lacunar and non-lacunar stroke patients and the remarkably wide range of coated-platelet levels in non-lacunar stroke [4,26]. The percentage of patients with early HT of a non-lacunar ischemic stroke reported here is slightly higher than in the data reported by Paciaroni et al. [12] in a large cohort of consecutive patients with acute ischemic stroke and by Lindley et al. [27] in a systematic review of HT, but lower than the percentage reported by other authors [13]. Potential explanations for these differences include the focus of our study on non-lacunar stroke patients, differences both in sample size and timing of imaging studies, and the exclusion of patients who received thrombolysis. The underlying mechanism for early HT following brain infarction is believed to consist of an initial ischemic injury, followed by a transient increase in vascular permeability and secondary hemorrhage [7,10,28–30]. Our study suggests that a decreased coated-platelet production, and by extrapolation a decrease in prothrombinase activity, is also present in these patients. This is an attractive premise for explaining the hemorrhage present within the infarcted brain tissue but problematic in explaining the actual thrombi responsible for the stroke. Perhaps this is another example of the complex and overlapping pathophysiologies capable of leading to a common end point of ischemic stroke. From a clinical standpoint, the presence of early HT changes in patients with ischemic stroke may represent a worrisome complication [12,13,27]. Although prior studies showed that parenchymal hematoma, in contrast to petechial changes, was more likely to be associated with a less favorable neurological outcome [10–13,27,31,32], it has been suggested that a complete understanding of the impact of asymptomatic HT may require larger studies [33]. Thus, while some authors emphasize differences between subtypes of HT [11], these differences may be Ôof degree rather than of kindÕ [10] and the presence of early HT changes may have an impact on the choice of medical therapy [27]. 2010 International Society on Thrombosis and Haemostasis
Limitations of this pilot study include a relatively modest sample size, cross-sectional data and the exclusion of patients who received thrombolysis or were treated with anticoagulants for the current stroke prior to enrollment. A similar study is currently underway investigating coated-platelet formation and hemorrhagic complications in patients with lacunar stroke, while a longitudinal study is addressing the potential connection between coated-platelet levels and the risk of recurrent cerebrovascular events. Larger prospective studies are needed to confirm these findings, address the limitations listed above and better establish the potential connection between altered coated-platelet formation, cerebral infarction and complications of ischemic stroke. Acknowledgements This work was supported by the Department of Veterans Affairs and grants from the Oklahoma Center for Advancement of Science and Technology (C. I. Prodan) and the American Heart Association (G. L. Dale). The authors are indebted to R. Cox, P. Friese, E. Nagode, P. Joseph and R. Padmanabhan for their assistance with this project. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Summary of the distribution of coated-platelet levels between patients with and without early hemorrhagic transformation in subgroups of subjects defined by baseline demographics and relevant comorbidities and medications. Table S2. Coated-platelet levels in all patients (n = 115) with or without relevant chronic use medications (antiplatelet drugs, SSRI and statins). The use of a particular medication is indicated as yes or no. Data S1. Coated-platelet assay: materials and methods. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. References 1 Dale GL. Coated-platelets: an emerging component of the procoagulant response. J Thromb Haemost 2005; 3: 2185–92. 2 Remenyi G, Szasz R, Friese P, Dale GL. Role of mitochondrial permeability transition pore in coated-platelet formation. Arterioscler Thromb Vasc Biol 2005; 25: 467–71. 3 Dale GL, Remenyi G, Friese P. Quantitation of microparticles released from coated-platelets. J Thromb Haemost 2005; 3: 2081–8. 4 Prodan CI, Joseph PM, Vincent AS, Dale GL. Coated-platelets in ischemic stroke: differences between lacunar and cortical stroke. J Thromb Haemost 2008; 6: 609–14.
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