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CLINICAL RESEARCH: HEART RHYTHM DISORDER

Platelet Surface CD62P and CD63, Mean Platelet Volume, and Soluble/Platelet P-Selectin as Indexes of Platelet Function in Atrial Fibrillation

A Comparison of "Healthy Control Subjects" and "Disease Control Subjects" in Sinus Rhythm

Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, United Kingdom.

Manuscript received November 27, 2006; revised manuscript received January 29, 2007, accepted February 5, 2007.

^_* Reprint requests and correspondence: Prof. Gregory Y. H. Lip, University Department of Medicine, Dudley Road, City Hospital, Birmingham B18 7QH, United Kingdom. (Email: g.y.h.lip{at}bham.ac.uk).

	Abstract

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Objectives: The aim of this work was to comprehensively study the role of platelets in atrial fibrillation (AF), in relation to the underlying cardiovascular diseases and type of AF, and to analyze the effect of antithrombotic treatment on different aspects of platelet activation.

Background: Platelet activation is present in nonvalvular AF, but there is debate whether this is due to AF itself and/or to underlying cardiovascular diseases.

Methods: A total of 121 AF patients were compared with 65 "healthy control subjects" and 78 "disease control subjects" in sinus rhythm. Platelet activation was assessed using 4 different aspects of platelet pathophysiology: 1) platelet surface expression of CD62P (P-selectin) and CD63 (a lysosomal glycoprotein) (by flow cytometry); 2) mean platelet volume (MPV) (by flow cytometry); 3) plasma levels of soluble P-selectin (sP-selectin, enzyme-linked immunoadsorbent assay); and 4) total amount of P-selectin per platelet (pP-selectin) ("platelet lysis" assay).

Results: Both AF patients and "disease control subjects" had higher levels of CD62P (p < 0.001), CD63 (p < 0.001), and sP-selectin (p < 0.001) compared with "healthy control subjects," with no difference between AF patients and "disease control subjects." Patients with permanent AF had higher levels of sP-selectin (p = 0.014) and MPV (p = 0.025) compared with those with paroxysmal AF. The presence of AF independently affected the levels of CD62P expression, while "high-risk" AF patients (CHADS score 2) had higher levels of CD62P compared with those with "low risk." Introducing warfarin resulted in a reduction of pP-selectin (p = 0.013).

Conclusions: There is a degree of excess of platelet activation in AF compared with "healthy control subjects," but no significant difference between AF patients and "disease control subjects" in sinus rhythm. Platelet activation may differ according to the subtype of AF, but this is not in excess of the underlying comorbidities that lead to AF. Platelet activation in AF may be due to underlying cardiovascular diseases, rather than due to AF per se.

Abbreviations and Acronyms   AF = atrial fibrillation   CAD = coronary artery disease   ELISA = enzyme-linked immunoadsorbent assay   MPV = mean platelet volume   PAF = paroxysmal atrial fibrillation   PBS = phosphate-buffered solution   PFP = platelet-free plasma   pP-selectin = total amount of P-selectin per platelet   PRP = platelet-rich plasma   sP-selectin = soluble P-selectin   ßTG = beta-thromboglobulin

Platelets play a role in rendering AF more "prothrombotic" by interacting with the endothelium, proteins of the coagulation cascade, and inflammatory cells (e.g., monocytes) (4). On activation, the normal platelet undergoes a series of characteristic changes that include shape change, membrane budding, adhesion, aggregation, release of granular contents, and thromboxane synthesis. Methods aimed at measuring platelet activation detect these changes either directly or indirectly. For example, direct assays (e.g., electron microscopy, flow cytometry) allow platelets to be assessed in their physiological milieu of whole blood, and are extremely sensitive, with the ability to detect as few as 1% of activated platelets (5). Hence, such methods are particularly useful in monitoring rapid changes of activation status, but, in order to prevent/minimize artifactual platelet activation, blood has to be collected with minimal stasis and analyzed promptly (6). In contrast, "indirect" assays mostly measure the metabolites of activated platelets in the plasma or urine (e.g., enzyme-linked immunoadsorbent assay [ELISA]). In general, as these metabolites are modified in vivo before assay, and these assays are arguably not as sensitive in detecting dynamic changes as the "direct" assays, but at the same time are less prone to be affected by artifactual platelet activation during blood collection. Hence, they are more useful in measuring "baseline" platelet activation.

Thus, different platelet assays assess different aspects of platelet pathophysiology, and the results depend on the clinical condition being investigated and the aspect of platelet activation being measured. Unfortunately, there is no consensus on the best method of measuring platelet activation, and an "ideal" study of platelet activation should include at least 1 of each of the "direct" and "indirect" assays. Although there are numerous studies assessing platelet activation in AF, the results are (unsurprisingly) inconsistent, thus reflecting difference between the assays and the different aspects of platelet function measured (7–12).

The possibility also arises that platelet activation differs in AF depending on the type of AF and the use of antithrombotic therapy (13–15). It also remains unclear to what extent platelet activation in AF is due to AF itself and/or to the underlying cardiovascular diseases (e.g., coronary artery disease [CAD], hypertension, diabetes, and stroke).

The aim of the present study was to comprehensively study the role of platelets in AF, especially in relation to the underlying cardiovascular diseases, type of AF, and to analyze the effect of antithrombotic treatment on different aspects of platelet activation. To achieve this, AF patients were compared not only with "healthy control subjects," but also with "disease control subjects" (in sinus rhythm). Platelet activation was assessed by looking into 4 different aspects of platelet pathophysiology: 1) platelet surface expression of CD62P (P-selectin) and CD63 (lysosomal glycoprotein) (using flow cytometry); 2) mean platelet volume (MPV) (flow cytometry); 3) plasma levels of soluble P-selectin (sP-selectin) (ELISA); and 4) total amount of P-selectin per platelet (pP-selectin) (as quantified with a "platelet lysis" assay).

	Methods

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Outpatients attending our AF clinic in our hospital were invited to participate in our study. All had AF diagnosed on electrocardiogram before recruitment. 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, and any concomitant "active/acute" cardiovascular disease. "Disease control subjects" were recruited from patients attending our CAD, hypertension, and general cardiology clinics. "Healthy control subjects" were normotensive and in sinus rhythm, who were not taking regular medications and had no evidence of disease on careful history, clinical examination, and basic blood tests. To assess the effect of antithrombotic therapy, we also recruited AF patients whose antithrombotic treatment was changed from aspirin to warfarin. Paired data from these patients were used to determine the effect of antithrombotic treatment on indexes of platelet activation.

Blood collection.   Blood was collected according to the method described in the European Working Group on Clinical Analysis Consensus Protocol on platelet flow cytometry (6): 25 ml of blood was taken from an antecubital vein with minimal stasis into vacutainers tubes (BD Biosciences, Oxford, United Kingdom). Aliquots of blood were collected into citrated (containing 0.5 ml of 3.8% sodium citrate) and CTAD (containing citrate, theophylline, adenosine, and dipyridamole) vacutainers. The citrated samples were to be used for platelet lysate assay and measuring plasma levels of sP-selectin, while the CTAD samples were used for flow cytometric work.

Flow cytometry.   Blood for platelet flow cytometry was collected as described in the preceding text (6). CaliBRITE 3-color beads and FACSComp software (BD Biosciences) were used to set photomultiplier tube voltages, fluorescence compensation, spectral overlap, and sensitivity. Size-dependent forward scatter and 90° side scatter were set at logarithmic gain. Mouse IgG_2a and IgG₁ antikeyhole limpet hemocyanin monoclonal antibodies conjugated to phyco-erythrin were used as isotype-negative controls to define nonspecific binding. Monoclonal antibodies were obtained from BD Biosciences. Data acquisition and analysis were performed with CELLQuest software version 3.1 in the flow cytometer (FACScan, BD Biosciences).

Flow cytometric analysis was commenced within 15 min of blood collection, to prevent artificial elevated levels secondary to in-vitro platelet activation; 100 µl of the CTAD sample was diluted with 800 µl of phosphate-buffered solution (PBS). Two separate aliquots of 15 µl of this diluted sample were then incubated with 10 µl of anti–CD42b-FITC (binds to platelet glycoprotein 1b) and 10 µl of anti–CD62P-PE (binds to platelet membrane bound P-selectin) or anti–CD63-PE (binds to platelet lysosomal membrane glycoprotein). After incubation for 30 min, the samples were further diluted with 800 µl of PBS immediately before flow cytometric analysis. Results were expressed as the percentage of CD62P- or CD63-positive events compared with the total number of CD42b-positive events. The inter- and intra-assay coefficients of variation were <5% and <8%, respectively.

Platelet lysate.   The mass of pP-selectin per platelet (i.e., platelet P-selectin/pP-selectin) was measured using a "platelet lysate assay," as follows: platelet-rich plasma (PRP) was obtained by centrifugation of citrated blood within 15 min of collection, at 1,000 rpm for 10 min; 1 ml of the PRP was centrifuged for a further 20 min at 3,000 rpm to obtain a platelet pellet. The supernatant (platelet-free plasma [PFP]) was stored at –70°C for measurement of plasma levels of sP-selectin by ELISA.

The concentration of platelets (from the PRP) was determined using ADVIA 120 hematology system (Bayer, Newbury, United Kingdom). Accordingly, the platelet pellet was resuspended in saline to achieve a fixed concentration of 200 x 10⁶ platelets per ml, and then lysed with an equal volume of 0.1% Triton X-100 (Sigma/Aldrich, Poole, Dorset, United Kingdom). Aliquots were stored at –70°C to allow batch analysis of pP-selectin by ELISA. The inter- and intra-assay coefficients of variation were <5% and 10%, respectively.

ELISA for P-selectin.   Levels of sP-selectin (1/5 dilution of plasma) from the PFP and pP-selectin (1/10 dilution) from the lysed platelet sample were derived from standard curves using the commercial ELISA kit from R&D Systems (Abingdon, Oxon, United Kingdom). The inter- and intra-assay coefficient 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 markers of platelet activation reveal that some are normally distributed (14–17) while others have a non-normal distribution (13,15,18). We hypothesized that indexes of platelet activation will be elevated by: 1) 0.25 of a SD for indexes normally distributed and by 50% of the median values in indexes non-normally distributed, in "disease control subjects" compared with "healthy control subjects"; and by 2) 0.5 SD and 100% of the median in AF patients compared with "healthy control subjects." Using sP-selectin as the reference molecule, to achieve a difference of p < 0.05 and a power of 0.80, we required 60 recruits in each group. We therefore recruited consecutive patients and controls until we had 60 in each group and then exceeded it for extra confidence. We also hypothesized that levels of indexes of platelet activation will intercorrelate with sP-selectin, to a degree of a Spearman’s rank correlation of at least 0.4, which we consider as significant.

Data analysis and statistics.   After application of Anderson-Darling test, normally distributed data were expressed as mean (SD), while non-normally distributed data were expressed as median (interquartile range). Categorical data were analyzed by chi-square test. Differences between groups were analysed by Kruskal-Wallis test (for non-normally distributed data) or 1-way analysis of variance (for normally distributed data). For intergroup differences, Tukey’s post hoc test was used, using log-transformed data where appropriate. As all research indexes other than MPV were non-normally distributed, all correlations were according to Spearman’s rank correlation method. Stepwise multiple regression analysis on the combined cohort of AF and "disease control subjects" was performed to determine whether the presence/absence of AF and other clinical features (again, on log-transformed data where appropriate) was independently predictive of the levels of the platelet markers under investigation. A probability of 0.05 was considered statistically significant.

	Results

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We invited 150 consecutive patients with AF to participate in our study. Of these, 9 declined our invitation while another 20 could not be recruited because of our exclusion criteria. Thus, we recruited a total of 121 patients with AF (58 paroxysmal AF [PAF] and 63 permanent AF) and compared them with 78 "disease control subjects" and 65 "healthy control subjects" (Table 1). There were no significant differences in underlying cardiovascular comorbidities and antiplatelet drug usage between the AF patients and "disease control subjects."

Stepwise multiple regression analysis on the combined cohort of AF and "disease control subjects" revealed that presence of AF was independently associated with CD62P expression (p = 0.029), but not CD63 expression (p = 0.166), sP-selectin (p = 0.097), pP-selectin (p = 0.397), or MPV (p = 0.288).

	Discussion

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Our results suggest that there is excess of platelet activation in AF patients compared with "healthy control subjects" as measured by sP-selectin levels and expression of CD62P and CD63, but not in terms of total amount of pP-selectin and MPV. However, there was no excess of platelet activation (as measured by 4 different methods) in AF patients compared with "disease control subjects." This is consistent with the view that platelet activation in AF is more related to the associated vascular diseases, rather than the arrhythmia itself. On a stepwise multiple regression analysis, presence of AF did not significantly affect CD63, sP-selectin, pP-selectin, or MPV levels.

The role of platelets in atherothrombotic diseases is well established (21). However, the relative significance of platelet activation in the overall atherothrombotic risk in different cardiovascular diseases seems to vary. Although there is evidence suggesting that platelets are activated in AF, the evidence that it relates directly to the increased thrombotic risk in AF is uncertain. Initial studies had reported associations between beta-thromboglobulin (ßTG) (a marker of platelet activation) (22) and CD62P with intra-atrial thrombus and echo-contrast (23). However, a subsequent much larger study (7) found no association between plasma ßTG levels and subsequent thromboembolic events. Also, plasma levels of sP-selectin were unrelated to estimated stroke risk (14), despite associations between sP-selectin levels and atherothrombotic risk factors, such as smoking and peripheral vascular disease. Moreover, plasma markers that seem to have prognostic importance in identifying high-risk AF patients (e.g., fibrin D-dimer and von Willebrand factor) (14,24) are directly or indirectly linked to the coagulation cascade rather than platelet activation per se. Unsurprisingly, the efficacy of warfarin in preventing the likelihood of strokes in AF is significantly superior not only to aspirin (25) but also compared with the combination of aspirin and clopidogrel (26). Of note, the 22% risk reduction (of strokes) with aspirin just reaches statistical significance, and it has been suggested to be secondary to the beneficial role of aspirin on the underlying vascular disease rather than on AF itself (25).

Over the last decade, the MPV has been established as a simple and practical method of assessing platelet activation in hypertension, CAD, diabetes, and stroke (16,17,27,28), which are all diseases that frequently coexist with AF. Among patients with established cardiovascular diseases, higher levels of MPV have been shown to identify "high-risk" patients and those more likely to have poorer outcome after an acute vascular event (29–35). Until now, there have been no previous reports on the behavior of MPV in patients with nonvalvular AF, but the lack of significant difference in MPV between AF patients and "healthy control subjects" is in keeping with platelet activation playing a secondary role in rendering AF more "pro-thrombotic."

The finding of no significant differences in pP-selectin among patients and control subjects could potentially be explained by 2 reasons. First, activated platelets result in fusion of -granules with the platelet surface, followed by degranulation. This would explain higher levels of CD62P and sP-selectin in AF patients and "disease control subjects" compared with "healthy control subjects." However, this very process might result in depletion of the total amount of P-selectin in the platelets, sufficient to render the difference in pP-selectin statistically insignificant between the 3 groups. Second, it has been suggested that P-selectin, expressed on the surface of the platelet, changes its configuration upon platelet activation (36,37). Indeed, Semenov et al. (38) demonstrated that the polyclonal antibody used in their study to detect sP-selectin in platelets had a lower reactivity toward membrane P-selectin. As we have used the ELISA that detects sP-selectin, it is possible that the assay might have detected granular P-selectin, but not all of the membrane expressed P-selectin.

Our finding of higher levels of sP-selectin in patients with permanent AF compared with those with PAF is in keeping with previous reports (13). We also found higher values of MPV in permanent AF compared with PAF, but no difference between the 2 groups with regards to platelet membrane expression of CD62P and CD63, or pP-selectin levels. It has been suggested that sP-selectin reflects background chronic platelet activation, while CD62P expression is associated with acute changes (39,40). Patients with permanent AF in our study had increased associated underlying cardiovascular disease compared with those with PAF, and it is possible that the associated comorbidities might have resulted in excess of background platelet activation, as manifested by higher levels of sP-selectin and MPV in permanent AF.

Among the different platelet markers, CD62P and CD63 were strongly correlated in all the 3 groups supporting the concept that platelet activation results in migration and fusion of and lysosomal granules with the platelet membrane with subsequent expression of CD62P and CD63, respectively. The negative correlation noted between CD62P expression and pP-selectin suggests expression of CD62P (and its subsequent release in the plasma) results in depletion of pP-selectin. However, no correlation was noted between CD62P and sP-selectin. There can be 3 potential explanations for this. First, other than active cleavage or shedding of platelet membrane-bound P-selectin, there is evidence that sP-selectin may be released from the platelets by "direct" secretion (36,37) or passive diffusion (41). Moreover, some contribution from the endothelium is possible, although remote (42–44). Second, Ferroni et al. (40), in the setting of cardiopulmonary bypass, reported no correlation of CD62P and sP-selectin in view of the different time course of the rise and fall (normalization) of these molecules. Similar lack of correlation between CD62P and sP-selectin has been reported in patients presenting with acute chest pain (45) and in patients with myocardial infarction after thrombolysis (46) leading to suggestions that CD62P might reflect platelet activation in response to acute stimuli, while sP-selectin is a better marker in detecting circulating activated platelets in clinical settings where more "chronic" stimuli are present (39). Third, there is also experimental evidence to suggest that activated platelets might completely loose surface P-selectin, but continue to function and circulate (47).

We also found that AF patients whose antithrombotic therapy was changed from aspirin to warfarin resulted in reduction in pP-selectin along with nonsignificant reductions in 3 out of 4 other platelet markers. Aspirin has been previously shown to have no significant effect on sP-selectin levels (48–50), as P-selectin release from platelets is adenosine-diphosphate-dependent and independent of the cyclooxygenase pathway. There are also reports of excess platelet expression of P-selectin after stimulation by thrombin receptor-activating peptide (51). Thus, it is possible that warfarin by reducing circulating thrombin might result in reduction of overall platelet activation on AF.

Among the different platelet markers, presence of AF independently affected the levels of CD62P expression. "High-risk" AF patients had significantly higher levels of CD62P compared with the "low-risk" patients. Pongratz et al. (23) has previously reported an association between higher levels of CD62P with intra-atrial echocardiography contrast and thrombus. Whether this is a chance finding or higher levels of CD62P can truly identify AF patients with a higher stroke risk can only be ascertained in longitudinal follow-up studies.

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