Altered profile of circulating microparticles in nonvalvular atrial fibrillation

Background Nonvalvular atrial fibrillation (AF) is the most common cardiac arrhythmia, and it is associated with the prothrombotic state. Circulating microparticles (cMPs) are membrane vesicles that are shed from many cell types in response to cell activation and cell apoptosis. Several studies reported that cMPs may play a role in the hypercoagulable state that can be observed in patients with AF. The aim of this study was to determine the levels of total cMPs and characterize their cellular origins in AF patients. Methods Atotal of 66 AF patients and 33 healthy controls were enrolled. This study investigated total cMP levels and their cellular origin in AF patients using polychromatic flow cytometry. Results AF patients had significantly higher levels of total cMPs (median 36.38, interquartile range [IQR] 21.16‐68.50 × 105 counts/mL vs median 15.21, IQR 9.91‐30.86 × 105 counts/mL; P = 0.004), platelet‐derived MPs (PMPs) (median 10.61, IQR 6.55‐18.04 × 105 counts/mL vs median 7.83, IQR 4.44‐10.26 × 10/mL; P = 0.009), and endothelial‐derived MPs (EMPs CD31+ CD41−) (median 2.94, IQR 1.78‐0.60 × 105 counts/mL vs median 1.16, IQR 0.71‐2.30 × 105 counts/mL; P = 0.001) than healthy controls after adjusting for potential confounders. Phosphatidylserine positive MP (PS + MP) levels were similar compared between AF patients and healthy controls. Conclusion The results of this study revealed a marked increase in total cMP levels, and evidence of elevated endothelial damage and platelet activation, as demonstrated by increased PMP and EMP levels, in AF patients. Additional study is needed to further elucidate the role of cMPs (PMPs and EMPs) in the pathophysiology of and the complications associated with AF.

corroborated by their involvement in the increased risk of thromboembolic events in various diseases, including thalassemia, 7 atherosclerosis, 8 and venous thrombosis. 9 Endothelial-derived MP (EMP) levels have been reported to impact the severity, lesion volume, and outcome of acute ischemic stroke. 10 Platelet-derived MP (PMP) levels have been proposed as a potent marker for cerebral injury in ischemic stroke. 11 Elevated levels of procoagulant MPs, like PMPs and EMPs, have been observed in persistent and permanent AF, which suggests that sustained and enhanced production of circulating procoagulant MPs may reflect a prothrombotic state with an increased risk of atrial thrombosis and thromboembolism. [12][13][14] However, no study has investigated the role of MPs in the pathophysiology of AF. Accordingly, the aim of this study was to use polychromatic flow cytometry to investigate total circulating MP (cMP) levels and their cellular origin in AF.
The results of this study may yield important clues regarding the association between cMPs and the thrombogenicity and pathophysiology of AF. Sixty-six AF patients (28 with paroxysmal AF, 7 with persistent AF, and 31 with permanent AF) and 33 healthy individuals were recruited. All subjects underwent thorough historical investigation and 12-lead electrocardiography to confirm cardiac rhythm. The exclusion criteria were refusal of provide consent, history of myeloproliferative disorders, thrombocytopenia, ischemic stroke within the previous 3 months, malignancy, rheumatic mitral valve disease, acute infectious or inflammatory disease, and pregnancy. Control subjects had no cardiovascular risk factors, no history of atrial arrhythmias, no clinical evidence of disease, and no current cardiovascular treatment. Controls also underwent electrocardiography to document sinus rhythm.

| Creatinine assessment
Serum was used to analyze creatinine levels using an automatic biochemistry analyzer (Roche Cobas 8000 modular analyzer Series, Roche Diagnostics GmbH, Mannheim, Germany).

| Circulating microparticle determination
Serum was obtained from clotted whole blood, and then centrifuged at 1500g for 15 minutes to remove all cells. We modified the method from Choudhury et al study which determined microparticle levels by flow cytometry from one centrifugation step plasma. 12 Aliquots of each centrifuged serum sample were immediately subjected to polychromatic flow cytometric analysis. Twenty-five microliters of each serum sample was incubated with appropriate monoclonal antibodies (mAbs) in 300 μL of filtered Annexin-V Binding Buffer (BD Biosciences, Franklin Lakes, New Jersey) for 15 minutes at room temperature in the dark. The staining panels of mAbs for enumeration of cMPs were modified from previous study. 15 Flow cytometric gating strategy for circulating microparticles analysis are shown in Figure S1. Data analysis was performed using FlowJo software version 10 (FlowJo, LLC, Ashland, Oregon). The absolute count, expressed as number of cMPs per mL, was obtained by dividing the number of positive cMP events or cell-derived MP events by the number of bead events, and then multiplying that figure by TC bead concentration. Red blood cells derived microparticles (RMP) were detected by CD235a or glycophorin-A.

| Statistical analysis
All data analyses were performed using PASW Statistics version 18 (SPSS, Inc., Chicago, Illinois). Kolmogorov-Smirnov test was used to evaluate the distribution of data. Since circulating MP levels were not normally distributed, comparisons between groups were performed using Mann-Whitney U test. Kruskal-Wallis test was used to compare independent samples from more than two groups. cMP levels were expressed as log-transformed counts per mL (log MPs/mL). Unpaired t test was used to compare normally distributed continuous variables.
Proportion comparisons were performed using χ 2 test. Analysis of covariance (ANCOVA) was used to adjust for potential confounders.
MP levels were transformed on a logarithmic scale before entering ANCOVA analysis. Data are presented at number and percentage or mean ± SD. All statistical tests were two-tailed, and a P-value less than 0.05 was considered statistically significant.

| Patient Characteristics
The baseline characteristics of 66 AF patients and 33 control subjects are summarized in Table 1. Significant differences were observed for age (P < 0.0001), gender (P = 0.001), and body mass index (BMI) (P < 0.0001) between AF patients and controls. Worsening of renal function was presented by increasing creatinine levels in AF patients.
Baseline characteristics of AF patients are shown in Table 2. Abbreviations: AF, atrial fibrillation; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure. Data presented as mean ± SD and 25% to75% interquartile range unless otherwise indicated. A P-value<0.05 indicates statistical significance.

| cMP levels and their cellular origins analysis
The cellular origins of cMPs in peripheral blood were identified by staining cMPs with cell-specific surface markers. PMPs were the most abundant population of cMPs in both AF patients and healthy controls. AF patients had significantly higher levels of total cMPs (P < 0.001), PMPs (P = 0.002), EMPs (CD31+ CD41−) (P < 0.001), red blood cell-derived microparticle (RMPs) (P = 0.020), and tissue factor bearing microparticle (TF-MPs) (P = 0.014) than healthy controls.
There were no significant differences in the levels of EMPs (CD144+), leukocyte-derived microparticle (LMPs), or phosphatidylserine expressing microparticle (PS + MPs) between AF patients and healthy controls (Table 3). ANCOVA was performed to examine for differences in cMPs between groups after adjusting for potential confounders. After adjusting for age, gender, and BMI, the total cMP, PMP, and EMP (CD31+ CD41−) levels were still higher in the AF group than in the control group. Comparisons of total cMP, PMP, and EMP (CD31 + CD41−) levels between AF patients and control subjects are shown in Figure 1. No significant differences were observed for the level of any type of MP among paroxysmal, persistent, and permanent AF patients.   breakage, strong caspase-3 (CASP-3) expression, and decreased antiapoptotic BCL-2 proteins. 16 The possible roles of apoptosis and cell activation in MP generation have been described in AF, possibly through low shear stress, hypoxia, stretch, inflammation, and oxidative stress in the atrium, the combination of which promotes cell apoptosis and the release of MPs. 2 We found higher EMP levels (CD31+ CD41−) in AF patients than in healthy controls. It is known that EMPs play a pivotal role in vascular homeostasis. Several stimuli, such as shear stress, inflammation, proapoptotic stimulation, and vascular damage, have been described as promoting EMP secretion. 17 EMP levels are suggested to be a potential marker of endothelial dysfunction. 18  Increased CD62E+ EMP levels are observed in AF patients after treatment by digoxin, suggesting the use of digitalis in AF is associated endothelial activation. 29 Moreover, EMP levels have been shown the potential relationship with worsening renal dysfunction in AF patients with chronic kidney disease (CKD), as presented by a significant negative correlation with estimated glomerular filtration rate (eGFR) and a positive correlation with serum creatinine. 30 High levels of PMPs were observed in AF patients with no observed differences among the different types of AF, which is consistent with previous finding. 12 That study found PMP levels to be significantly higher in both AF patients and disease controls when compared with healthy controls. However, they found no significant difference between AF patients and disease control subjects. The authors of that study suggested that high PMP levels are related to  This observation is consistent with our data; we found that no effect of warfarin and aspirin on cMP levels and all cellular origins. Nowadays, non-vitamin K antagonist oral anticoagulants (NOAC) have been approved as alternatives for warfarin in patients with nonvalvular AF.  Although the reduction of protein C and protein S reflects the high consumption of activated protein C (APC) and was associated with an increase in MP levels in some disease state, 41 the relation of reduction in APC and protein S during warfarin treatment and MP levels is still unclear. Moreover, APC has been demonstrated to induce the release of microparticle-associated EPCR. 42 Moreover, we found RMP and TF-MP levels to be significantly higher in AF patients than in controls, but these differences did not persist after adjusting for potential confounders. These results suggest that RMP and TF-MP levels are more related to age, gender, and BMI than to the AF itself. The number of RMPs was reported to correlate positively with age in women, but not in men. 43 Correlations were identified between the numbers of various TF-positive MP subpopulations and body mass index in type 2 diabetes. 44 LMPs play an important role in both proinflammatory and anti-inflammatory processes. In our study, we found LMP levels not to be significantly different between AF patients and controls, which suggesting that LMPs may not be the main effector of inflammation in AF. It remains to be determined whether the above findings are also observed if the control subjects have cardiovascular risk factors.

| CONCLUSION
This is the first report that found a marked increase in total cMP levels in AF patients. The evidence of elevated endothelial damage and platelet activation were demonstrated by increased PMP and EMP levels in AF patients. Additional study is needed to further elucidate the role of cMPs (PMPs and EMPs) in the pathophysiology and the complications associated with AF.