Platelet reactivity and mean platelet volume as risk markers of thrombogenesis in atrial fibrillation

    Platelet reactivity and mean platelet volume as risk markers of thrombogenesis in atrial fibrillation Marcin Makowski, Ireneusz Smorag, Joanna Makowska, Andrzej Bissinger, Tomasz Grycewicz, Jarek Pa´snik, Michal Kidawa, Andrzej Lubi´nski, Marzenna Zieli´nska, Zbigniew Baj PII: DOI: Reference:

S0167-5273(17)31518-8 doi:10.1016/j.ijcard.2017.03.023 IJCA 24702

To appear in:

International Journal of Cardiology

Received date: Revised date: Accepted date:

3 July 2016 2 March 2017 8 March 2017

Please cite this article as: Makowski Marcin, Smorag Ireneusz, Makowska Joanna, Bissinger Andrzej, Grycewicz Tomasz, Pa´snik Jarek, Kidawa Michal, Lubi´ nski Andrzej, Zieli´ nska Marzenna, Baj Zbigniew, Platelet reactivity and mean platelet volume as risk markers of thrombogenesis in atrial fibrillation, International Journal of Cardiology (2017), doi:10.1016/j.ijcard.2017.03.023

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Platelet reactivity and mean platelet volume as risk markers of thrombogenesis in atrial fibrillation

T

Marcin Makowski1, Ireneusz Smorag2, Joanna Makowska3, Andrzej Bissinger1, Tomasz

IP

Grycewicz1, Jarek Paśnik4, Michal Kidawa5, Andrzej Lubiński1, Marzenna Zielińska5,

1

SC R

Zbigniew Baj2

Department of Interventional Cardiology and Cardiac Arrhythmias, Medical University of

Lodz, Poland

Department of Pathophysiology and Immunology, Medical University of Lodz, Poland

3

Department of Rheumatology, Medical University of Lodz, Poland

4

Department of Paediatrics, Preventive Cardiology and Immunology of Developmental Age,

Medical University of Lodz, Poland

Intensive Cardiac Therapy Clinic, Medical University of Lodz, Poland

TE

D

5

MA

NU

2

Abstract

CE P

Atrial fibrillation (AF) is associated with increased risk of thromboembolic complications. One of the markers of the increased risk of hypercoagulable state is platelet hyperreactivity. The aim of the study was to assess impact of arrhythmia on platelet reactivity. Methods

AC

The study included 36 (mean age 48,3; range 21-60) male patients with lone atrial fibrillation, with exclusion of concomitant diseases known to trigger hypercoagulable state. The AF patients underwent cardioversion to restore sinus rhythm and were subsequently under observation for 1 month. Echocardiography, ECG and blood collection was performed before cardioversion (T0) and 4 weeks after successful cardioversion (T1). During the study period patients have been contacted and examined every week and 24 hour ECG monitoring was performed. Platelet reactivity was assessed based on changes of CD62 and CD42b expression on platelet surface after stimulation with thrombin. Also changes in MPV were assessed. Results In all patients sinus rhythm was maintained at the end of the study period, however in 14 patients recurrences of AF were observed, confirmed by 24 hour ECG monitoring (atrial fibrillation recurrence group – AFR) and 22 patients maintained sinus rhythm throughout whole study period (SR group). 1

ACCEPTED MANUSCRIPT Mean fluorescence intensity (MFI) of CD62 on thrombin stimulated platelets decreased significantly 4 weeks after electrical cardioversion as compared to T0 (48.04+/-22.42 vs 41,47+/-16.03; p<0.01). Also MFI of CD42b on thrombin stimulated platelets decreased

T

significantly 4 weeks after electrical cardioversion as compared to T0 (22.16+/-10.82 vs

IP

12,06+/-5.99; p<0.0001).

Platelets reactivity estimated by CD 62 expression in SR group decreased significantly after 4

SC R

weeks observation (58.01+/-15.26 vs 46.57+/-13.44; p<0.001) opposite to AFR group 35.66+/-21.87 vs 34.54+/-16.4; p-ns). Moreover there were significant differences between basal reactivity during AF between SR and AFR groups (58.01+/-15.26 vs 35.66+/-21.87; p-

NU

0.01). MFI of CD42b on thrombin stimulated platelets decreased significantly both in AFR and SR groups (22.05+/-11.36 vs 13.8+/-6.03; p<0.001 and 21.87+/-14.18 vs 10.04+/-5.09;

MA

p<0005). MPV decreased significantly 4 weeks after electrical cardioversion as compared to T0 (8.81 +/- 0.19 vs 8.42 +/- 0.14; p<0.0001). Conclusion

D

The changes of platelet reactivity to thrombin observed after restoration of sinus rhythm in

CE P

TE

patients proves that arrhythmia intrinsically leads to increased reactivity of platelets.

AC

Key words: atrial fibrillation, platelet reactivity, mean platelet volume.

2

ACCEPTED MANUSCRIPT Introduction

Atrial fibrillation (AF), the most common clinically significant arrhythmia, is known to be

T

associated with an increased risk of thromboembolic complications such as severe stroke

IP

which affects even up to 8%1 of AF patients annually. The pathophysiology of thromboembolic complications in course of AF is multifactorial. One of the possible

SC R

mechanisms resulting in hypercoagulable state in AF patients is the increased platelet activation2-4. For a long time, there has been a dispute regarding if the platelet activation was a result of arrhythmia intrinsically or appeared due to other concomitant cardiovascular

NU

allergic or metabolic diseases2-9 as the group of atrial fibrillation patients is very heterogeneous and the risk of thromboembolic complication is increased by concomitant

MA

disorders like diabetes, hypertension and congestive heart failure10. Several studies5,6,11-13 suggest that platelet activation in AF patient develops as a result of the concomitant diseases mentioned above due their inherent influence on platelet function. On the other hand the

D

recent studies7,14 supports hypothesis that also arrhythmia per se results in platelet activation.

TE

Platelet reactivity is one of the factors increasing risk of thromboembolic complication in patients with coronary artery disease as related to effectiveness of antiplatelet therapy15,16. Patients with high on-treatment platelet reactivity after percutanous coronary intervention

CE P

(PCI) had increased risk of ischemic events15,16, particularly stent thrombosis15,17,18. Platelet reactivity examination is perceived to be an useful method in stratification and prevention of thrombosis in high risk patients, when PCI is recommended19. The IDEAL-PCI registry using

AC

multiple electrode aggregometry to estimate platelet reactivity confirmed utility of this method in reducing thrombotic events due to adjusting of antiplatelet therapy without increasing bleeding20. However, the role of platelet reactivity in increased risk of thromboembolic complications in AF has not been elucidated by now. There is only one paper assessing platelet reactivity after stimulation with ADP and TRAP-6 in atrial fibrillation21. The authors observed decreased platelet reactivity in patients who maintained sinus rhythm. One of the markers of platelet reactivity proposed is mean platelet volume (MPV), as this parameter increased correlates with higher platelet reactivity22 and platelet activation (betathromboglobulin release, increased number of adhesion molecules and thromboxane synthesis, amplified platelet aggregation23 and high platelet turnover23. The aim of the study was to assess if atrial fibrillation itself, independent of other risk factors leads to increased platelet reactivity. To confirm the hypothesis group of patients with lone AF without other concomitant diseases, known to influence the platelet activation, have been 3

ACCEPTED MANUSCRIPT included to the study. We also assessed changes in mean platelet volume as a proposed marker of platelet activation in patient during arrhythmia and after its termination.

IP

T

Methods

Patients

SC R

The study involved 36 (mean age 48,3; range 21-60) male patients with lone atrial fibrillation who were hospitalized in the Interventional Cardiology Department of Medical University of Lodz. The exclusion criteria were: age >60, coronary artery disease, left ventricular

NU

dysfunction (EF < 40%), congenital or acquired heart defects, artificial heart valve, diabetes, thyroid disease, inflammatory diseases (including also allergic diseases), cancer, renal

MA

disease, active smoking. Persistent AF was confirmed electrocardiographically on at least two separate occasions.

The protocol was approved by the Medical University of Lodz Ethics Committee and all

D

participants gave written informed consent to the study. The AF patients underwent

TE

cardioversion to restore sinus rhythm and were subsequently under observation for 1 month (Fig. 1). All patients were fully anticoagulated with dose-adjusted Acenocumarol achieving an

CE P

International Normalized Ratio (INR) of 2.0-3.0 during the study period. Mean time to reach therapeutic INR were 6 weeks. No changes in the treatment was allowed during the study period. Echocardiography, ECG and blood collection was performed before cardioversion (T0) and 4 weeks after the successful cardioversion (T1). During the study period patients

AC

have been contacted and examined every week and 24 hour ECG monitoring was performed. In all patients sinus rhythm was maintained at the end of the study period. Transthoracic echocardiography was performed in all the patients using the Sequoia C512 echocardiography system with 3.5 MHz probe (Acuson/Siemens, Mountain View, CA, USA) in the left lateral decubitus position according to standard protocol. Left atrium (LA) diameters were measured on the parasternal long axis view at end-systole. The measurements of the LA dimensions and areas were acquired from 3 consecutive beats and subsequently averaged. LV ejection fraction (EF) were assessed by Simpson’s method. Transmitral pulsed Doppler was recorded from the apical 4-chamber view, with the sample volume positioned between the tips of the mitral leaflets. To confirm left atrium contractility recovery 4 weeks after electric cardioversion the early filling peak (E) and late filling (A) velocities were recorded and the E/A ratio was calculated. The criterion of left atrium mechanical function restoration was E/A ratio > 0.33.

4

ACCEPTED MANUSCRIPT Platelet analysis We used the flow cytometry (FACScan, Becton Dickinson, San Jose, USA) to measure the expression of P-selectin (CD62P) and GP Ibα glycoprotein (CD42b), on thrombin activated

T

blood platelets. Blood samples were taken into tubes from forearm vein of patients without

IP

stasis to avoid activation of platelets. The tube containing 0,5ml of blood and 0,5ml of EDTA reflected the platelet reactivity to 0.08U bovine thrombin for 4 min. The platelet tests

SC R

were performed within 90 min. after blood withdrawal. The reaction mixture was incubated at room temperature for 30 min in a dark room. Then, the antibody-bound platelets were fixed with 200 µl l FACS flow liquid and analyzed.

NU

A fluorescein isothiocyanate (FITC) - conjugated antibody to glycoprotein IIIa (anti-CD61FITC; DAKO) was use as a marker of platelets. The platelets were subtracted from other

MA

blood cells and identified by flow cytometry based on size and platelet specific CD61 surface expression. Antibodies anti CD62P (Becton Dickinson) and anti-CD42b-PE (DAKO) conjugated with a phycoerythrin (PE) were use to assess platelet release reaction and GPIbα

D

to assess expression of the von Willebrand receptor. To exclude the non-specific association

TE

of protein with platelets, a control tube containing anti-CD61-FITC and nonfractionated PEconjugated IgG (Becton Dickinson) was used for each blood sample.

CE P

All data are presented as the percentage of platelets expressing CD62P or CD42b and its median of fluorescence intensity (MFI) reflecting density of the molecules on platelet surface. Data collected with flow cytometry were analyzed using WinMDI 2.8. Complete blood count, total cholesterol level, glucose, C-reactive protein (CRP) and

AC

fibrinogen measurement were performed in a fasting state in all patients. Total cholesterol level was determined enzymatically with OLIMPUS AU 6400 analyser. CRP was detected by nephelometric method using N latex high-sensitivity test Hs-CRP (Dade Behring, Marburg, Germany). Fibrinogen was detected by Clauss` methods (Multifibren U; Dade Behring Diagnostics; Behring Fibrintimer).

Statistical analysis

Data in table are shown as mean and standard error of the mean (SEM). Statistical analysis to compare data in the same group was performed by Willcoxon test. Statistical analysis to compare data between the subgroups (SR and AFR) was conducted by Mann-Whitney test. Fisher test was used to compare categorical data. Statistical significance was defined as p<0.05. 5

ACCEPTED MANUSCRIPT

Results

T

In all patients sinus rhythm was maintained at the end of the study period, however in 14

IP

patients recurrence of AF were observed, confirmed by 24 hour ECG monitoring (atrial fibrillation recurrence group – AFR). In 12 patients the episodes of arrhythmia were

SC R

asymptomatic, while only 2 patients complained of arrhythmia symptoms. In 22 patients no recurrence of AF in 24 hours ECG monitoring was observed and patient were free of

NU

arrhythmia symptoms (sinus rhythm group – SR group).

MA

Platelet reactivity in patients with AF 4 weeks after electrical cardioversion

Mean fluorescence intensity (MFI) of CD62 on thrombin stimulated platelets decreased significantly 4 weeks after electrical cardioversion as compared to T0 (48.04+/-22.42 vs

D

41.47+/-16.03; p<0.01). Also MFI of CD42b on thrombin stimulated platelets decreased

TE

significantly 4 weeks after electrical cardioversion as compared to T0 (22.16+/-10.82 vs 12.06+/-5.99; p<0.0001). MPV decreased significantly after 4 weeks after electrical

CE P

cardioversion as compared to T0 (8.81 +/- 0.19 vs 8.42 +/- 0.14; p<0.0001).

Platelet reactivity in patients with AF 4 weeks after electrical cardioversion in AF recurrence (AFR) and sinus rhythm group (SR).

AC

In SR group platelet reactivity measured by CD 62 MFI decreased significantly 4 weeks after electrical cardioversion as compared to T0. There was no significant change in mean fluorescence intensity of CD62 on thrombin stimulated platelets in AFR group 4 weeks after electrical cardioversion as compared to T0 . However there were significant higher platelet reactivity in T0 in SR group compared to T0 AFR (Tab. 2). However there were significant higher platelet reactivity during AF between SR and AFR groups (Tab 2). MFI of CD42b on thrombin stimulated platelets decreased significantly 4 weeks after electrical cardioversion as compared to T0 in both groups of patient, in AFR group (Tab. 2). MPV decreased significantly 4 weeks after electrical cardioversion as compared to T0 in both groups of patient. No significant differences between AFR and SR group both in T0 and T1 point were observed (Tab. 2).

6

ACCEPTED MANUSCRIPT Echocardiography In all patients (n=36) the restoration of left atrium mechanical function was observed. In all subjects left ventricle diastolic diameter LVdD (5.51+/-0.9 vs 5.2+/-0.3; p<0.001) and left

T

atrium cavity diameter (4.42+/-0.8 vs 4.14 +/-0.4; p<0.001) decreased significantly 4 weeks

IP

after sinus rhythm restoration (T1) in comparison to baseline values (T0) (Table 2). Ejection fraction increased significantly in T1 in comparison to T0 (49.6+/-6.03 % vs 57.08+/- 3.3%;

SC R

p<0.001) (Table 3). There were no significant changes between AFR and SR groups in examined ECHO parameters.

NU

Discussion:

MA

To our knowledge it is the first paper documented the increased platelet reactivity in the group of patients with lone atrial fibrillation. Up till now only one study assessed the reactivity of platelets in patients with atrial fibrillation showing that reactivity of platelets to ADP and

D

TRAP-6 is increased in AF patients and decreases after sinus rhythm restoration, which is in

TE

line with our results21.

Atrial fibrillation is the most common cardiac arrhythmia and usually occurs in patients with concomitant disease like hypertension and chronic heart failure. Lone atrial fibrillation occurs

CE P

very rarely24,25. It is assessed that lone atrial fibrillation patients have lower risk of thromboembolism than other AF patients and according to current guidelines they do not require treatment with oral anticoagulants26.

AC

Most of the studies suggest that platelet activation observed in AF patients develop as a result of concomitant disease5,6,12,27,28 however, some previous studies have confirmed that AF itself could activate platelets7,14,29. To exclude potential impact of other pro-thrombotic factors, patients in our study were selected very carefully, excluding any disease that could probably lead to thromboembolism. Our results show that presence of arrhythmia, independently of other concomitant diseases leads to increased platelet reactivity. Increased platelet reactivity can have potentially important clinical implications. Available papers studied platelet reactivity in response to antiplatelet treatment in patients with coronary heart disease15,16. Low platelet reactivity during dual antiplatelet therapy has been shown to decrease the major adverse cardiovascular events rate after percutanous coronary interventions30,31. Moreover, there is evidence that to weak inhibition of platelets could increase the risk of thrombosis in such patients15,17,18 and the platelet reactivity examination

7

ACCEPTED MANUSCRIPT could be additional, useful method in clinical practice to optimization doses of antiplatelet treatment. Up till now there is no confirmed biomarker which could predict the thromboembolic

T

complications in patients. Platelet reactivity examination using flow cytometry is problematic

IP

in the routine clinical practice and finding a cheap, reliable and widely available marker of increased thrombotic risk in patient with atrial fibrillation is very important. Recently mean

SC R

platelet volume has been proposed to serve as the biomarker of increased risked of thromboembolic complications. In present study we showed that MPV decreases after sinus rhythm restoration, although it did not correlate directly with platelet reactivity and AF

NU

recurrences. In our study MPV decreased significantly after sinus rhythm restoration in both AFR and SR groups. The previous study confirmed that platelet size, measured as mean platelet volume (MPV), correlates with their reactivity22 and increased platelet aggregation ex

MA

vivo32. Large platelets expressed higher levels of P-selectin33 and glycoprotein IIb-IIIa34 than small platelets. MPV is also positively correlated with platelet turnover23. MPV could be a

D

cheap and easily accessible marker predicting thromboembolic complication in AF35. MPV is

TE

an independent predictor of mortality in STEMI36 patient and risk factor for stroke37. Higher MPV correlated with increased number of both platelet-platelet and platelet-leukocyte

CE P

aggregates38. MPV remains very useful marker for indirect platelet monitoring and estimated platelets activation in vivo.

Platelet are activated even in patients with lone atrial fibrillation without any diseases which could be a risk factor for TE. The lack of effectiveness of antiplatelet treatment in patients

AC

with AF implies that the dominant role of prothrombotic activity is driven by plasma factors, which could in response activated platelets. Increased reactivity of platelets could serve as a marker of increased risk of TE complications. Interesting finding in our study was the differences between subgroup of patients with recurrence of AF during study period and patient who maintained sinus rhythm throughout the whole study. Only patients without silent episodes of AF presented decreased platelet reactivity assessed by changes of expression of CD62 on platelet surface. On the other hand there was significantly higher platelet reactivity during AF in SR group compared to AFR. One possible explanation is duration of AF before cardioversion21. Platelet activity could be decreased due to chronic platelet activation. Reactivity of the platelets assessed as expression of receptor for von Willebrand factor (CD42b) decreases after sinus rhythm restoration independently on recurrence of AF. It

8

ACCEPTED MANUSCRIPT suggests the increased adhesion properties of platelets and supports chronic anticoagulant treatment irrespective to maintenance of sinus rhythm or not10,39,40. Although we did not observe any thromboembolic complications during the study period

T

there is need for further studies on the group with silent recurrence of AF and their impact on

IP

ischaemic complications to assess the potential risk of stroke in this subgroup of patient. Study limitation

SC R

We cannot exclude the influence of oral anticoagulants on platelet function41. However all patients included to the study were on continuous anticoagulant treatment with INR values controlled and remaining without normal limits throughout the whole study period thus the

NU

observed changes of reactivity cannot be attributed to this factor. Moreover it would be unethical to conduct the study without providing patients with optimal antithrombotic therapy before and after cardioversion. Arrhythmia duration potentially can also influence the platelet

MA

reactivity21.

Also we cannot exclude the influence of other drugs taken by the patients on platelet

D

reactivity, but there was no change in treatment schemes during the study.

TE

Conclusion

The changes of platelet activation and volume observed after restoration of sinus rhythm in

CE P

patients without concomitant diseases proves that arrhythmia intrinsically leads to increased reactivity of platelets. Increased platelets expression of adhesion molecules, despite sinus rhythm restoration, suggests need for anticoagulation treatment continuation. Remaining high platelet reactivity may contribute in hypercoagulable state, atrial thrombosis and

AC

thromboembolism.

Acknowledgements

The study has been supported by Ministry of Science and Education Republic of Poland nr N402439833.

9

ACCEPTED MANUSCRIPT References

2

8 9

10

11 12 13 14 15

16

17

NU

MA

D

7

TE

6

CE P

5

AC

4

SC R

IP

3

Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation as an independent risk factor for stroke: the Framingham Study. Stroke 1991; 22:983-988 Yip H, Chang L, Sun C, et al. Platelet activation in patients with chronic nonvalvular atrial fibrillation. Int Heart J 2006; 47:371-379 Conway DS, Pearce LA, Chin BS, et al. Plasma von Willebrand factor and soluble pselectin as indices of endothelial damage and platelet activation in 1321 patients with nonvalvular atrial fibrillation: relationship to stroke risk factors. Circulation 2002; 106:1962-1967 Goette A, Ittenson A, Hoffmanns P, et al. Increased expression of P-selectin in patients with chronic atrial fibrillation. Pacing Clin Electrophysiol 2000; 23:18721875 Minamino T, Kitakaze M, Sanada S, et al. Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation: role of nitric oxide. Circulation 1998; 98:1721-1727 Li-Saw-Hee FL, Blann AD, Gurney D, et al. Plasma von Willebrand factor, fibrinogen and soluble P-selectin levels in paroxysmal, persistent and permanent atrial fibrillation. Effects of cardioversion and return of left atrial function. Eur Heart J 2001; 22:1741-1747 Akar JG, Jeske W, Wilber DJ. Acute onset human atrial fibrillation is associated with local cardiac platelet activation and endothelial dysfunction. J Am Coll Cardiol 2008; 51:1790-1793 Potaczek DP. Links between allergy and cardiovascular or hemostatic system. Int J Cardiol. 2014; Jan 1;170(3):278-285 Nastałek M PD, Wojas-Pelc A, Undas A. Plasma platelet activation markers in patients with atopic dermatitis and concomitant allergic diseases. J Dermatol Sci. 2011; Oct;64(1):79-82. Gage BF, Waterman AD, Shannon W, et al. Validation of clinical classification schemes for predicting stroke: results from the National Registry of Atrial Fibrillation. JAMA 2001; 285:2864-2870 Gawaz M, Ott I, Reininger AJ, et al. Agglutination of isolated platelet membranes. Arterioscler Thromb Vasc Biol 1996; 16:621-627 Sims PJ, Wiedmer T. The response of human platelets to activated components of the complement system. Immunol Today 1991; 12:338-342 Ruggeri ZM. Structure and function of von Willebrand factor. Thromb Haemost 1999; 82:576-584 Makowski M, Smorag I, Bissinger A, et al. Effect of sinus rhythm restoration on platelet function in patients with lone atrial fibrillation. Int J Cardiol; 172:e22-23 Gurbel PA, Bliden KP, Hiatt BL, et al. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation 2003; 107:2908-2913 Matetzky S, Shenkman B, Guetta V, et al. Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction. Circulation 2004; 109:3171-3175 Cuisset T, Frere C, Quilici J, et al. High post-treatment platelet reactivity identified low-responders to dual antiplatelet therapy at increased risk of recurrent cardiovascular events after stenting for acute coronary syndrome. J Thromb Haemost 2006; 4:542-549

T

1

10

ACCEPTED MANUSCRIPT 18

25

26

27

28

29

30

31

SC R

NU

MA

24

D

23

TE

22

CE P

21

AC

20

IP

T

19

Buonamici P, Marcucci R, Migliorini A, et al. Impact of platelet reactivity after clopidogrel administration on drug-eluting stent thrombosis. J Am Coll Cardiol 2007; 49:2312-2317 Holmes DR, Jr., Dehmer GJ, Kaul S, et al. ACCF/AHA clopidogrel clinical alert: approaches to the FDA "boxed warning": a report of the American College of Cardiology Foundation Task Force on clinical expert consensus documents and the American Heart Association endorsed by the Society for Cardiovascular Angiography and Interventions and the Society of Thoracic Surgeons. J Am Coll Cardiol; 56:321341 Christ G, Siller-Matula JM, Francesconi M, et al. Individualising dual antiplatelet therapy after percutaneous coronary intervention: the IDEAL-PCI registry. BMJ Open; 4:e005781 Milovanovic M, Fransson E, Hallert C, et al. Atrial fibrillation and platelet reactivity. Int J Cardiol; 145:357-358 van der Loo B, Martin JF. A role for changes in platelet production in the cause of acute coronary syndromes. Arterioscler Thromb Vasc Biol 1999; 19:672-679 Bath PM, Butterworth RJ. Platelet size: measurement, physiology and vascular disease. Blood Coagul Fibrinolysis 1996; 7:157-161 Brand FN, Abbott RD, Kannel WB, et al. Characteristics and prognosis of lone atrial fibrillation. 30-year follow-up in the Framingham Study. JAMA 1985; 254:3449-3453 Kopecky SL, Gersh BJ, McGoon MD, et al. The natural history of lone atrial fibrillation. A population-based study over three decades. N Engl J Med 1987; 317:669-674 Camm AJ, Kirchhof P, Lip GY, et al. Guidelines for the management of atrial fibrillation: the Task Force for the Management of Atrial Fibrillation of the European Society of Cardiology (ESC). Europace; 12:1360-1420 Choudhury A, Chung I, Blann AD, et al. Elevated platelet microparticle levels in nonvalvular atrial fibrillation: relationship to p-selectin and antithrombotic therapy. Chest 2007; 131:809-815 Choudhury A, Freestone B, Patel J, et al. Relationship of soluble CD40 ligand to vascular endothelial growth factor, angiopoietins, and tissue factor in atrial fibrillation: a link among platelet activation, angiogenesis, and thrombosis? Chest 2007; 132:19131919 Sohara H, Amitani S, Kurose M, et al. Atrial fibrillation activates platelets and coagulation in a time-dependent manner: a study in patients with paroxysmal atrial fibrillation. J Am Coll Cardiol 1997; 29:106-112 Wright RS, Anderson JL, Adams CD, et al. 2011 ACCF/AHA focused update incorporated into the ACC/AHA 2007 Guidelines for the Management of Patients with Unstable Angina/Non-ST-Elevation Myocardial Infarction: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines developed in collaboration with the American Academy of Family Physicians, Society for Cardiovascular Angiography and Interventions, and the Society of Thoracic Surgeons. J Am Coll Cardiol; 57:e215-367 Anderson JL, Adams CD, Antman EM, et al. 2011 ACCF/AHA Focused Update Incorporated Into the ACC/AHA 2007 Guidelines for the Management of Patients With Unstable Angina/Non-ST-Elevation Myocardial Infarction: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation; 123:e426-579

11

ACCEPTED MANUSCRIPT

36

37

38 39

T

CE P AC

41

TE

D

40

IP

35

SC R

34

NU

33

Sharp DS, Bath PM, Martin JF, et al. Platelet and Erythrocyte Volume and Count: Epidemiological Predictors of Impedance Measured ADP-Induced Platelet Aggregation in Whole Blood. Platelets 1994; 5:252-257 Breimo ES, Osterud B. Studies of biological functions in blood cells from individuals with large platelets. Platelets 2003; 14:413-419 Pathansali R, Smith NM, Bath PM. Prothrombotic megakaryocyte and platelet changes in hypertension are reversed following treatment: a pilot study. Platelets 2001; 12:144-149 Ha SI, Choi DH, Ki YJ, et al. Stroke prediction using mean platelet volume in patients with atrial fibrillation. Platelets; 22:408-414 Huczek Z, Kochman J, Filipiak KJ, et al. Mean platelet volume on admission predicts impaired reperfusion and long-term mortality in acute myocardial infarction treated with primary percutaneous coronary intervention. J Am Coll Cardiol 2005; 46:284290 Bath P, Algert C, Chapman N, et al. Association of mean platelet volume with risk of stroke among 3134 individuals with history of cerebrovascular disease. Stroke 2004; 35:622-626 Tsiara S, Elisaf M, Jagroop IA, et al. Platelets as predictors of vascular risk: is there a practical index of platelet activity? Clin Appl Thromb Hemost 2003; 9:177-190 Petersen P, Godtfredsen J. Embolic complications in paroxysmal atrial fibrillation. Stroke 1986; 17:622-626 Hart RG, Pearce LA, Rothbart RM, et al. Stroke with intermittent atrial fibrillation: incidence and predictors during aspirin therapy. Stroke Prevention in Atrial Fibrillation Investigators. J Am Coll Cardiol 2000; 35:183-187 Lip GY, Lowe, G.D., Rumley, A. and Dunn, F.G. Increased markers of thrombogenesis in chronic atrial fibrillation: effects of warfarin treatment. Br Heart J 1995; 73:527-533

MA

32

12

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Figure 1. Study protocol.

13

ACCEPTED MANUSCRIPT Table 1 Baseline characteristics of AF patients

49.36+/-9.6 29.6+/-5.16 13.35+/-8.5

52.7+/-7.2 26.3+/-4.7 13.5+/-14.5

T

AFR group n=14

D

MA

TE

22 14 10 6 1

AC

14

p value

124.2+/-4.2 80.8+/-4.2 1.46+/-0.8 5.16+/-0.8 5.36+/-0.4 231.7+/-67.3 6.92+/-1.7 14.95+/-0.91 44.33+/-2.67 198.6+/-149.6 342.56+/-127.8

ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

14 10 6 1

ns ns ns ns ns

IP

NU

123.7+/-3.8 79.8+/-3.6 2.15+/-1.35 4.67+/-0.6 5.47+/-0.3 226.34+/-51.4 7.1+/-1.5 15.17+/-1.3 45.11+/-3.61 205.44+/-136.44 364.22+/-108.43

CE P

Patients demographics Age, yr BMI, kg/m2 Duration of AF (month) Clinical measurements SBP, mm Hg DBP, mm Hg CRP, mg/L Cholesterol, mmol/L Glucose, mmol/L Platelets, x103 cell/uL WBC count, x103 cell/uL Hb, g/dL Hct, % D-Dimer Fibrynogen Treatment Acenocumarol Amiodarone β-blocker Propafenon Statin

SR group n=22

SC R

Variables

ACCEPTED MANUSCRIPT Table 2 The results of flow cytometric assessment, MPV and platelets count in the whole group of patients with AF including AFR and SR group (n=36).

AFR T1

p-value

SR T0

n=14

n=14

CD62 (MFI)

35.66+/-21.87*

34.54+/-16.4

ns

58.01+/-15.26* 46,57+/-13.44

<0.001

CD42b (MFI)

22.05+/-11.36

13.8+/-6.03

<0.001

21.87+/-14.18

10.04+/-5.09

<0.0005

MPV (fl)

8.59 +/- 0.09

8.29+/- 0.08

0.01

8.97 +/- 0.14

8.37 +/- 0.12

<0.001

PLT x103(uL)

234.9+/-10.26

225.9+/-7.22

ns

233.5+/-13.92

234.9+/-12.71

ns

SC R

NU MA D TE CE P AC

p-value

n=22

IP

n=22

15

SR T1

T

AFR T0

ACCEPTED MANUSCRIPT Table 3. The echocardiographic changes in SR and AFR groups 4 weeks after electric cardioversion.

T1

T0

IP

T0

SR

T

AFR 5.62+/-0.2

5.34+/-0.26 *

LA (cm)

4.46+/-0.34

4.14+/-0.48 *

4.6+/-0.45

4.2+/-0.4 *

EF %

49.8+/-3.64

56.9+/-3.67 *

50.2+/-2.7

58.6+/-3.4 *

NU

SC R

LVdD (cm)

AC

CE P

TE

D

MA

* - p <0.001 T1 as compared to T0

5.66+/-0.44

T1

16

5.52+/-0.24 *