A cross-sectional and diurnal study of thrombogenesis among patients with chronic atrial fibrillati

A Cross-Sectional and Diurnal Study of Thrombogenesis Among

Patients With Chronic Atrial Fibrillation

Foo Leong Li-Saw-Hee,MRCP,Andrew D.Blann,P H D,MRCP ATH ,Gregory Y.H.Lip,MD,FRCPE,FACC Birmingham,United Kingdom

OBJECTIVES

First,we sought to determine whether there is diurnal variation in hemostatic factors related to thrombogenesis and hypercoagulability among patients with chronic atrial ?brillation (AF).Second,we sought to determine whether levels of soluble thrombomodulin (sTM),a marker of endothelial function,or soluble P-selectin (sP-sel),an index of platelet activation,are altered in patients with AF as compared with subjects in sinus rhythm.

BACKGROUND Atrial ?brillation is associated with an increased risk of stroke and thromboembolism and is

known to confer a hypercoagulable state,with abnormalities of thrombosis,platelet activation and endothelial cell function.Many cardiovascular events,such as acute myocardial infarction,have thrombosis as an underlying process,and they undergo diurnal variation.METHODS

Fifty-two patients (45men,mean [?SD]age 66?6years)with chronic AF,none of whom received antithrombotic therapy,were studied.Baseline levels of ?brinogen,sP-sel,sTM and von Willebrand factor (vWF)were compared to those levels in matched healthy control subjects in sinus rhythm.In a subgroup of 20patients,?ve venous blood samples were collected through an indwelling cannula at 6-h intervals from 12PM to 12PM the following day and were analyzed for the same markers.

RESULTS

Patients with chronic AF had higher plasma sP-sel,sTM,vWF and ?brinogen levels as compared with control subjects in sinus rhythm.Signi?cant correlations were found between ?brinogen and sP-sel in patients with AF (r ?0.567[Spearman],p ?0.001)and in control subjects (r ?0.334,p ?0.016).There was no signi?cant diurnal variation in plasma levels of sP-sel,sTM,vWF or ?brinogen over the 24-h study period (repeated measures analysis of variance,p ?NS).

CONCLUSIONS There is no circadian or diurnal variation in the hypercoagulable state seen in AF,as

assessed by plasma ?brinogen and markers of platelet (sP-sel)and endothelial function (vWF and sTM).The persistent hypercoagulable state,together with the loss of diurnal variation in various hemostatic markers,in chronic AF may contribute to the high risk of stroke and thromboembolic complications in these patients.(J Am Coll Cardiol 2000;35:1926–31)?2000by the American College of Cardiology

Atrial ?brillation (AF)is a common cardiac arrhythmia and is associated with a substantial risk of stroke and thrombo-embolism (1).This is probably because AF confers a hypercoagulable state,with abnormalities of hemostasis,thrombosis and platelet function (2–4).For example,in a cross-sectional study of 73patients with chronic AF and 21patients in sinus rhythm,Kumagai et al.(4)reported increased ?brin D -dimer levels,a marker of intravascular

thrombogenesis,irrespective of the presence of underlying heart disease.The bene?cial role of oral anticoagulation therapy in reducing the risk of stroke and thromboembolic in patients with nonvalvular AF has been con?rmed by recent large-scale studies (5).

Recent research has identi?ed two more plasma markers that may be of use in understanding the pathophysiology of thrombogenesis.Soluble thrombomodulin (sTM),another marker of endothelial cell damage,is increased in patients with systemic hypertension,peripheral artery disease and coronary artery disease,and raised levels predict adverse events even among patients receiving long-term anticoagu-lation (6–9).It has been suggested that increased levels of the soluble adhesion molecule,P-selectin,implies platelet activation (10,11),as may be the case in atherosclerosis,

From the Haemostasis,Thrombosis and Vascular Biology Unit,University De-partment of Medicine,City Hospital,Birmingham,United Kingdom.Dr.Li-Saw-Hee is supported by a nonpromotional research fellowship from Merck Sharpe and Dohme.We acknowledge the support of the City Hospital Research and Develop-ment program for the Haemostasis,Thrombosis and Vascular Biology Unit.

Manuscript received December 22,1998;revised manuscript received December 16,1999,accepted February 9,2000.Journal of the American College of Cardiology Vol.35,No.7,2000?2000by the American College of Cardiology ISSN 0735-1097/00/$20.00Published by Elsevier Science Inc.PII S0735-1097(00)00627-6

hypertension and thrombotic consumptive disorders(12–14).

Many variables within biophysical systems,such as the onset of acute vascular events,show peaks and troughs in the course of a24-h period(15–19).For example,promi-nent clustering of cardiovascular events,such as acute myocardial infarction,between6AM and12PM has been demonstrated(20–23).Many such events have thrombosis as an underlying pathologic process,and some hemostatic factors have also been shown to demonstrate a circadian variation that may in part contribute to this diurnal inci-dence(24–26).Furthermore,paroxysmal AF,which is also associated with a risk of stroke,exhibits a unique circadian variation that differs from the well-known pattern in acute cardiovascular events(27).As the mechanisms of the increased thromboembolic risk in patients with AF have not been fully elucidated,abnormalities in indexes of hyperco-agulability indicative of a prothrombotic state could account for this risk(1–4,28,29),which may demonstrate diurnal variation.

It is unclear whether there is diurnal variation in these hemostatic factors related to thrombogenesis and hyperco-agulability in patients with chronic AF that may perhaps relate to the occurrence of clinical events.It has also not yet been determined whether levels of sTM or soluble P-selectin(sP-sel)are altered in patients with AF as compared with subjects in sinus rhythm.The aim of the present study was to test both these hypotheses prospec-tively.

METHODS

Patients and control subjects.We recruited patients with chronic AF,which was seen on the electrocardio-gram on at least two occasions at least six weeks apart, either by a general practitioner or at hospital or outpa-tient clinic review.Exclusion criteria were other acute causes of AF(e.g.,thyrotoxicosis,pneumonia),acute cardiovascular or cerebrovascular events(e.g.,myocardial infarction,congestive heart failure,stroke),use of aspirin or anticoagulant agents,in?ammatory or connective tis-sue disease and chronic renal or hepatic disease.A random subgroup of20patients was admitted for36h for clinical assessment before initiation of anticoagulation therapy.Five venous blood samples were collected through an indwelling cannula at6-h intervals from12 PM to12PM the following day.

Control subjects were found among the healthy hos-pital staff and among those patients who were in the

hospital for hernia repair,varicose veins or minor oper-

ations.All were free of diabetes and were without signs or

symptoms of cardiovascular,neoplastic or connective

tissue disease.Systolic and diastolic blood pressure were

recorded in each subject after a minimum of5min of rest,

and the subjects’smoking status was determined.The

project had the approval of the research Ethics Commit-

tee of the West Birmingham Health Authority,and

written,informed consent was obtained from each par-

ticipant.

Laboratory measures.Citrated plasma was obtained from

venous blood by centrifugation at2,500rpm for15min at

4°C.Aliquots were stored at?70°C to allow batch analysis.

Soluble P-selectin,sTM and von Willebrand factor(vWF)

were measured by the enzyme-linked immunosorbent assay

(ELISA)technique using commercial reagents(R&D Sys-

tems,Abingdon,United Kingdom;Diagnostica Stago,

Asnieres-sur-Seine,France;and Dako-Patts,Ely,United

Kingdom,respectively).The unit for vWF is IU/dl and was

standardized by the reference vWF from the National

Institute for Biological Standards and Controls(Hertford-

shire,United Kingdom).Other indexes(ng/ml)were stan-

dardized by the recombinant product supplied by the

manufacturer.Intra-assay coef?cients of variation for all

ELISA assays were?5%;interassay variances were?10%.

Plasma?brinogen(g/liter)was measured by the Clauss

technique on a Paci?c Hemostasis(Hunterville,North

Carolina)coagulometer and with reagents from Alpha

Laboratories(Eastleigh,Hants,United Kingdom). Power calculation and statistics.Data were initially ana-

lyzed by using the Shapiro-Wilks test to determine normal-

ity of distribution.In the cross-sectional study,data for

vWf,sTM and?brinogen are presented as the mean

value?SD and analyzed by using the unpaired t test;

however,sP-sel levels were nonparametrically distributed

and expressed as the median value with interquartile range

and analyzed using the Mann-Whitney U test.Correlations

between indexes were performed by the Spearman rank

correlation method.We hypothesized that sP-sel and sTM

would be increased by one-half of a standard deviation

among patients with AF as compared with control subjects.

To prove this,we would need to recruit a minimum of40

patients and40control subjects for a one-sided p value ?0.05with80%power.Two other studies of diurnal variation recruited only9and10subjects,respectively

(24,25).Therefore,we aimed to recruit at least20subjects.

In the diurnal study,the results were analyzed by repeated

measures analysis of variance.All statistical calculations

were performed on a microcomputer using a commercially

available statistical package(Minitab Release12,Minitab

Inc.,State College,Pennsylvania).A p value?0.05was

considered statistically signi?cant.

Abbreviations and Acronyms

AF?atrial?brillation

sP-sel?soluble P-selectin

sTM?soluble thrombomodulin vWF?von Willebrand factor 1927

JACC Vol.35,No.7,2000Li-Saw-Hee et al. June2000:1926–31Diurnal Variation and Hypercoagulability in AF

RESULTS

Cross-sectional data.Demographic data are presented in Table 1.There were no differences in age,gender,propor-tion of current smokers or blood pressure between the groups.Patients with AF had higher plasma sP-sel,sTM,?brinogen and vWF levels as compared with control sub-jects in sinus rhythm (Table 2).There were no signi?cant differences for the measured levels of plasma sP-sel,sTM,vWF and ?brinogen between patients with lone AF (n ?16)and patients with AF associated with underlying disease (n ?36)(Table 2).

Correlations with clinical variables.Among patients with AF,there was a signi?cant correlation between plasma ?brinogen and sP-sel levels (r ?0.567[Spear-man],p ?0.001).Among the control subjects,the correlation between plasma ?brinogen and sP-sel was also signi?cant (r ?0.334,p ?0.016).There were no other signi?cant correlations between various indexes in patients or control subjects.

Diurnal study.We studied 20patients with chronic AF who were not receiving antithrombotic therapy.Demo-graphic data for these patients are summarized in Table 3.

Table 1.Demographic Data for Total Study Group

Control Subjects

(n ?60)

Patients With AF

(n ?52)p Value Age (years)66?668?130.639Male gender 45(75%)42(80%)0.779Smokers

7(11.2%)8(15.4%)0.842Systolic blood pressure (mm Hg)145?21143?230.883Diastolic blood pressure (mm Hg)

80?1182?120.735

Known hypertension (?160/90mm Hg)013(25%)Lone AF

016(30.8%)Coronary artery disease

012(23.1%)Previous thromboembolic stroke 03(5.8%)Diabetes mellitus

04(7.7%)Peripheral vascular disease

4(7.7%)

Data are presented as the mean value ?SD or number (%)of patients or subjects.AF ?atrial ?brillation.

Table 2.Plasma Levels of Soluble Adhesion Molecule P-Selectin,Soluble Thrombomodulin,von Willebrand Factor and Fibrinogen in Patients With Chronic Atrial Fibrillation and in Control Subjects

a)Patients With AF Versus Healthy Control Subjects in Sinus Rhythm Control Subjects

(n ?60)

Patients With AF

(n ?52)

p Value Soluble P-selectin (ng/ml)1261850.0002(91–160)(134–317)Soluble thrombomodulin (ng/ml)44?1352?170.015von Willebrand factor (IU/dl)103?33137?27?0.0001Fibrinogen (g/liter)

2.6?0.8

2.9?0.9

0.025

b)Lone AF Compared With Atrial Fibrillation Associated With Underlying Disease Patients With Lone AF

(n ?16)

Patients With AF ?Underlying Disease

(n ?36)

p Value Soluble P-selectin (ng/ml)1601850.74(139–322)(133–317)Soluble thrombomodulin (ng/ml)49?1255?180.24von Willebrand factor (IU/dl)136?7139?50.80Fibrinogen (g/liter)

2.7?0.7

3.0?0.9

0.24

Data are presented as the mean value ?SD (analyzed by the unpaired t test),except for soluble P-selectin levels,which are expressed as the median value (interquartile range),analyzed using the Mann-Whitney U test.AF ?atrial ?brillation.

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Diurnal Variation and Hypercoagulability in AF

June 2000:1926–31

There was no signi?cant diurnal variation in plasma levels of sP-sel,sTM,vWF or ?brinogen over the ?ve sampling points (12PM ,6PM ,12AM ,6AM and 12PM )over the 24-h period (Table 4).

DISCUSSION

In the present study,we con?rmed previous observations of increased vWF and ?brinogen levels in patients with chronic AF as compared with healthy control subjects in sinus rhythm (2),and we provide new evidence of a hypercoagulable or thrombogenic state in chronic AF,with increased levels of the endothelial cell product,sTM,as well as a new marker of platelet activation—soluble adhesion molecule P-selectin.Importantly,this hypercoagulable state does not appear to be subject to any signi?cant diurnal variation,nor is it related to whether or not the patient had lone AF or not,suggesting that chronic AF confers a constant prothrombotic state per se over the 24-h day,which was independent of underlying heart disease or etiology (3).

Endothelial dysfunction in AF.The increase in vWF levels parallels the increased levels of sTM,another index of endothelial dysfunction (6,30–32).The latter molecule is of interest because thrombomodulin is a constitutive mem-

brane protein that is an important regulator of activated thrombin,converting thrombin from a procoagulant (cleav-ing ?brinogen)to an anticoagulant by altering its substrate speci?city,so that it activates protein C (30).However,thrombomodulin probably needs to be cleaved for soluble forms to be found in the circulation.In vitro experiments suggest that the presence of sTM in tissue culture superna-tant is probably the result of damage to the endothelial cells (31).Unlike vWF,however,levels of sTM appear to be independent of the in?ammatory cytokines interleukin-1and tumor necrosis factor (6,32).Our ?nding of high sTM levels in patients with chronic AF in the present study is in contrast to a recent preliminary report from a much smaller cohort of patients with chronic AF in whom this marker may not have been elevated (33).The precise mechanism for the increased markers of endothelial dysfunction or activa-tion in cardiovascular disorders is uncertain,but may nev-ertheless include a cytokine-mediated increase in synthesis,increased secretion from stored pools,increased synthesis de novo or release from damaged endothelial cells.In the case of AF,abnormalities in blood ?ow may be partly responsi-ble,resulting in ?ow abnormalities and adding to endothe-lial disturbance in the pulmonary vasculature.

Platelet activation in AF.The presence of platelet activa-tion has been recognized in chronic AF,which has usually been indicated by high plasma levels of beta-thromboglobulin (28),although this ?nding is controversial (34,35).In the present study,we have measured levels of sP-sel,which is a new marker of platelet activation (10,11).Our sP-sel data therefore complement those of Pongratz et al.(35),who found increased expression of membrane-bound P-selectin on platelets of patients with AF.Like Pongratz et al.(35),we interpret this as further evidence of inappropriate platelet activation in these patients,which is correlated with plasma ?brinogen,an established index of hemorheology and clotting.Increased platelet activation,in combination with endothelial dysfunction and abnormal hemostatic factors,is thus in keeping with the hypercoag-ulable state in AF (3,4).

Table 3.Demographic Data for Diurnal Study Group (n ?20,All With Atrial Fibrillation)Age (years)73?7Male gender 16(80%)Smokers

3(15%)Systolic blood pressure (mm Hg)144?22Diastolic blood pressure (mm Hg)82?12Hypertension only 5(25%)Lone AF

10(50%)Coronary artery disease

2(10%)Previous thromboembolic stroke 1(5%)Diabetes mellitus

1(5%)Peripheral vascular disease

1(5%)

Data are presented as the mean value ?SD or number (%)of patients.AF ?atrial ?brillation.

Table 4.Diurnal Changes in Markers of Thrombogenesis in Chronic Atrial Fibrillation*

Time Points

p Value for Main Effect 12

PM

6

PM

12

AM

6

AM

12

PM

Soluble P-selectin (ng/ml)

113(82–168)107(71–139)104(85–139)105(74–149)114(100–157)0.690Soluble thrombomodulin (ng/ml)

49?1948?1546?1350?1351?170.231von Willebrand factor (IU/dl)

134?33135?27134?27133?31135?270.990Fibrinogen (g/liter)

2.62?0.57

2.69?0.56

2.74?0.59

2.73?0.66

2.89?0.60

0.240

*There was no signi?cant variation in these data (repeated measures analysis of variance)for diurnal changes in patients with atrial ?brillation (all p ?NS).Data are presented as the median value (interquartile range)for soluble P-selectin and the mean value ?SD for the other markers.

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June 2000:1926–31

Diurnal Variation and Hypercoagulability in AF

Diurnal variation in markers of thrombogenesis.Previ-ous studies in subjects without AF have suggested diurnal or circadian variation in certain clotting factors.For example, the study by Jafri et al.(25)reported that circadian changes occurred in beta-thromboglobulin(p?0.05)and platelet factor-4(p?0.06,NS)in nine normal healthy subjects.In another small study involving only10normal healthy subjects,Bridges et al.(24)demonstrated that signi?cant circadian variation occurred with tissue plasminogen activa-tors(p?0.001),plasminogen activator inhibitor(p?0.04) and11-dehydro-thromboxane B2(p?0.005),with mea-surements taken at4-h intervals from12PM to8AM the following day.These?ndings are thus consistent with the clinical observations of a diurnal pattern to acute vascular events.Nevertheless,in the present study,we report that the hypercoagulable state in chronic AF does not seem to undergo any signi?cant diurnal or circadian rhythm.This may be a re?ection of the high risk of stroke and thrombo-embolism associated with chronic AF,suggesting that such patients may be at a constant high prothrombotic risk throughout the day,necessitating adequate antithrombotic therapy.Although there appears to be a diurnal pattern to the onset of stroke in general(17),we are not aware of any published studies speci?cally investigating whether there is a diurnal variation in stroke onset among patients with AF. Nevertheless,preliminary observations from an ongoing project in our unit do not suggest a signi?cant diurnal pattern to the onset of stroke in patients with AF.The absence of a diurnal variation in the hypercoagulable state in patients with AF would be in keeping with this. Clinical study implications.Although the use of anti-thrombotic therapy may reduce the risk of thrombosis,even administration of anticoagulation with a constant infusion of intravenous heparin is associated with a diurnal variation in the intensity of anticoagulation(36).If chronic AF did show signi?cant diurnal variation in the hypercoagulable or prothrombotic state,there will be periods when anticoagu-lation intensity may be insuf?cient to provide prophylaxis against thromboembolism,and other periods when antico-agulation exceeds that needed therapeutically,with a corre-sponding increase in the risk of bleeding.These problems are highly clinically relevant when considering thrombopro-phylaxis for patients with AF.Our?nding of a constant hypercoagulable state in chronic AF,therefore,provides further reassurance that anticoagulation with warfarin,aim-ing for a consistent target International Normalized Ratio of 2.0to3.0,is likely to provide adequate anticoagulation “cover”for each24-h period.

Study limitations.This study is limited by its case-controlled,cross-sectional design and the association of AF with other pathologic processes,such as hypertension and coronary artery disease,or risk factors for atherosclerosis.It has previously been shown that the hypercoagulable state in AF is independent of underlying etiology or associated heart disease(2–4).In addition,hypertension and coronary artery disease would result in relatively smaller changes in these markers as compared with those seen in the present study of AF.However,the main objective of our study was to assess the circadian or diurnal variation in the hypercoagulable state in AF,rather than to reproduce many previous analyses of the effects of heart disease on the markers of hyperco-agulability,which are suggestive of a continuum that exists between health,“statistically”increased hemostatic abnor-malities as a prethrombotic or hypercoagulable state and “overtly”increased clotting in acute thrombosis(or some-times in acute extravascular?brin formation)which follows injury or operation(2–4).We also accept that the present study was neither designed nor adequately powered to speci?cally compare differences in the hypercoagulable state between patients with lone AF and patients with AF associated with underlying disease;however,previous data (3)suggest that the hypercoagulable state in AF is indepen-dent of underlying etiology and structural heart disease. Conclusions.We suggest that there is no circadian or diurnal variation in the hypercoagulable state seen in AF,as assessed by plasma?brinogen and markers of platelet (sP-sel)and endothelial function(vWF and sTM).The persistent hypercoagulable state,together with the loss of diurnal variation in various hemostatic markers,in chronic AF may contribute in part to the high risk of stroke and thromboembolic complications in these patients.

Reprint requests and correspondence:Dr.Gregory YH Lip, Haemostasis,Thrombosis and Vascular Biology Unit,University Department of Medicine,City Hospital,Dudley Road,Birming-ham B187QH,United Kingdom.E-mail:G.Y.H.LIP@ https://www.360docs.net/doc/fd2936530.html,.

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