Open Access

Prognostic implications and procoagulant activity of phosphatidylserine exposure of blood cells and microparticles in patients with atrial fibrillation treated with pulmonary vein isolation

  • Authors:
    • Huan Meng
    • Junjie Kou
    • Ruishuang Ma
    • Wenbo Ding
    • Yan Kou
    • Muhua Cao
    • Zengxiang Dong
    • Yayan Bi
    • Hemant S. Thatte
    • Jialan Shi
  • View Affiliations

  • Published online on: October 11, 2017     https://doi.org/10.3892/mmr.2017.7763
  • Pages:8579-8588
  • Copyright: © Meng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The present study aimed to evaluate the procoagulant effects of phosphatidylserine (PS) exposure on blood cells and microparticles (MPs), and examine its role in predicting early recurrence atrial fibrillation (ERAF) in patients with atrial fibrillation (AF) treated with pulmonary vein isolation (PVI). Blood samples were obtained from 40 healthy controls and 56 patients with AF at baseline (prior to PVI), and 0, 1 h, 1 day, 3 days and 7 days following PVI. The exposure of PS (PS+) to blood cells (platelets, erythrocytes and leukocytes) and MPs was detected using flow cytometry. The procoagulant activity was evaluated by coagulation time, and the formation of factor Xa (FXa) and thrombin. In addition, independent factors associated with PS+ blood cells and MPs, and significant predictors of ERAF following PVI were investigated by statistical analyses. The numbers of PS+ blood cells and MPs were significantly increased by PVI (P<0.01). A significant decrease in coagulation time, and increases in FXa and thrombin were exhibited in the PS+ blood cells and MPs from patients with AF treated with PVI, whereas these alterations were inhibited by either lactadherin or anti‑tissue factor (P<0.01). The maximum power of the PVI was significantly associated with platelet‑derived MPs, and high‑sensitivity C‑reactive protein (hs‑CRP) was closely associated with leukocyte‑derived MPs and endothelial‑derived MPs (EMPs) (P<0.01). In addition, hs‑CRP and EMPs >355/µl were identified as independent predictors of ERAF (P<0.05). The increased numbers of PS+ platelets, erythrocytes, leukocytes and MPs contributed to the procoagulant state of AF, and hs‑CRP and EMPs were able to predict ERAF following PVI.

Introduction

Atrial fibrillation (AF) is a common and serious type of arrhythmia, which is characterized by an irregular and rapid heartbeat (1). Clinically, pulmonary vein isolation (PVI) is a feasible, safe and effective treatment method for patients with AF; however, it is associated with the potential risk of peri-procedural stroke, despite appropriate anticoagulation (2). As a high risk factor in the treatment of AF, thrombosis can seriously affect the effects of PVI treatment on AF, and appropriated anticoagulation is always used to reduce the risk of thrombosis (3,4). It has been reported that P-selection+ platelets, platelet-derived microparticles (PMPs) and leukocyte-derived MPs (LMPs) are increased immediately following PVI and persist in the subsequent 24 h, which can significantly increase the risk of thrombosis (57). In terms of the predominant underlying mechanisms, increased thromboembolic risk during PVI may be explained by endocardium denaturation and hemodynamic alterations due to energy applied in catheterization and electrical cardioversion (810). However, associated mechanisms underlying how procoagulant activity (PCA) is affected by PVI require further investigation.

As an anionic lipid, phosphatidylserine (PS) is an important phospholipid membrane component located between the inner and cytoplasmic leaflet of the bilayer (11). Exposure of PS on the outer leaflet and microparticles (MPs) are found in the process of apoptosis (12,13). In addition, PS exposure on the outer membrane surface can function as a docking site for various coagulation proteins, including factor (F)VII, FIX, FV, FVIII, FX and prothrombin, and promote the formation of thrombin (14,15). As a primary cellular initiator of blood coagulation via interaction with coagulation FVII, tissue factor (TF) is generally quiescent unless it resides on a cell membrane containing PS (16). It has been reported that TF is involved in the generation of MPs, cell-associated PCA in diabetes mellitus and disseminated intravascular coagulation associated with severe infections (17,18). Therefore, PS and TF are considered to contribute to PCA and outcomes for patients with AF following PVI. However, whether and how PS exposure of blood cells and MPs contributes to PCA following PVI requires further investigation.

The post-ablation recurrence of AF remains a major clinical problem, occurring in 20–60% of patients during follow-up (19,20). In addition, there is a paucity of data on the correlations of early recurrence AF (ERAF) following PVI with PS+ blood cells and MPs. In the present study, the exposure of PS on blood cells (platelets, erythrocytes and leukocytes) and MPs were detected in patients with AF treated with PVI, and the PCA was evaluated by determining coagulation time, and the formation of FXa and thrombin. Subsequently, independent factors associated with PS+ blood cells and MPs, and the predictors of ERAF following PVI were investigated. The findings may reveal the mechanisms underlying the promoting effects of PS+ blood cells and MPs on PCA following PVI, and its clinical prognostic value on ERAF.

Materials and methods

Subjects

A total of 56 patients with AF, including 48 patients with paroxysmal AF and 8 with non-paroxysmal AF, who had undergone a transseptal PVI procedure were selected from the Second Hospital of Harbin Medical University (Harbin, China) between November 2013 and April 2015. AF was identified according to the Heart Rhythm Society expert consensus statement (21). An additional 40 healthy subjects were recruited from the preoperative clinic, which were included as the control group. Patients with a history of previous myocardial infarction, surgery or ablation procedure within 3 months, congenital heart disease, history of connective tissue diseases or chronic inflammatory conditions, acute/chronic infection or chronic renal/liver failure were excluded from the study. The study was approved by the local ethics committee of the Second Hospital of Harbin Medical University and performed with written informed consent from the patients.

PVI

The PVI was performed on patients with AF, as previously described (9). In brief, warfarin was first titrated to an international normalized ratio of 1.8–2.5 2 days prior to PVI by oral anticoagulation. Trans-septal puncture was guided by transesophageal echocardiography to exclude thrombi in the left atrium. Subsequently, 50 IU/kg unfractionated heparin was administered to maintain an activated clotting time between 300 and 350 sec. Ablation of the pulmonary veins was performed with a delivered power of 30–35 W and irrigation rates of 17–30 ml/min. All patients were scheduled for 24 h Holter recording at baseline (prior to PVI), and at follow-up at 1, 3 and 6 months. ERAF was defined as AF occurring at any time beyond the 3-month blanking period following ablation.

Flow cytometric analysis of PS-exposed blood cells

Peripheral vein blood samples were collected from the patients with AF prior to PVI (baseline), at the end of transseptal puncture (0 h), 1 h post-PVI, and 1, 3 and 7 days post-PVI. Platelets (300 × g for 15 min at 20°C), erythrocytes (1,500 × g for 10 min at room temperature) and leukocytes (300 × g for 5 min at room temperature) were isolated by centrifugation and Percoll density gradients (22,23). These blood cells, at a density of 1×106, were adjusted to a final volume of 45 µl in Tyrode's buffer, and then incubated with 4 nM Alex Fluor 488-lactadherin (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 15 min at room temperature in the dark (diluted with 150 µl Tyrode's buffer). Finally, the exposure of PS on the blood cells was detected using flow cytometry (FACSAria, BD Biosciences, Franklin Lakes, NJ, USA).

Flow cytometric analysis of PS-exposed MPs

The MPs were isolated from platelet-free plasma of peripheral vein blood samples by centrifugation (350 × g for 15 min at 4°C) and Tyrode's buffer (10). The phenotype of the MPs was identified, as previously described (22), and MPs were bound by lactadherin (Al ex Fluor 488-lactadherin) to evaluate the exposure of PS. In detail, platelet-derived MPs (PMPs), endothelial-derived MPs (EMPs), leukocyte-derived MPs (LMPs), erythrocyte-derived MPs (RMPs) and tissue factor-MPs (TF-MPs) were identified by lactadherin+ CD41a+, CD31+/CD41a, CD45+, CD235a+ and CD142, respectively (14,24). The numbers of various MPs were calculated using a Trucount Tube (BD Biosciences) with a precise number of fluorescent beads (48,678; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) (22).

Subcellular localization of PS by microscopy

The subcellular localizations of PS on platelets, leukocytes and erythrocytes were observed via microscopic fluorescence observation. Briefly, 50 µl suspensions of platelets, leukocytes and erythrocytes at a density of 1×106 were collected at baseline and 1 day post-PVI. These cells were then incubated with 128 nM Alex Fluor 488-lactadherin and propidium iodide (PI) for 10 min at room temperature in the dark. Following removal of the unbound dye by PBS containing 0.02% Triton X-100 (v/v), images were captured using an LSM 510 Meta confocal microscope (Carl Zeiss AG, Jena, Germany) (22).

PCA assays

The PCAs of the platelets, leukocytes, erythrocytes and MPs at baseline, and 0, 1 h, 1 day, 3 days and 7 days post-PVI were measured in 100 µl citrate plasma using a single-stage recalcification time assay with an Amelung KC4A-coagulometer (Labcon, Heppenheim, Germany) as previously described (14). The effects of 128 nM lactadherin and 40 µg/ml anti-TF on the PCAs of the platelets, erythrocytes and MPs (1 day post-ablation) and leukocytes (3 day post-ablation) were also analyzed (22).

FXa and thrombin formation assays

FXa and thrombin formation assays were performed, as previously described (22). A universal microplate spectrophotometer (PowerWave XS; Bio-Tek, Instruments, Inc., Winooski, VT, USA) was used to detect the quantities of FXa and thrombin at 405 nm. The effects of lactadherin (128 nM) and anti-TF (40 µg/ml) on blood cells and MPs were also evaluated.

Statistical analyses

Continuous variables were examined for normal distribution using a Shapiro-Wilk test. Normally distributed variables are expressed as the mean ± standard deviation and significance was analyzed using Student's t-test or repeated-measures analysis of variance. Non-normally distributed variables are expressed as the median with interquartile range and significance was analyzed using a Mann-Whitney U-test. Categorical variables are expressed as numbers (frequency) and were compared using a Χ2 test or Fischer's exact test. Stepwise multivariate linear regression analyses were used to assess independent factors associated with PS+ blood cell and MPs. Receiver operating characteristic curve analysis (ROC) was used to determine the optimum cut-off levels of PS+ blood cells and MPs in the prediction of EARF following PVI. Univariate and multivariate Cox proportional hazard analyses were performed to investigate the predictors of ERAF post-PVI. Among the above analytical methods, P<0.05 was considered to indicate a statistically different difference. All statistical analyses were performed by SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA).

Results

Clinical characteristics of patients with AF who underwent PVI

The clinical characteristics of the patients with AF who underwent PVI were evaluated. As shown in Table I, no significant differences were found in age, male/female, body mass index, diabetes mellitus, coronary artery disease, hypertension, previous stroke/transient ischemic attack, smoking, dyslipidemia, leukocyte counts, prothrombin time or D-dimer in the patients with AF, compared with the controls. However, significantly shorter activated partial thromboplastin time and higher fibrinogen were found in the patients with AF, compared with the control group. Among these patients, the levels of high-sensitivity C-reactive protein (Hs-CRP), CHA2DS2-VASc score, left ventricular ejection fraction (LVEF) and left atrial diameter (LAD) were 2.5 (1.3–5.3) mg/l, 1.2±0.3, 61.18±10.22% and 34.97±5.84 mm (23.21% >40 mm), respectively. In addition, between one and three types of antiarrhythmic medications were used by these patients, including angiotensin converting enzyme inhibitor/angiotensin receptor blockers (53.57%) and statins (35.71%).

Table I.

Clinical characteristics of patients with AF treated with pulmonary veins isolation.

Table I.

Clinical characteristics of patients with AF treated with pulmonary veins isolation.

CharacteristicControl (n=40)AF (n=56)
Age (years)54.23±7.1055.25±7.70
Male/female25/1534/22
BMI (kg/m2)23.43±3.2323.75±3.82
DM, n (%)  4 (10%)  6 (10.71%)
CAD, n (%)  3 (7.5%)5 (8.92%)
Hypertension, n (%)12 (30%)19 (33.92%)
Previous stroke/TIA, n (%)  3 (7.5%)4 (7.14%)
Smoking, n (%)  4 (10%)7 (12.5%)
Dyslipidemia, n (%)  3 (7.5%)4 (7.14%)
Leukocyte counts (103/µl)6.74±1.35   6.91±1.62
PT (sec)11.71±1.0211.62±0.93
APTT (sec)44.23±9.15   42.61±7.82a
Fibrinogen (g/l)  2.73 [1.73–4.24]3.43 [2.13–5.31]a
D-dimer (ng/ml)98 [45–172]110 [32–145]

{ label (or @symbol) needed for fn[@id='tfn1-mmr-16-06-8579'] } Data are presented as the mean ± standard deviation, number (%) and mean [range].

a P<0.05, compared with the control. AF, atrial fibrillation; BMI, body mass index; DM, diabetes mellitus; CAD, coronary artery disease; TIA, transient ischemic attack; PT, prothrombin time; APTT, activated partial thromboplasting time.

In the treatment of AF, segmental PVI (5.38%), circumferential pulmonary vein ablation (48.39%), left atrium linear ablation (16.13%), right atrium linear ablation (20.43%) and electrogram-based ablation (9.68%) were performed. During these procedures, the total ablation time was 86.20±39.01 min and the maximum power was 28.82±6.71 W. Following ablation, the curative rate was 74.73% and ERAF was detected in 42.65% patients at the 3 month follow-up. Late recurrence of AF occurred in 15.27% patients, whereas 62.5% patients were confirmed to have delayed curing at subsequent follow-up.

PS exposure of blood cells

The platelets, leukocytes and erythrocytes exposed to PS were detected by lactadherin binding using flow cytometry. At baseline, PS+ platelets, leukocytes and erythrocytes, and TF+ leukocytes were significantly higher in patients with AF, compared with those in the control (P<0.01), whereas no significant differences were revealed in the patients with paroxysmal and non-paroxysmal AF (data not shown). Following treatment with PVI, the number of PS+ platelets increased with time until a peak at 1 day (17.53±5.23×109/l; P<0.01), as shown in Fig. 1A. PS+ erythrocytes and leukocytes were increased with a peak at 1 h (26.23±5.31×109/l; P<0.001) and 3 days (6.63±2.90×108/l; P<0.01), respectively (Fig. 1B and C). TF+ leukocytes were also increased with time, which peaked at 1 h (7.23±3.20×108/l; P<0.01), as shown in Fig. 1D.

To further identify the presence of PS on platelets, leukocytes and erythrocytes in the patients with AF following PVI, the subcellular localizations of PS on these cells were observed. No fluorescence was observed on the membranes of platelets, leukocytes or erythrocytes in the patients with AF at baseline, whereas fluorescence was observed in the platelets, leukocytes and erythrocytes from the patients with AF at 1 day post-PVI treatment (Fig. 1E-H).

Numbers of MPs

The effects of PVI on the numbers of MPs were evaluated. As shown in Table II, the PS+ MPs predominantly originated from platelets, leukocytes and endothelial cells. The numbers of total MPs, PMPs, LMPs, EMPs and TF-MPs were all significantly higher in the patients with AF, compared with those in the control (P<0.01), whereas no significant differences were found in the RMPs. No significant differences were found in the patients with paroxysmal and non-paroxysmal AF (data not shown). Following PVI, all types of MPs, with the exception of TF-MPs (peak at 3 days) were significantly elevated with a peak at 1 day, followed by a decrease with intervention time (P<0.01; Table II).

Table II.

Numbers of PS+ MPs in patients with AF treated with pulmonary veins isolation.

Table II.

Numbers of PS+ MPs in patients with AF treated with pulmonary veins isolation.

Patients with AF (n=56)

PS+ (/µl)Control (n=40)Baseline0 h1 h1 day3 days7 days
Total MPs 1,560±143 1,800±276b 1,999±289a 2.399±407a 3,201±552a 2,998±473a 2,300±398a
PMPs 835±170 1,143±245b 1,276±247a 1,528±279a 2,023±376a 1,846±287a 1,485±268a
LMPs 205±43 266±52b 283±6a 514±124a 671±189a 488±101a 411±98a
EMPs 136±23 174±34b 226±56a 302±74a 367±99a 340±86a 277±67a
RMPs 29±19 31±19 45±25a 54±33a 72±37a 67±35 60±29a
TF-MPs 33±12 43±13b 54±14a 68±22a 84±32a 104±78a 66±27a

{ label (or @symbol) needed for fn[@id='tfn3-mmr-16-06-8579'] } Data are presented as the mean ± standard deviation.

a P<0.01, compared with different time points

b P<0.01, compared with the control. PS, phosphatidylserine; MPs, microparticles; AF, atrial fibrillation; PMPs, platelets-MPs; LMPs, leukocyte-MPs; EMPs, endothelial-MPs; RMPs, erythrocyte-MPs; TF-MPs, tissue factor-MPs.

PCAs of PS+ blood cells and MPs

In order to reveal the PCA of PS in the patients with AF, the coagulation time of PS+ platelets, leukocytes, erythrocytes and MPs were evaluated. The coagulation times of these factors were significantly shortened by PVI, compared with those at baseline (P<0.01). The shortest coagulation time of the PS+ platelets, erythrocytes and MPs was observed at 1 day post-PVI, and at 3 days post-PVI for leukocytes (Fig. 2A). The effects of lactadherin and anti-TF on the coagulation times of these factors were also evaluated. As shown in Fig. 2B and C, the coagulation times of PS+ platelets, leukocytes, erythrocytes and MPs were all prolonged by lactadherin (P<0.01), which was close to baseline levels. Anti-TF also inhibited the coagulation times, although the inhibitory efficacy was lower, compared with that of lactadherin (P<0.01).

The formation of FXa and thrombin in PS+ platelets, leukocytes, erythrocytes and MPs were found to reveal alterations in PCA. The production of intrinsic and extrinsic FXa and thrombin in the PS+ platelets, leukocytes, erythrocytes and MPs were significantly increased and then reduced with intervention time post-PVI (P<0.01). At peak concentrations, the production of FXa and thrombin were 2–3-fold higher than at baseline (Fig. 3A-C). However, the contents of FXa and thrombin in platelets, leukocytes, erythrocytes and MPs were significantly inhibited by lactadherin (Fig. 3D-F). Anti-TF also inhibited the activity of extrinsic FXa in leukocytes and MPs (Fig. 3E).

Associations between the clinical characteristics of patients with AF and the numbers of PS+ blood cells/MPs

The associations between the numbers of PS+ blood cell/MPs and clinical characteristics of AF were analyzed using multiple linear regression analysis. As shown in Table III, the maximum power was significantly associated with PMPs (β=0.101; P=0.0009), and Hs-CRP was as a significant predictor of LMPs (β=0.325; P=0.038) and EMPs (β=0.114; P=0.004).

Table III.

Multiple linear regression analysis of the associations between the number of PS+ blood cell/MPs and clinical characteristics of patients with AF.

Table III.

Multiple linear regression analysis of the associations between the number of PS+ blood cell/MPs and clinical characteristics of patients with AF.

VariableCoefficientPLTLEUERYPMPsLMPsRMPsEMPs
Age β-coefficienta0.0390.2530.1570.0440.266−0.0270.001
P-value0.8770.2330.5810.7510.3760.9170.221
Maleβ-coefficient0.3540.2760.006−0.018−0.2810.028−0.047
P-value0.1260.7120.4780.8990.1050.8560.727
DMβ-coefficient0.063−0.091−0.105−0.0280.000−0.087−0.071
P-value0.6210.4820.3600.8430.9880.5440.567
CADβ-coefficient0.0200.1420.0100.047−0.0290.0090.152
P-value0.8810.2920.8940.2900.8310.1430.239
Non-paroxysmal AFβ-coefficient−0.063−0.128−0.0280.021−0.087−0.1520.189
P-value0.6210.3360.1430.1980.5540.2390.155
CHA2DS2-VASc ≥2β-coefficient0.0010.167−0.042−0.0360.374−0.276−0.035
P-value0.2460.8680.1620.2160.3210.4270.793
Hs-CRPβ-coefficient−0.1260.1150.103−0.1720.325−0.1900.114
P-value0.4160.4560.4280.4570.038b0.1690.004b
Total ablation timeβ-coefficient0.1400.0270.020−0.207−0.189−0.0600.611
P-value0.2930.8500.8930.8370.1550.6630.167
Maximum powerβ-coefficient0.1030.158−0.0390.101−0.0140.0960.003
P-value0.4280.2270.7540.009b0.9020.4950.982
Baseline PS+ PLTβ-coefficient0.142
P-value0.292
Baseline PS+ LEUβ-coefficient0.128
P-value0.322
Baseline PS+ ERYβ-coefficient−0.006
P-value0.968
Baseline PS+PMPsβ-coefficient0.178
P-value0.130
Baseline PS+LMPsβ-coefficient−0.195
P-value0.183
Baseline PS+RMPsβ-coefficient0.036
P-value0.812
Baseline PS+EMPsβ-coefficient0.206
P-value0.108

a Numbers are standardized β-coefficients.

b P<0.05. AF, atrial fibrillation; PS, phosphatidylserine; PLT, platelets; LEU, leukocytes; ERY, erythrocytes; MPs, microparticles; PMPs, platelets-MPs; LMPs, leukocyte-MPs; EMPs, endothelial-MPs; RMPs, erythrocyte-MPs; DM, diabetes mellitus; CAD, coronary artery disease; Hs-CRP, high sensitivity C-reactive protein.

Prediction of ERAF by PS+ blood cells and MPs

The ROC was used to identify the role of PS+ blood cells and MPs in the prediction of ERAF at 1 day post-PVI treatment. As shown in Fig. 4A and B, the area under the curve (AUC) was >0.7 for PS+ leukocytes, LMPs and EMPs. The optimal cut-off values for the PS+ leukocytes, LMPs and EMPs were 568, 639 and 355/µl, respectively (Fig. 4A-C). In the prediction of ERAF, a sensitivity of 76.92% and specificity of 75% were exhibited for the PS+ leukocytes (AUC, 0.760; 95% CI, 0.587–0.932; P=0.001). Sensitivities of 69.23 and 92.31%, and specificities of 80 and 75% were exhibited for the LMPs (AUC, 0.792; 95% CI, 0.628–0.957; P<0.001) and EMPs (AUC, 0.835; 95% CI, 0.686–0.983; P<0.001), respectively.

The prognostic factors for ERAF were further analyzed using univariate and multivariate analyses. The results showed that hypertension (HR 1.96; 95% CI 0.75–5.14; P=0.047), non-paroxysmal AF (HR 2.35; 95% CI, 1.08–5.11; P=0.031), hs-CRP (HR 5.47; 95% CI, 3.26–9.20; P=0.0001), PS+ leukocytes >568/µl (HR 2.17; 95% CI, 1.06–4.41; P=0.033), LMPs >639/µl (HR 1.29; 95% CI, 1.10–1.53; P=0.002) and EMPs >355/µl (HR 4.28; 95% CI, 2.03–9.03; P=0.0001) were risk factors for ERAF in the multivariate model. Furthermore, hs-CRP (HR 4.89; 95% CI, 2.37–6.28; P=0.0001), non-paroxysmal AF (HR 1.64; 95% CI, 1.03–2.61; P=0.03) and EMPs >355/µl (HR 4.92; 95% CI, 2.25–10.74; P=0.0001) were identified as independent predictors of ERAF (Table IV).

Table IV.

Univariate and multivariate analyses of predictors of early AF recurrence in patients with AF treated with pulmonary veins isolation.

Table IV.

Univariate and multivariate analyses of predictors of early AF recurrence in patients with AF treated with pulmonary veins isolation.

Univariate analysisMultivariate analysis


VariableHazard ratio (95% confidence interval)P-valueHazard ratio (95% confidence interval)P-value
Age0.99 (0.96–1.02)0.464
Male1.25 (0.63–2.51)0.526
History
  Hypertension1.96 (0.75–5.14)0.047a0.98 (0.96–1.01)0.167
  CAD0.63 (0.26–1.90)0.461
  Non-paroxysmal AF2.35 (1.08–5.11)0.031a1.64 (1.03–2.61)   0.030a
  CHA2DS2-VASc ≥21.00 (1.00–1.01)0.255
Transthoracic echocardiography
  LAD >40 mm1.04 (0.98–1.11)0.156
  LVEF1.62 (0.83–3.17)0.161
Laboratory data
  Hs-CRP5.47 (3.26–9.20) <0.001a4.89 (2.37–6.28) <0.001a
  PS+ leukocytes >568/µl2.17 (1.06–4.41)0.033a0.84 (0.32–2.21)   0.721
  PS+ LMPs >639/µl1.29 (1.10–1.53)0.002a  3.06 (0.62–14.98)   0.168
  PS+ EMPs >355/µl4.28 (2.03–9.03) <0.001a  4.92 (2.25–10.74) <0.001a

a P<0.05. AF, atrial fibrillation; CAD, coronary artery disease; LAD, left atrial diameter; LVEF, left ventricular ejection fraction; Hs-CRP, high sensitivity C-reactive protein; PS+, phosphatidylserine exposed; LMPs, leukocyte microparticles; EMPs, endothelial microparticles.

Discussion

PS is known to be important in the process of coagulation, and thrombosis is a risk factor in the treatment of AF by ablation (25,26). It has been reported that PS+ MPs exhibit marked procoagulant effects in patients with AF following PVI (27). Similarly, the PCA of MPs in the patients with AF treated with PVI were significantly promoted by PS in the present study, which exhibited decreased coagulation time, and increased levels of FXa and thrombin. The present study is the first, to the best of our knowledge, to evaluate the procoagulant role of PS on blood cells. The results showed that PS exposure significantly reduced the coagulation time, and increased the production of FXa and thrombin in the blood cells of patients with AF treated with PVI. In addition, 80% of the PCA of the blood was inhibited by the intervention of lactadherin. These findings demonstrated that exposure of blood cells to PS increased the risk of thrombosis, and this effect may be caused by an additional procoagulant phospholipid surface for the assembly of thrombase complexes and thrombin generation in the circulation (28). In the clinical treatment of AF by PVI, the inhibition of PS+ blood cells and MPs may bean an effective method in the prevention of hypercoagulable states. TF on blood cells and MPs has also been suggested to be associated with increased PCA during PVI (29). In the present study, TF+ leukocytes were significantly increased at 1 h post-PVI, and anti-TF treatment significantly prolonged the coagulation time of the blood cells and MPs. This result indicated that thermally injured leukocytes induced by ablation increased the expression of TF (30). TF was also be activated by PS residing on cell membranes and promoted the coagulation reaction (31).

Clinically, higher levels of MPs are found in patients with AF, and ablation is considered to affect the distribution of MPs (32). It has been reported that GPIb+ PMPs and CD11a+ LMPs are increased in patients with AF within 48 h following radiofrequency ablation or cryoablation (6). Consistent with previous findings, the present study showed that the numbers of all types of MPs were significantly increased by PVI in the patients with AF, which further indicated the activation of apoptosis in blood cells. However, associated studies on the associations between elevated PS+ MPs and clinical characteristics of patients with AF during PVI are limited. In the present study, the maximum power of PVI was an independent predictor of PS+ PMPs, which indicated the role of PMPs on the extent of tissue damage. In addition, the level of hs-CRP was correlated with PS+ LMPs and EMPs independently. This correlation suggested that the procoagulant state was associated with inflammation (33), potentially due to the activation of associated complements. For example, the activation of cyclosporine 3 has been shown to induce MP shedding (34). However, non-paroxysmal AF and CHA2DS2-VASc scores ≥2, which are particularly sensitive to disease severity in AF, were not found to be associated with MPs.

Although PVI is regarded as an effective and safe therapeutic option for patients with symptomatic and drug-refractory AF, ERAF occurs during follow-up (19). In the present study, PS+ EMPs >355/µl was revealed to be a significant predictor of ERAF. This result supports the hypothesis that transient inflammation and procoagulant state following PVI-induced tissue damage contribute to the occurrences of ERAF (35). The preoperative hs-CRP level was also found to be an independent predictor of ERAF within 3 months following PVI. A high hs-CRP level has been associated with abnormal left atrial substrate and a high incidence of nonpulmonary vein AF sources, which contribute to the recurrence of AF (36), and the preoperative hs-CRP level has been associated with ERAF within 3 days following catheter ablation in patients with paroxysmal or persistent AF (37). The results of the present were consistent with previous studies, further demonstrating the predictive value of hs-CRP on ERAF during follow-up, and its association with inflammation and procoagulant activity. Therefore, the high prognostic values of hs-CRP and PS+ EMPs in ERAF were considered to be beneficial to the determination of AF in those not benefiting from PVI.

In conclusion, the present study demonstrated significant procoagulant effects exhibited by PS+ blood cells and MPs in patients with AF treated with PVI. Hs-CRP and EMPs >355/µl were significant prognostic factors of ERAF during the follow-up period in patients with AF treated with PVI. However, limitations of the study included insufficient subjects, whereas bolus heparin and continuous aspirin treatment in patients may underestimate the activation of the coagulating cascade post-PVI. Therefore, further investigations on the procoagulant effect and long-term prognostic implications of PS+ blood cells and MPs in AF are required.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (grant nos. 81270588 and 81470301), the Natural Science Foundation of Heilongjiang Province (grant no. ZD2015020) and the Graduate Innovation Fund of Harbin Medical University (grant no. YJSCX2014-41HYD). The authors would like to thank Professor Bo Yu, Professor Jie Yuan, Professor Yong Sun, Ms. Xueqin Gao, Mr. Qinlong Tao and Ms. Na Han from the Department of Cardiology, The First Hospital, Harbin Medical University (Harbin, China) for sample collection.

References

1 

Lip GY and Lane DA: Stroke prevention in atrial fibrillation: A systematic review. JAMA. 313:1950–1962. 2015. View Article : Google Scholar : PubMed/NCBI

2 

Calkins H, Kuck KH, Cappato R, Brugada J, Camm AJ, Chen SA, Crijns HJ, Damiano RJ Jr, Davies DW, DiMarco J, et al: 2012 HRS/EHRA/ECAS expert consensus statement on catheter and surgical ablation of atrial fibrillation: Recommendations for patient selection, procedural techniques, patient management and follow-up, definitions, endpoints, and research trial design. J Interv Card Electrophysiol. 33:171–257. 2012. View Article : Google Scholar : PubMed/NCBI

3 

Noel P, Gregoire F, Capon A and Lehert P: Atrial fibrillation as a risk factor for deep venous thrombosis and pulmonary emboli in stroke patients. Stroke. 22:760–762. 1991. View Article : Google Scholar : PubMed/NCBI

4 

Kiedrowicz RM, Kazmierczak J and Wielusinkski M: Left atrial massive thrombus formation on the transseptal sheath despite adequate anticoagulation with warfarin and heparin during pulmonary vein isolation. J Cardiovasc Electrophysiol. 24:11852013.PubMed/NCBI

5 

Lim HS, Schultz C, Dang J, Alasady M, Lau DH, Brooks AG, Wong CX, Roberts-Thomson KC, Young GD, Worthley MI, et al: Time course of inflammation, myocardial injury, and prothrombotic response after radiofrequency catheter ablation for atrial fibrillation. Circ Arrhythm Electrophysiol. 7:83–89. 2014. View Article : Google Scholar : PubMed/NCBI

6 

Siklódy C Herrera, Arentz T, Minners J, Jesel L, Stratz C, Valina CM, Weber R, Kalusche D, Toti F, Morel O and Trenk D: Cellular damage, platelet activation, and inflammatory response after pulmonary vein isolation: A randomized study comparing radiofrequency ablation with cryoablation. Heart Rhythm. 9:189–196. 2012. View Article : Google Scholar : PubMed/NCBI

7 

Stazi A, Scalone G, Laurito M, Milo M, Pelargonio G, Narducci ML, Parrinello R, Figliozzi S, Bencardino G, Perna F, et al: Effect of remote ischemic preconditioning on platelet activation and reactivity induced by ablation for atrial fibrillation. Circulation. 129:11–17. 2014. View Article : Google Scholar : PubMed/NCBI

8 

Haeusler KG, Kirchhof P and Endres M: Left atrial catheter ablation and ischemic stroke. Stroke. 43:265–270. 2012. View Article : Google Scholar : PubMed/NCBI

9 

Gaita F, Caponi D, Pianelli M, Scaglione M, Toso E, Cesarani F, Boffano C, Gandini G, Valentini MC, De Ponti R, et al: Radiofrequency catheter ablation of atrial fibrillation: A cause of silent thromboembolism? Magnetic resonance imaging assessment of cerebral thromboembolism in patients undergoing ablation of atrial fibrillation. Circulation. 122:1667–1673. 2010. View Article : Google Scholar : PubMed/NCBI

10 

Bulava A, Slavik L, Fiala M, Heinc P, Skvarilova M, Lukl J, Krcová V and Indrák K: Endothelial damage and activation of the hemostatic system during radiofrequency catheter isolation of pulmonary veins. J Interv Card Electrophysiol. 10:271–279. 2004. View Article : Google Scholar : PubMed/NCBI

11 

Leventis PA and Grinstein S: The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys. 39:407–427. 2010. View Article : Google Scholar : PubMed/NCBI

12 

Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A and Grinstein S: Membrane phosphatidylserine regulates surface charge and protein localization. Science. 319:210–213. 2008. View Article : Google Scholar : PubMed/NCBI

13 

Rysavy NM, Shimoda LM, Dixon AM, Speck M, Stokes AJ, Turner H and Umemoto EY: Beyond apoptosis: The mechanism and function of phosphatidylserine asymmetry in the membrane of activating mast cells. Bioarchitecture. 4:127–137. 2014.PubMed/NCBI

14 

Tan X, Shi J, Fu Y, Gao C, Yang X, Li J, Wang W, Hou J, Li H and Zhou J: Role of erythrocytes and platelets in the hypercoagulable status in polycythemia vera through phosphatidylserine exposure and microparticle generation. Thromb Haemost. 109:1025–1032. 2013. View Article : Google Scholar : PubMed/NCBI

15 

Tormoen GW, Recht O, Gruber A, Levine RL and McCarty OJ: Phosphatidylserine index as a marker of the procoagulant phenotype of acute myelogenous leukemia cells. Phys Biol. 10:0560102013. View Article : Google Scholar : PubMed/NCBI

16 

Chen VM and Hogg PJ: Encryption and decryption of tissue factor. J Thromb Haemost. 11 Suppl 1:S277–S284. 2013. View Article : Google Scholar

17 

Diamant M, Nieuwland R, Pablo RF, Sturk A, Smit JW and Radder JK: Elevated numbers of tissue-factor exposing microparticles correlate with components of the metabolic syndrome in uncomplicated type 2 diabetes mellitus. Circulation. 106:2442–2447. 2002. View Article : Google Scholar : PubMed/NCBI

18 

Geisbert TW, Young HA, Jahrling PB, Davis KJ, Kagan E and Hensley LE: Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: Overexpression of tissue factor in primate monocytes/macrophages is a key event. J Infect Dis. 188:1618–1629. 2003. View Article : Google Scholar : PubMed/NCBI

19 

Joshi S, Choi AD, Kamath GS, Raiszadeh F, Marrero D, Badheka A, Mittal S and Steinberg JS: Prevalence, predictors, and prognosis of atrial fibrillation early after pulmonary vein isolation: Findings from 3 months of continuous automatic ECG loop recordings. J Cardiovasc Electrophysiol. 20:1089–1094. 2009. View Article : Google Scholar : PubMed/NCBI

20 

Takahashi Y, Takahashi A, Kuwahara T, Fujino T, Okubo K, Kusa S, Fujii A, Yagishita A, Miyazaki S, Nozato T, et al: Clinical characteristics of patients with persistent atrial fibrillation successfully treated by left atrial ablation. Circ Arrhythm Electrophysiol. 3:465–471. 2010. View Article : Google Scholar : PubMed/NCBI

21 

Calkins H, Kuck KH, Cappato R, Brugada J, Camm AJ, Chen SA, Crijns HJ, Damiano RJ Jr, Davies DW, DiMarco J, et al: 2012 HRS/EHRA/ECAS expert consensus statement on catheter and surgical ablation of atrial fibrillation: Recommendations for patient selection, procedural techniques, patient management and follow-up, definitions, endpoints, and research trial design: A report of the heart rhythm society (HRS) task force on catheter and surgical ablation of atrial fibrillation. Developed in partnership with the European heart rhythm association (EHRA), a registered branch of the European society of cardiology (ESC) and the European cardiac arrhythmia society (ECAS); and in collaboration with the American college of cardiology (ACC), American heart association (AHA), the Asia pacific heart rhythm society (APHRS) and the society of thoracic surgeons (STS). Endorsed by the governing bodies of the American college of cardiology foundation, the American heart association, the European cardiac arrhythmia society, the European heart rhythm association, the society of thoracic surgeons, the Asia pacific heart rhythm society and the heart rhythm society. Heart Rhythm. 9:632–696.e21. 2012. View Article : Google Scholar : PubMed/NCBI

22 

Gao C, Xie R, Yu C, Wang Q, Shi F, Yao C, Xie R, Zhou J, Gilbert GE and Shi J: Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome. Thromb Haemost. 107:681–689. 2012. View Article : Google Scholar : PubMed/NCBI

23 

Gao C, Xie R, Li W, Zhou J, Liu S, Cao F, Liu Y, Ma R, Si Y, Liu Y, et al: Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity. Thromb Haemost. 109:1079–1090. 2013. View Article : Google Scholar : PubMed/NCBI

24 

van Ierssel SH, van Craenenbroeck EM, Conraads VM, van Tendeloo VF, Vrints CJ, Jorens PG and Hoymans VY: Flow cytometric detection of endothelial microparticles (EMP): Effects of centrifugation and storage alter with the phenotype studied. Thromb Res. 125:332–339. 2010. View Article : Google Scholar : PubMed/NCBI

25 

Spronk HM, ten Cate H and van der Meijden PE: Differential roles of tissue factor and phosphatidylserine in activation of coagulation. Thromb Res. 133 Suppl 1:S54-S562014. View Article : Google Scholar

26 

Kakkar AK, Mueller I, Bassand JP, Fitzmaurice DA, Goldhaber SZ, Goto S, Haas S, Hacke W, Lip GY, Mantovani LG, et al: Risk profiles and antithrombotic treatment of patients newly diagnosed with atrial fibrillation at risk of stroke: Perspectives from the international, observational, prospective GARFIELD registry. PLoS One. 8:e634792013. View Article : Google Scholar : PubMed/NCBI

27 

Jesel L, Morel O, Pynn S, Radulescu B, Grunebaum L, Freyssinet JM, Ohlmann P, Bareiss P, Toti F and Chauvin M: Radiofrequency catheter ablation of atrial flutter induces the release of platelet and leukocyte-derived procoagulant microparticles and a prothrombotic state. Pacing Clin Electrophysiol. 32:193–200. 2009. View Article : Google Scholar : PubMed/NCBI

28 

Puddu P, Puddu GM, Cravero E, Muscari S and Muscari A: The involvement of circulating microparticles in inflammation, coagulation and cardiovascular diseases. Can J Cardiol. 26:140–145. 2010. View Article : Google Scholar : PubMed/NCBI

29 

Lwaleed BA, Breish MO, Birch BR, Chowdhary AP, Saad RA, Perigo O, Kazmi RS, Dusse LM and Cooper AJ: Tissue factor and tissue factor pathway inhibitor in women with a past history of preeclampsia: Implication for a hypercoagulable state postpregnancy. Blood Coagul Fibrinolysis. 25:671–674. 2014. View Article : Google Scholar : PubMed/NCBI

30 

Rao LV and Pendurthi UR: Regulation of tissue factor coagulant activity on cell surfaces. J Thromb Haemost. 10:2242–2253. 2012. View Article : Google Scholar : PubMed/NCBI

31 

Bach RR: Tissue factor encryption. Arterioscler Thromb Vasc Biol. 26:456–461. 2006. View Article : Google Scholar : PubMed/NCBI

32 

Jesel L, Abbas M, Toti F, Cohen A, Arentz T and Morel O: Microparticles in atrial fibrillation: A link between cell activation or apoptosis, tissue remodelling and thrombogenicity. Int J Cardiol. 168:660–669. 2013. View Article : Google Scholar : PubMed/NCBI

33 

Kallergis EM, Manios EG, Kanoupakis EM, Mavrakis HE, Kolyvaki SG, Lyrarakis GM, Chlouverakis GI and Vardas PE: The role of the post-cardioversion time course of hs-CRP levels in clarifying the relationship between inflammation and persistence of atrial fibrillation. Heart. 94:200–204. 2008. View Article : Google Scholar : PubMed/NCBI

34 

Renner B, Klawitter J, Goldberg R, McCullough JW, Ferreira VP, Cooper JE, Christians U and Thurman JM: Cyclosporine induces endothelial cell release of complement-activating microparticles. J Am Soc Nephrol. 24:1849–1862. 2013. View Article : Google Scholar : PubMed/NCBI

35 

Grubman E, Pavri BB, Lyle S, Reynolds C, Denofrio D and Kocovic DZ: Histopathologic effects of radiofrequency catheter ablation in previously infarcted human myocardium. J Cardiovasc Electrophysiol. 10:336–342. 1999. View Article : Google Scholar : PubMed/NCBI

36 

Lin YJ, Tsao HM, Chang SL, Lo LW, Tuan TC, Hu YF, Udyavar AR, Tsai WC, Chang CJ, Tai CT, et al: Prognostic implications of the high-sensitive C-reactive protein in the catheter ablation of atrial fibrillation. Am J Cardiol. 105:495–501. 2010. View Article : Google Scholar : PubMed/NCBI

37 

Koyama T, Sekiguchi Y, Tada H, Arimoto T, Yamasaki H, Kuroki K, Machino T, Tajiri K, Zhu XD, Kanemoto M, et al: Comparison of characteristics and significance of immediate versus early versus no recurrence of atrial fibrillation after catheter ablation. Am J Cardiol. 103:1249–1254. 2009. View Article : Google Scholar : PubMed/NCBI

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December 2017
Volume 16 Issue 6

Print ISSN: 1791-2997
Online ISSN:1791-3004

2016 Impact Factor: 1.692
Ranked #19/128 Medicine Research and Experimental
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APA
Meng, H., Kou, J., Ma, R., Ding, W., Kou, Y., Cao, M. ... Shi, J. (2017). Prognostic implications and procoagulant activity of phosphatidylserine exposure of blood cells and microparticles in patients with atrial fibrillation treated with pulmonary vein isolation. Molecular Medicine Reports, 16, 8579-8588. https://doi.org/10.3892/mmr.2017.7763
MLA
Meng, H., Kou, J., Ma, R., Ding, W., Kou, Y., Cao, M., Dong, Z., Bi, Y., Thatte, H. S., Shi, J."Prognostic implications and procoagulant activity of phosphatidylserine exposure of blood cells and microparticles in patients with atrial fibrillation treated with pulmonary vein isolation". Molecular Medicine Reports 16.6 (2017): 8579-8588.
Chicago
Meng, H., Kou, J., Ma, R., Ding, W., Kou, Y., Cao, M., Dong, Z., Bi, Y., Thatte, H. S., Shi, J."Prognostic implications and procoagulant activity of phosphatidylserine exposure of blood cells and microparticles in patients with atrial fibrillation treated with pulmonary vein isolation". Molecular Medicine Reports 16, no. 6 (2017): 8579-8588. https://doi.org/10.3892/mmr.2017.7763