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Original Research: ANTITHROMBOTIC THERAPY |

Elevated Platelet Microparticle Levels in Nonvalvular Atrial Fibrillation*: Relationship to P-Selectin and Antithrombotic Therapy FREE TO VIEW

Anirban Choudhury, MBBS; Irene Chung, MBChB; Andrew D. Blann, PhD; Gregory Y. H. Lip, MD
Author and Funding Information

*From the Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, UK.

Correspondence to: Gregory Y. H. Lip, City Hospital, Haemostasis, Thrombosis and Vascular Biology Unit, Dudley Rd, Birmingham B18 7QH, UK; e-mail: g.y.h.lip@bham.ac.uk



Chest. 2007;131(3):809-815. doi:10.1378/chest.06-2039
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Background: Platelet microparticles (PMPs), are procoagulant membrane vesicles that are derived from activated platelets, the levels of which are elevated in patients with hypertension, coronary artery disease (CAD), diabetes, and stroke, all of which are conditions that lead to (and are associated with) atrial fibrillation (AF). We hypothesized the following: (1) PMP levels are elevated in patients with AF compared to levels in both healthy control subjects (ie, patients without cardiovascular diseases who are in sinus rhythm) and disease control subjects (ie, patients with hypertension, CAD, diabetes or stroke, but who are in sinus rhythm); (2) PMP levels correlate with levels of soluble P-selectin (sP-selectin) [a marker of platelet activation]; and (3) PMP levels are related to the underlying factors in patients with AF that contribute to the overall risk of stroke secondary to AF.

Methods: We performed a case-control study of 70 AF patients, 46 disease control subjects and 33 healthy control subjects. Peripheral venous levels of PMP and sP-selectin were analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively.

Results: Both AF patients and disease control subjects had significantly higher levels of PMPs (p < 0.001) and sP-selectin (p = 0.001) compared to healthy control subjects, but there was no difference between AF patients and disease control subjects. There was no difference in PMP levels between patients with paroxysmal and permanent AF (p = 0.581), and between those receiving therapy with aspirin and warfarin (p = 0.779). No significant correlation was observed between PMP and sP-selectin levels (p = 0.463), and the clinical characteristics that contribute to increased stroke risk in patients with AF. On stepwise multiple regression analysis in the combined cohort of AF patients plus disease control subjects, the presence/absence of AF was not an independent determinant of PMP and sP-selectin levels.

Conclusion: There is evidence of platelet activation (ie, high PMP and sP-selectin levels) in AF patients, but this is likely to be due to underlying cardiovascular diseases rather than the arrhythmia per se.

Nonvalvular atrial fibrillation (AF) is the most common sustained cardiac rhythm disturbance encountered in clinical practice, the presence of which substantially increases the risk of stroke and thromboembolism.13 The pathophysiology of thromboembolism is multifactorial, but increasing evidence points to the fulfillment of the Virchow triad with flow abnormalities, endothelial/endocardial abnormalities, and abnormal blood constituents (ie, platelets and clotting factors), resulting in a prothrombotic or hypercoagulable state in patients with AF.4

Platelets are an important procoagulant moiety in the plasma, and by interacting with the proteins of the coagulation cascade and the vascular endothelium they render AF hypercoagulable. Evidence supporting increased platelet activation in patients with AF has been provided by numerous studies510 with conflicting results, reflecting differences between the assays and the different aspects of platelet activation measured by them. There also seems to be some variation in the degree of platelet activation, depending on the subtype of AF and the type of antithrombotic therapy.1113

Platelet microparticles (PMPs) are defined as membranous vesicles derived from activated platelets that are < 1 μm in diameter and bind to monoclonal antibodies directed against platelet antigens, such as CD61 and CD42b. PMPs have been shown to have many pathophysiologic effects, including effects on thrombosis, cell signaling, and angiogenesis.1416 Up to two thirds of the plasma tissue factor activity is carried on PMPs.17PMPs also have a role in the inhibition of fibrinolysis by expressing plasminogen activator inhibitor-1 on their surface.18Indeed, experimental evidence points to the role of PMPs in causing further platelet activation19and endothelial dysfunction,20 and in generating an inflammatory state,15 all of which feature in the pathophysiology of thrombogenesis in AF. Abnormal PMP levels have been shown in patients with coronary artery disease (CAD),2122 diabetes,23hypertension,24peripheral vascular disease,2526 and stroke,27which are conditions that are clinically associated with AF. One small study28 investigated the relationship of digoxin use to platelet expression of P-selectin (CD62P), PMPs, and endothelial microparticles in AF patients, comparing digoxin users (n = 16) and nonusers (n = 14); no significant difference in PMP levels was found, although this may be due to small numbers and thus insufficient power. Thus, the behavior of PMPs in AF patients compared to control subjects who were in sinus rhythm, their relation to the associated/underlying cardiovascular diseases and other markers of platelet activation (eg, soluble P-selectin [sP-selectin]), and their role in the generation of the prothrombotic state in AF patients has yet not been adequately investigated.

We hypothesized the following: (1) PMP levels are elevated in AF patients compared to both healthy control subjects (ie, patients without cardiovascular diseases who are in sinus rhythm) and disease control subjects (ie, patients with hypertension, CAD, diabetes, or stroke, but who are in sinus rhythm); (2) PMP levels correlate with levels of sP-selectin, which is an established marker of platelet activation; (3) PMP levels are related to the underlying factors in AF that contribute to the overall risk of stroke secondary to AF; and (4) introducing antithrombotic therapy reduces the levels of PMPs. We tested these hypotheses in a case-control study of AF patients, who were compared to disease control subjects and healthy control subjects.

Patients attending our outpatient AF clinic were invited to participate in the study. All had ECG-defined AF prior to study inclusion. Exclusion criteria for the study included refusal of consent, any history of malignancy, connective tissue disease, thyroid disease, renal disease, use of steroids, hormone replacement therapy, active smoking (or those who were smokers in the last 3 months), and any major concomitant active cardiovascular disease (eg, uncontrolled hypertension [≥ 160/100 mm Hg], left ventricular systolic dysfunction [ejection fraction < 40%], or a history of unstable angina, myocardial infarction, or stroke in the previous 3 months). Disease control subjects were recruited from patients attending our outpatient hypertension and general cardiology clinics, while healthy control subjects were recruited from hospital staff, patient relatives, or those attending the hospital preoperative clinic for minor procedures. The latter were not receiving regular therapy with medications and had no evidence of vascular, metabolic, neoplastic or inflammatory disease after a careful history and clinical examination. They were normotensive (BP < 140/85 mm Hg) and in sinus rhythm. The protocol was approved by the West Birmingham Research Ethics Committee, and all study participants gave written informed consent to the study.

To assess the effect of antithrombotic treatment on PMP levels, we identified and recruited 20 consecutive patients who were referred to our outpatient AF clinic for the initiation of appropriate antithrombotic therapy. Half of these patients (n = 10) were started on therapy with aspirin (150 mg once daily), while the remaining patients (n = 10) received anticoagulation with warfarin (international normalized ratio, 2 to 3). We also recruited another 10 AF patients whose antithrombotic treatment was changed from aspirin to warfarin (based on clinical risk stratification protocols). Paired data (baseline vs 2 months after being established on therapy) from these 30 patients were used to determine the effect of antithrombotic treatment on PMP levels in AF patients.

PMP Determination

Blood for PMP testing was collected according to the method described in the study by the European Working Group on Clinical Cell Analysis on platelet flow cytometry.29 Venous blood was drawn from an antecubital vein with minimal stasis into vacutainer tubes (BD Biosciences; Oxford, UK). Subsequent 4.5-mL aliquots of whole blood were collected into citrated tubes (containing 0.5 mL of a 3.8% sodium citrate solution) and vacutainers (Diatube-H; BD Biosciences) containing citrate as well as platelet activation inhibitors theophylline, adenosine, and dipyridamole (ie, CTAD tubes).

Within 15 min of venipuncture, blood samples collected in the CTAD tubes were centrifuged at 2,000g for 20 min to obtain platelet-poor plasma. One hundred microliters of the platelet-poor plasma was incubated for 30 min in the dark with 10 μL each of anti-CD42b-fluorescein isothiocyanate and anti-CD61PerCP (BD Biosciences). Then 600 μL of phosphate-buffered saline and 2 μL of a known concentration of red latex beads (L-2778; Sigma; Poole, UK) measuring 1.01 μm in diameter were added to the sample immediately prior to flow cytometry. PMPs were defined as particles less than the mean diameter of the latex beads and showing positive binding to both anti-CD42b and anti-CD61. The number of PMPs was calculated from the number of events in comparison to a fixed number of events from the known concentration of the latex beads. Data acquisition and analysis was performed with a dedicated flow cytometry software (CELLQuest, version 3.1; BD Biosciences) in the flow cytometer (FACScan; BD Biosciences). The interassay and intraassay coefficients of variation were < 8% and < 5%, respectively.

Enzyme-Linked Immunosorbent Assay for sP-Selectin

Blood collected in citrated tubes were centrifuged at 1,500g for 20 min. The supernatant was aspirated and stored at −70°C for batch analysis by enzyme-linked immunosorbent assay. Reagents and recombinant human P-selectin (as a standard) were obtained (R&D Systems UK Ltd; Abingdon, Oxon, UK). The interassay and intraassay coefficients of variation were < 5% and < 10%, respectively. The lower limit of sensitivity of the assay was 0.8 ng/mL.

Power Calculation

Previous data on PMPs has revealed a nonnormal distribution.22,2526 Our power calculation was based on our previous report26 of a threefold and twofold rise, respectively, of PMP levels in patients with critical limb ischemia (n = 23) and intermittent claudication (n = 36) compared to healthy control subjects (n = 30). We hypothesized an equivalent rise in AF patients (threefold) and disease control subjects (twofold) compared to healthy control subjects. We therefore recruited consecutive patients and control subjects until we had 30 individuals in each group and then exceeded it for extra confidence. We also hypothesized that PMP levels would correlate with sP-selectin levels with a correlation coefficient of ≥ 0.4, which we regard as meaningful. To achieve this at p < 0.05 and 1 − β = 0.85, 53 data points were required.30

Statistical Analysis

Continuous data were subjected to an Anderson-Darling test to determine their distribution. Normally distributed data were represented as the mean (± SD) and were analyzed by one-way analysis of variance. Nonnormally distributed data were presented as the median (first to third interquartile range) and were analyzed by the Kruskal-Wallis test. Categoric data were analyzed by the χ2 test. Differences among multiple groups were analyzed by analysis of variance and Tukey post hoc test where appropriate (data were log-transformed if nonnormally distributed). As PMPs are nonnormally distributed, all correlations were performed according to the Spearman rank correlation method. Stepwise multiple regression analysis was used to determine whether the presence/absence of AF and other clinical features were predictive of PMP and sP-selectin levels. All analyses and power calculations were performed using a statistical software package (Minitab 13; Minitab Inc; State College, PA). A probability of < 0.05 was considered to be statistically significant.

We recruited 70 patients with AF (35 with paroxysmal AF [PAF] and 35 patients with permanent AF), who were compared to 46 disease control subjects and 33 healthy control subjects. Clinical and demographic data for the three groups are presented in Table 1 . There were no significant differences in total platelet count, underlying cardiovascular comorbidities, and antiplatelet drug usage between AF patients and disease control subjects.

PMP Levels in AF Patients and Control subjects

AF patients and disease control subjects had significantly higher levels of PMPs (p < 0.001) and sP-selectin (p = 0.001) compared to healthy control subjects. There was no significant difference in PMP and sP-selectin levels between AF patients and disease control subjects (Table 1).

There was no significant difference in PMP levels between PAF and permanent AF patients (p = 0.581) [Table 2 ]. As expected, patients with permanent AF were older (mean age, 64.48 ± 6.65 vs 60.14 ± 8.98 years, respectively; p = 0.025), had higher mean diastolic BP (82.18 ± 5.94 vs 77.42 ± 10.47 mm Hg, respectively; p = 0.026), and had increased associated/underlying cardiovascular diseases (85.71% vs 60.00%, respectively; p = 0.015) compared to PAF patients. There were no significant differences in PMP levels among AF patients who were receiving therapy with aspirin and warfarin (p = 0.779) [Table 2].

Effects of Introducing Antithrombotic Therapy

Neither aspirin nor warfarin use significantly altered PMP levels in patients who were previously not receiving antithrombotic therapy (Table 3 ). Similarly, in the AF patients in whom antithrombotic treatment was changed from aspirin to warfarin, there was no significant effect on PMP levels (Table 3).

Correlations and Multivariate Analysis

PMP levels did not correlate or relate significantly with the clinical characteristics of AF patients that contribute to increased stroke risk (ie, age, hypertension, CAD, diabetes, or history of stroke). Also, no significant correlation was found between PMP and sP-selectin levels (r = 0.089; p = 0.463) in AF patients. No significant correlations were noted in disease control subjects and healthy control subjects.

Stepwise multiple regression analysis on the combined cohort of AF patients and disease control subjects revealed a significant relationship between the presence of underlying cardiovascular disease (ie, hypertension, CAD, diabetes, and stroke) and sP-selectin levels (p = 0.029). In the same combined cohort, the presence/absence of AF (yes/no) was not an independent predictor of either PMP levels (p = 0.252) or sP-selectin levels (p = 0.065).

We have demonstrated significantly higher levels of PMPs and sP-selectin in both AF patients and disease control subjects when compared to healthy control subjects, but no difference in levels of these indexes between AF patients and disease control subjects; however, the degree of overlap among the three groups is noted. Our data are consistent with the presence of platelet activation in AF patients, which is more related to associated vascular disease rather than to the arrhythmia per se. Indeed, on stepwise multiple regression analysis, the presence/absence of AF was not an independent determinant of PMP or sP-selectin levels.

The three main pathologic features of excess platelet activation (ie, abnormal thrombosis, inflammation, and angiogenesis) are also features of AF.3134 However, there is limited evidence that the platelet activation in AF relates directly to an increased thrombotic risk or whether the platelet activation is due to associated vascular disease rather than AF per se. Heppell et al35 reported an independent association between β-thromboglobulin (a marker of platelet activation) and intra-atrial thrombus on transthoracic echocardiography. However, a much larger study5 found no association between β-thromboglobulin levels and subsequent thromboembolism. In addition, plasma levels of sP-selectin were unrelated to estimated stroke risk among AF patients in the Stroke Prevention in Atrial Fibrillation III trial36 despite associations between sP-selectin levels and atherothrombotic risk factors such as smoking and peripheral vascular disease in these patients. Thus, although platelets are activated in AF patients, they may not be directly related to thrombus formation but may perhaps play a more indirect role in rendering AF hypercoagulable.

While in patients with CAD and peripheral vascular disease platelet activation seems to play a central role in the generation of thrombus (called white thrombus), in AF patients it seems that the coagulation pathway has the predominant role (called red thrombus). Indeed, the markers that have been shown to have prognostic importance in AF patients (eg, fibrinogen, fibrin d-dimer, and von Willebrand factor)3637 are directly or indirectly linked to the coagulation cascade rather than platelet activation. Unsurprisingly, the efficacy of warfarin in preventing the likelihood of strokes in AF patients is significantly superior not only to that aspirin38but also to that of the combination of aspirin and clopidogrel.39 The 22% risk reduction (of strokes) with aspirin therapy just reaches statistical significance and has been suggested to be secondary to the beneficial effect of aspirin on the underlying cardiovascular diseases (eg, hypertension, CAD, diabetes, and peripheral vascular disease) rather than on AF itself.,38

PMPs are generated from activated platelets, and, apart from causing further platelet activation by a positive feedback mechanism, they have also been shown to activate the coagulation cascade, inhibit fibrinolysis, cause endothelial dysfunction, and generate an overall inflammatory state.15,1720 Though PMP levels have been shown to be raised in patients with the various cardiovascular diseases that often lead to AF (eg, hypertension, CAD, and diabetes), the behavior of PMPs in patients with AF itself has not been investigated until now.

Of note, we found no robust correlation between PMP and sP-selectin levels, which was in contrast to our previous findings in patients with peripheral vascular disease (a condition characterized by platelet-rich thrombus), in whom PMP levels correlated not only with sP-selectin levels but also with disease severity.26 We also found no significant differences in PMP levels between patients with PAF and those with permanent AF, and that neither aspirin nor warfarin therapy had any significant effect on PMP levels. The numbers of AF patients recruited to assess the effect of the introduction of anticoagulant therapy were modest and might be underpowered. However, there were reasonable numbers of patients (and sufficient power) in our cross-sectional study, and we found no difference in PMP levels between AF patients on aspirin and warfarin. This is in contrast to previous reports40 that antiplatelet treatment (using ticlopidine) reduces PMP levels in patients with peripheral vascular disease. These observations provide further evidence of the contrasting significance of platelet activation in peripheral vascular disease and AF. As there was no significant correlation between PMP levels and the presence of AF or the clinical factors that contribute to the overall risk of stroke in AF (ie, age, hypertension, CAD, diabetes, and history of stroke), it seems unlikely that elevated PMP levels directly contribute to the increased risk of stroke in AF patients.

This study is largely limited by its cross-sectional design, although we have included a disease control group to ascertain whether platelet abnormalities in AF patients were comparable to those in patients with vascular disease. We have also included a longitudinal component to ascertain the effects of introducing antithrombotic therapy. Ideally, a population of lone AF patients should also be studied, but as AF commonly coexists with vascular disease or its risk factors, a true lone AF population (which is a diagnosis of exclusion) is difficult to define in the age group of patients we have studied. However, we have shown that AF patients do not show abnormal platelet activation beyond what was seen in our disease control population.

It has been suggested that labeling PMPs with a single antigen (eg, with anti-CD41 or anti-CD61) resulted in higher PMP levels than dual-antigen labeling (eg, with anti-CD42 and anti-CD61).41 However, there are multiple reported studies22,26,4243 on PMPs that have used dual-antigen labeling. Analysis of the protein content of PMPs has identified multiple membrane proteins including glycoprotein (GP)1bα (bound by anti-CD42b) and GPIIIa (bound by anti-CD61),44justifying the dual labeling of PMPs with anti-CD42b and anti-CD61. Furthermore, it has been suggested that PMP subpopulations might reflect platelet activation status better than the total number of PMPs.45It has also been demonstrated that activated platelet surfaces support the activation of factor XI by thrombin through their interaction with GPIbα.4648 Hence, labeling PMPs with anti-CD42b and anti-CD61 should identify the subset of PMPs involved in the propagation phase of thrombin formation, the appropriate subset in AF. Although the large majority of microparticles are platelet derived,44,49 we recognize that there may be some contribution from the endothelium (ie, endothelial derived microparticles [EMPs]).,48 As raised levels of von Willebrand factor (a marker of endothelial damage/dysfunction) have been shown to identify high-risk AF patients, it would be interesting to ascertain the levels and types of EMPs in AF patients. As the main aim of our study was to assess the levels and role of PMPs (as a marker of platelet activation) in AF patients, measuring EMP was beyond the context of our study.

In conclusion, there is evidence of platelet activation (high PMP and sP-selectin levels) in AF patients, but this is likely to be due to underlying cardiovascular diseases rather than the arrhythmia per se. This would account in part for the similarity of the relative risk reduction in stroke with antiplatelet therapy among AF patients with that seen among vascular disease patients.

Abbreviations: AF = atrial fibrillation; CAD = coronary artery disease; EMP = endothelial derived microparticle; GP = glycoprotein; PAF = paroxysmal atrial fibrillation; PMP = platelet microparticle; sP-selectin = soluble P-selectin

The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Table Graphic Jump Location
Table 1. Clinical, Demographic, Routine Laboratory Indices, and Platelet Markers in Patients With Nonvalvular AF and Control Subjects*
* 

Values are given as the mean ± SD, median (interquartile range), or No. (%), unless otherwise indicated. BMI = body mass index; SBP = systolic BP; DBP = diastolic BP; Hb = hemoglobin; Hct = hematocrit; IHD = ischemic heart disease; HT = hypertension; CVA = cerebrovascular accident; TIA = transient ischemic attack; DM = diabetes mellitus; ACEI = angiotensin-converting enzyme inhibitor; ARB = angiotensin receptor blocker; CCB = calcium channel blocker.

 

Comparisons were made by χ2 test (categoric data) or one-way analysis of variance (continuous data), with the Tukey post hoc test performed where appropriate.

 

p < 0.05 for difference between healthy control subjects and disease control subjects.

§ 

p < 0.05 for difference between healthy control subjects and AF patients.

 

p < 0.05 for difference between disease control subjects and AF patients.

Table Graphic Jump Location
Table 2. PMP Levels Among Subsets of AF Patients

*Values are given as the median (interquartile range), unless otherwise indicated.

 

Comparisons were made by Mann-Whitney test.

Table Graphic Jump Location
Table 3. Effect of Aspirin or Warfarin Therapy on Levels of PMPs in AF Patients*
* 

Values are given as the median (interquartile range), unless otherwise indicated.

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Baglia, FA, Badellino, KO, Li, CQ, et al Factor XI binding to the platelet glycoprotein Ib-IX-V complex promotes factor XI activation by thrombin.J Biol Chem2002;277,1662-1668. [PubMed]
 
Keuren, JF, Magdeleyns, EJ, Govers-Riemslag, JW, et al Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation.Br J Haematol2006;134,307-313. [PubMed]
 
LL, Horstman, YS, Ahn Platelet microparticles: a wide-angle perspective.Crit Rev Oncol Hematol1999;30,111-142. [PubMed]
 

Figures

Tables

Table Graphic Jump Location
Table 1. Clinical, Demographic, Routine Laboratory Indices, and Platelet Markers in Patients With Nonvalvular AF and Control Subjects*
* 

Values are given as the mean ± SD, median (interquartile range), or No. (%), unless otherwise indicated. BMI = body mass index; SBP = systolic BP; DBP = diastolic BP; Hb = hemoglobin; Hct = hematocrit; IHD = ischemic heart disease; HT = hypertension; CVA = cerebrovascular accident; TIA = transient ischemic attack; DM = diabetes mellitus; ACEI = angiotensin-converting enzyme inhibitor; ARB = angiotensin receptor blocker; CCB = calcium channel blocker.

 

Comparisons were made by χ2 test (categoric data) or one-way analysis of variance (continuous data), with the Tukey post hoc test performed where appropriate.

 

p < 0.05 for difference between healthy control subjects and disease control subjects.

§ 

p < 0.05 for difference between healthy control subjects and AF patients.

 

p < 0.05 for difference between disease control subjects and AF patients.

Table Graphic Jump Location
Table 2. PMP Levels Among Subsets of AF Patients

*Values are given as the median (interquartile range), unless otherwise indicated.

 

Comparisons were made by Mann-Whitney test.

Table Graphic Jump Location
Table 3. Effect of Aspirin or Warfarin Therapy on Levels of PMPs in AF Patients*
* 

Values are given as the median (interquartile range), unless otherwise indicated.

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