Hemostatic risk factors and arterial thrombotic disease

Thromb Haemost 2001; 85: 584-95

Review Articles

Hemostdtic Rssk Factors and Arterlal TTuombotic Disease

Alexander R Reiner

1

, David S. Siscovick

1

, Frits R. Rosendaal

2

'Cardiovascular Health Research Unit, Departmenls of Medidno and

University of Washington, Seattle, WA, USA;

2

The Hemostasis and Thrombosis Research Center <

Department of Clinical Epidemiology, University Hospital Leiden, The Netl

The pathogenesis of arterial thrombotic disease involves multiple

genetic and environmental factors related to atherosclerosis and

throm-bosis. Acute thrombosis at the site of a ruptured, lipid-rich

atheroscle-rotic plaque is the usual precipitating event in the transition from stable

or subclinical atherosclerotic disease to acute myocardial infarction

(MI), stroke, or peripheral arterial occlusion (1). Pathologie studies of

coronary arteries in acute MI suggest that the acute thrombosis likely

involves activation of both platelets and the coagulation System.

Following atherosclerotic plaque rupture, platelets initially adhere to

exposed subendothelial von Willebrand factor (vWF) and Collagen

through the glycoprotein Ib/IX receptor complex and the glycoprotein

la/IIa receptor complex, respectively. The attachment of platelets to the

vascular subendothelium (platelet adhesion) induces a series of

intra-cellular signaling events that result in platelet activation. The binding of

fibrinogen and vWF to the conformationally active form of the

glyco-protein Hb/IIIa receptor results in platelet cohesion or aggregation, and

further propagation of the platelet thrombus, Plaque rupture also results

in exposure of subendothelial tissue factor, which initiales the

coagula-tion cascade, and leads to the generacoagula-tion of thrombin and formacoagula-tion of

a fibrin clot. While arterial thrombi traditionally are considered to be

composed predominantly of platelets, accumulating evidence suggests

that certain arterial thrombotic disorders (e.g., transmural or "Q-wave"

MI) are associated with greater activation of the coagulation system,

which results in occlusive thrombi that are relatively rieh in fibrin (2).^

Prevention of arterial thrombotic diseases, such äs myocardial

in-farction and ischemic stroke, has focused on identification and

mod-ification of traditional cardiovascular risk factors, such äs Smoking,

hyperlipidemia, hypertension, and obesity, that are related: to

athero-sclerosis. However, approximately 30% of MI occur in individuals

without traditional cardiovascular risk factors (3). Since antithrombotic

agents such äs aspirin have been shown to be beneficial in primary and

secondary prevention of arterial thrombosis (4), attention has shifted in

recent years to the characterization and evaluation of novel phenotypic

markers of cardiovascular disease, involving lipoprotein metabolism,

the coagulation and fibrinolytic Systems, and inflammation (5, 6). In

addition, the identification of molecular variants within the genes

en-coding proteins involved in the pathogenesis of thrombosis has led to

their evaluation äs genetic ^usceptibility markers in epidemiologic

studies of arterial thrombotic disease. In this review, we briefly discuss

some general issues related to the studies that examine the associations

-Correspondence to: Alexander P. Reiner, M. D., Harborview Medical

Center, 325 9

th

Avenue, Box 359756, Seattle, Washington 98104,

USA - Tel.: +1206-731-3291; Fax: +1206-341-5312; E-mail: apreiner@u.

washington.edu

of both genetic susccpiibiliiy markers and inlcrmcdiaic hemostadcffc

:

.J

notypes with arterial Ihroinhotic disease. \Ve then review thet

pertaining to the roie of specilic genetic markers and hemostaticpjfcjji

1

!

types in arterial thromhosis.

General Issues Related to Study Design and Interpretation of Results

Case-control und cross-scctional studies assess the rclaticmlip.'p

between phenotjpic markers (c.ü., plasiua coagulation facffif fe|jd$j

and the occurrence of arterial thrombutic disease. However;!

studies cannot esiablish a temporal order, und thercforecaoaote

the possibility thal a hemostalic phenotype is a c

disorder (e.g., due to aecompanying tissue injur>' or in

Even in prospectivc cohort sludics, the obscrved associatiöwi

confounded by subclinical atherosclerosis. which may <

both the hemostalic phenoiypes äs well äs the clinical ι

addition, plasma Icvels öl' hcmosuitic fuclors are sometimesc

with each other and-also are inlluenced by other<

factors (e.g., obesily, hyperlipidemia, smoking), that may tatet

found an apparenl relaiionship betwcen the hemostatic τΰαΛχίΛάΐ^Ά

of disease. Forthcsc rcasons, ine most convincing evidenceofae

relationship between a hemostatic phcnolypc and the ι

arterial thrombotic disease is inicrred from clinical

strate that alterin!! l he phenotype (e.g., lowering

hemostatic factor) reduces disease.

Another means of circiiinventing somc of the spurious (

that can result from Ihc use of inlermediate hemostatic

to analyze genetic variants that are known to be related tof

Variation. T h e advamage o f studying genetic varianls i s t

^

"fixed at birth", and thcrel'ore clearly preccde disease .onsetnijWj!^

confounded by oiher behavioral and environmental (

factors or subclinical atherosdcrosis. However, raolecutar fl

ologic studies evaluaiing potential genetic susce

their own set of limiuuions. and diftcrent studies

genetic marker ollen yield conflicting results. Con

among studies is liampcred by differences in sti

terisücs of case paiiems and control subjecls, Variation ofl

quencies among dift'erent populations, äs well äs ihe

heterogeneityof Ihe clinical endpoinisof coronary heart

and peripheral anerial occlusion. These complex

volve the

i

effect^ of multiple genes interacting wilhthe*Wj!

The effect of any single «enetic susceplibility factor riffjgj

thrombotic disease is likely lo hc rnodest, but may

Reiner et al.: Hemostatic Risk Factors and Arterial Thrombotic Disease

Äabetween a putative genctic suscepiibiliiy marker and

arte-pdisease may only he dcmoiisirablc wilhin a certain

sub-ι As

;

a result, large sample si/cs ma\ be ro(|uired to

demon-• stable esümaie of el'fcct. pariicularly for alleles or

; tlial arc uncommon within l ho population. >

ib'ootsnbject to conl'ouriding by olhcr behavioral and

envi-il risk factors, populalion siudics of «eneiic variants are subject

> by ethnicily, i.e.. population admixlure or "population

i". Thus, a spurious associalion may arise if the frequency

c.raarker and the frcquency öl anerial tlirombolic disease

»jccording to genetic anccstry. Thi.i is inosi likely to be the

jljtbuically hetcrogencous populalions, and tan be accounted for

i that asscss genetic linkage äs well äs association.

; reason for a positive association in some studies but

i is thal the genetic marker of inierest is not directly

in-idisease susceptibiliiy. bul radier is in linkage disequilibrium

Ifteactual causalive mutation locaied in the sanic gene or another

y. Depending on the populalion hisiory and the

recombina-ilinction, the genetie marker may be associatcd with the causative

i in some populalions bul not in olliers. Therefore, it is

prefer-iioassess genetic markers that. are wcll-characicri/ed with respect

fe ftfflctional consequences of the molecular defeci and that

con-tSgnificantly to phenotypic Variation uiiliin Ihe population.

ι Factors

Factor VII

FactorVII is a vitamin-K dependent coagulation protein, which

upon activation, binds to tissue factor present in damaged vascular

sub-endothelium and initiales coagulation. In Ihe Northwick Park Heart

Study, a prospective sludy of over 1,300 middle-aged men, plasma

factor, VII aclivity levels were associaled with fatal, but not non-fatal,

ischemic heart disease (20). However, seveVal subsequent prospective

studies have failed to confirm Ihe associalion between faclor VII levels

and risk of coronary heart disease (21-23). Factor VII is also unrelated

to risk of venous Ihrombotic disease (24). Plasma factor VII levels are

determined by multiple environmental faclors, including age, gender,

body mass index, dielary fal, and triglyceride level (25). In addilion,

several intragenic faclor VII polymorphisms which influence plasma

factor VII levels have been described. These include an Arg353GIn

Substitution that is associated with decreased factor VII secretion

in vitro and lower factor VII levels (26). An Italian case-control sludy

of youn'g adulls wilh familial MI observed an inverse associalion wilh

homozygosity of Ihe faclor VII Gln353 allele (OR = 0.08, 95% CI =

0.01-0.9) (27). In conlrasl, several olher case-control studies involving

either young men (28) or a broader age ränge (29-31) found no

associ-alion belween Ihe Arg353Gln polymorphism and risk of MI or stroke.

Thus; the evidence to date does not support faclor VII at eilher the

phenotypic or genotypic level äs an importanl risk factor for arterial

Ihrombotic disease.

i|!B

:

fibrinogen levels have been consisieniK ancf independenlly

"l.an increased risk öl' MI and Mroke in prospective

idiapparently hcallhy persons and paiienis with preexisting

:disease (7-9). Subjccts with plasma fibrinogen levels

t tertile have ~2-fold increased risk compared lo Ihöse in

ftftätile. Fibrinogen may conlribuie lo arterial thrombotic

|iaanurnber of mechanisms. including increased fibrin

forma-ia

:

viscosiiy, platelel aggregaiion, and \ascularendolhelial

imuscle proliferalion (7). On tlie olher band, fibrinogen is an

: reactant, and the associalion of clevatcd fibrinogen and

W protein levels wilh risk of arterial thrombolic disease

sug-jl'diatthe inflammation thal acconipanies alherosclcrosis may

con-ito increased fibrinogen levels (10). Plasma fibrinogen levels are

r correlated with olher cardiovascular risk tactors, such äs

, diabetes, and cslrogens (7.11). Be/afi brate, an agent that

sboth.fibrinogcn-lowcring and lipid-modilying properties, was

l with an overall rcduction ofarterial thrombotic events in

HfClinical trial of palients with coronary heart disease (12).

ι fibrinogen is composed of 3 pairs of pol\ peplide chains, et,

i encoded by 3 separale genes clusiered on chromosome 4q.

S.Öie synthesis of the fibrinogen ß-chain is raie-limiting (13),

i ß-chain polymnrphisnis have been associaled wilh

inter-R( Variation in plasma fibrinogen levels. The 455 G/A

substi-i the promoler regsubsti-iorsubsti-i of ι he fsubsti-ibrsubsti-inogen ß-chasubsti-in has been

texlensivcly. The -455A allele is present in about 20% of

, who have ~l()9r higher fibrinogen level> compared to

ι the GG genotypc (14). The relaiionship between the -455

(finogcn variant and risk of anerial thrombolic disease is

il. with sorne case-eonirol studies obsemng an association

fc'and other large siudies finding no associntion 118,19). These

nt finding cast doubl on ;i direct cause-and-efl'ect

relalion-wccn llbrinoüen le\els and anerial Ihrombotic disorders.

Factor VIII and von Willebrand Factor

Increased plasma levels of faclor VIII aclivily, von Willebrand

fac-lor (vWF) anligen, and vWF aclivity have been associated with incident

falal and non-falal arterial thrombotic events in prospective siudies

involving heallhy middle-aged (22,23,32,33) and elderly (34)

popula-tions. In some studies, the associations were independent of other

cardiovascular risk factors. In most prospective studies of patients

with underlying atherosclerolic disease, vWF levels have been

indepen-dently associated wilh risk of acule Ihrombotic events (10, 35, 36).

Factor VIII and vWF circulate äs a complex, and plasma levels of these

two factors are highly correlated wilh each olher. Thus, elevated

fac-tor VIII/vWF levels may be either a marker of atherosclerosis (i.e.,

endothelial dysfunction and vascular injury) or contribute directly lo

fibrin formation (factor VIII) or shear-induced platelet adhesion and

aggregaiion (vWF). The demonslralion lhat congenilal factor VIII

defi-ciency.is associated wilh decreased mortalily from ischemic heart

dis-ease (37, 38), äs well äs the association of elevated factor VIII levels

with venous thrombolic disease (39), support the hypothesis of a direct

effecl of faclor VIII concenlration on Ihe occurrence of thrombolic

events. Factor VIII levels are also influenced by ABO blood group, and

individuals with blood group non-0 have both higher factor VIII levels

and higher incidence of coronary heart disease compared to individuals

with blood group 0 (32, 40). Further evaluation of factor VIII and

cardiovascular risk awaits elucidalion of other genetic delerminanls of

increased faclor VIII levels (41).

Factor XIII

Faclor XIII is a Iransglulaminase lhal forms covalenl bonds between

adjacent fibrin monomers, and Ihus slabilizes a fibrin clol. Factor XIII

circulates äs a zymogen composed of 2 catalytic A subunits and 2

car-rier B subunits. Thrombin cleavage of a 37-amino-acid N-terminal

peptide results in activation of the catalytic subunit. A Val34Leu

Thromb H;HMW>>I 2 ' Ν ΐ ί : so: 58-1

moφhisnι ol'lhe caialyiic M.ibiinii i.·« locaied 3 ainino acid roiidues Iroiii

the thrombiii olca\ age site. and ihe los·« coinmon l.eu34 allcle h;i> Iven

associated uiih incroascd plasma lacior XIII acii\ity le\ds (42, 131.

Paradoxically. sevcral rcccnt smdies suggosi Ihm l ho "high acii\iiy"

Leu34 allclo may he associalod wiili a decreased ri.ik öl' MI (44. 45i.

ischemic ancrial siroko (46). and vonons ihromboiic disoasc (47l, äs

well äs an incrcascd risk öl' hemorrhauic siroke i4Si. Tho niochanism

for the apparent aniiihronihoiic effccl οΓ ιΐιο lacior XI11 Vall.L-u34

Substitution may involve an increased raie of convorsion οΓ lacior XIII

to activalcd lacior XIII by thrombin lhai resuli* in an aliered

cross-linkedfibrin clol simciure (49l.

Levels of oilicr (.'»amiltitiini l'aciim

Recenily. clcvaicd levels of lacior \'. bin not i'aclor X or

proihrorn-bin, were associated w ilh occurrence οΓ Ml aller adjusting l'or oihcr risk

factors (SOi. liocausc ol'the reirospeclive naiure oflhi.s siudy, onc can

not distiniiuish whoihor increased lacior V acii viiy is a caiise ralhcrihan

a consequence of Ml. To our knowlcdgc. Ihc a>socialions of lovcls of

coagulation laciors XI. X. and IX. V. and proihronibin with arlerial

thrombotic disease have noi heen assc.ised in pro^peciive Mudics.

Inherited Thrombophilic Disorders

Protein (', Protein S. Antiihroinhin Deßriency

It is \\cll-osiahlishod lhai congenital det'iciencics of protein (.'..

pro-tein S, and anliilirombin are associalecl wilh an increased ri.ik of venous

thrombotic disease. \Vhelhcr congeniial deficiencies of thesc

naturally-occurrin» coagulalion inhibitors are associalecl \\iih increaied risk of

arterial thromhoiic di.scasc is less clear öl. 52 i. In a pooled analysis

of 8 small series of anorial cerebral infarclion in predominanily young

patients i n = 3l 1 1. deficiencies of proiein S. proioin ('. and

anlithrom-bin were noied in 1 1.')' i. 2. 2' i. and 2.2'.'·; of cases. respecli\d\ (53 1.

However. in niaiiy -of ilu.se siudies. inicrpretalion of rcsuhs is limited

due to small mimhcr< of subjeeis. lack of controk lack of follou-up or

family siudic.i lo docinnenl inherilcd v>. acquiroil ddicicncy. and

pop-ulation adini.Miire. Morc recenl smdies of unselecied young adiills with

acute cerebral ischemia suggoit lhai aciile. iransioni or acquired

defi-ciencies of the naiurally-occurring anticoa.suilimis are noi iincommon.

but that iruo congoniial deticiencies are rare (54-.Ϊ6). Several recent

pediatric SCIUN siiajjcsi ι hat prolein C" or proioin S deficiency may be

present in äs many a*. 5-15'ί ofchildren wilh ischemic siroke (57-591.

However. in Mime case> inie con«enilal deficiency has not been clcarly

documeniod. and oilior |»xliairic siudies ha\c not confirmed these

asso-dations 1 60. 6l l. Taken logether. l ho evidena: suggests ι hat

deficien-cies of natural anticoaüulanls are likoly 10 be assoeiaied wilh arlerial

thromboiic disease. panicularly in very younu indiviüuals with acule

cerebral ischemia. Ho\vc\er. since ihe prevalences of theso deficicMicies

are low even amon;j younc palienls. ihey accouni for a small proporlion

of cases.

ActivatetI Protein (' Kevxmnre and l-'ucior \ ' Leiden

Resistance lo ihe anticoaguluni effect of acli\ated prolein C! is mosi

commonlj due lo a nucleoiide (Ί 1 69 1 Λ nunaiion in the szene encodinu

factor V (hat resulis in an .-\rs5()6(iln iiuiuiiion (factor V Leiden), "l'his

mutation occurs al Ihe initial acli\aled protein C cleava»e site wilhin

the factor Va heavy chain and rc.siilis in a prothn>mbotic siate becaibe

of a roducod MIO of facior \'a inaciivaiion. l-'acior V l .eiden is themojt

coiniDon iionoiio facior as.ioc.kned with risk of venous ihroinboenüxiBt;

disease, and is foinid in approximaiely 3-5'.i öl the Caucasian ρρρ&,«

·-''^

A relaiionship betwecn factor V Leiden and arlerial ihr

ease was first suggosiod by Hohn öl al. (64). who rcponed twoyöj

women lagos 33 and 34 years) with Ml and homo/ygosily forf

Gln5()6. However. nu>sl subscquenl siudies have failed to.-j

associalion boiween factor V Leiden and coronary disease on,

MI (65-71), even among patienls who dcveloped acutc coromey.'^

dromes al a young age (72-74). In contrasl, Roscndaal et al. (

öd a higher risk of non-falal Ml among young female cariieisoi

torV Leiden (OR 2.4, 95<i Cl 1.0-5.9) lhat was particulalyü

among smokers (32-fold increased risk comparcd lo nonsi

women withoul ihe facior V muiation). In a largc. casc-controlsta

middle-aged-to-elderly Duich men, Doggen el al. (76) alsore

inercased risk of MI assoeiaied with hetcro/ygosity for factor Vi

(OR l .4:95Ύ Cl 0.8 2.2) ihai was mosi pronounced in men wjtb.oj^v

cardiovascular risk faclors. ... .yi

:

y$

\Vhile facior V Leiden is noi a risk facior for ischemic sbobJll

middlo-agod lo elderly patienls. (66. 68. 71, 77), some studkti

youngor adults (<45-50 years) indicale a higher prevalenceoffkliirl

Leiden in siroke cases ihan conlrols (56), bul mosl shuwnoi

(78-80). In an Italian siudy (56). the risk of siroke associaledS

lacior V Leiden mutalion was sinniger in women Ihan men, j

smaller North American study of exclusively young womenί

association (80). In children. recent data suggesLs that i

may accouni for approximaiely 2()'.i. (if cases of is

(58-60.81).

The relaiionship beiwecn lacior V Leiden and

occlusixc disease has been examined in thrcc case-cpnöol-S

two siudies, Ihe fiequcncy of facior V Leiden carriershiJMiii

among eases. whiclns higher than the population ]

In conlrasl. a larger AuMrian siudy of 336 patients. with']

arterial occlusion and 300 conlrols observcd a similar f

facior V Leiden mulalion betwecn cases (8%) and controb(

Takcn together. ihe cumulaiive resulis of studiesexaminjflgl

tionship of facior V Leiden and incident arterial throrabotic'i

suggest lhat facior V Leiden is noi a major cardiovascülar.lill

but may assumc increased imponancc in cenain patienti

panicularly children wrih ancrial siroke and young womeiM

cardiovascular risk factors. Allernatively. the positive findingl»

only a l'ew of Ihe puhlished siudies suggesls the possibilityofi

positive associalion due lo population admixture. AddiöofHll

tion siudies involving larger numbers of subjects i

siudies lhat assess geoelic linkago will bc required to ί

issues.

Thcre is some ovidcncc lhat aclivaied proiein Cfesislaeeil

ence the risk of arlerial ihroinboiic disease even in!

factor V Leiden mutalion (85, 86). In a cross-!

men and women. carolid and feinoral artcry atherosderasBi

prevalent athero-ihromhoiic disease were associated '

response lo APC (86). In ihis study. less than 50% of tbe«

APC resislance were carriers of the factor V Leiden i

ing lhai oihcr gcnelic and environmental factors raay <

APC resislance phenolype äs well äs to Ihc riskof art«?W<

disease (86.87). I Iowever. the e\ aluation of a phenotypcr"

contexl of a cross-seclional study raises Ihe issue of *

resislance is a consequence rather Ihan a cause of ί

thrombosis.

Kcincr ci al.: l liimosiaiic Ri^k I-adors and Ancrial Throinhoiic

fG20210A Mutation

i is.the circulating precursor öl' ihronibin. which pliiys ;i

sinfibrin fonnalion and ihrombosis. A G to A iransition in

520210 »ithin ihe .V untranslaled region of ihe proihromhin

l with elcvalcd circulating prothronibin levcK, and

identified äs a risk lacior l'or venous thromboembolic

fe(88)..This mutalion is presem in about 2'i of Caucasians. (>ιιι

—

i;

--«increases froni Nonhcrn lo Southern F.nmpc (89).

V leiden, the rclationship bclween ihe prothrombin

fc Imitation and risk of arlerial ihronibolic (iisease is

conirover-jiiost of the published studies. ihcre was no as.iociation beiween

!0f the 202ΙΟΛ allele and risk of acuie MI or coronary

: (90-94). Whilc ihcsc rcsults suggest llial ihi; prothrombin

W is not a major risk facior for coronary hcarl discase. llic sial is ·

rof many of ihcse studies lo deiect an association \* limhcd

flriatively Iow frequcncy of ihe imnalion in ihc gencral

popula-I pooled analysis of 1.115 palicnis and 1.888 conirols l'roin

[.Franco et al. nolcd a significani association in l he Mudies lhat

itootifioed to patients wilh MI (OR = 2.5: 95',?· CI l .5 4..?). bin no

ι in die pooled analysis of Mudies lhat iricludcd eases wiih a

f ränge of athero-thrombolic disease (95). In the: largest single

ii'icase-control study of patienls with MI (560 paticnis under

ί70)

(

the prothrombin 202 1 OA allele was associalcd wiih a

6) incrcascd risk of MI (hat incrcascd substaniially 10

e-pfesencc of tradiiional cardiovascular risk factors such äs

Jhypertension (76). Similarlv. in a small case-conirol siudv

|:<45 years old with acute MI, Roscndaal et al. reponed a

l risk of MI associatcd wilh prothrombin 202 1 ΟΛ thai

'enhanced in the presencc of smokin» or other meiabolie

of studies examining the rclalionship bciween

pro-»;"620210A and ischemic artcrial siroke in young adults (56.

dolder subjccts (90, 94) have bcen negali\ e. The exceplion is

öl sludy of 72 Italian young inen and womcn (<5() \ears

h ischemic arlerial strokc u ho did not have tradilional

cardio-rrisk factors (OR = 5.1. 95';;. CI = 1.6-16.3) (78). In a

casc-istudy of 148 pediairic palicnis. the prolhrombin 202 IOA allele

with a 5-fold increased risk of sponlaneons ischemic

oke (58), but iwo srnallcr pediairic siudies found no

associa-it,

i

8l). A recenl case-control study found no association of ihe

1 2021 OA allele with risk of periphcral arlerial occlusion in

f patient population (84).

i-together. the resulis of these studies suggest lhat the

pro-ΪΪ20210Α mulalion may bc associaied with a modesily

in-trisk of arterial thronibotic disease thal may as.Miine (tanicular

• in certain subgroups. such äs young wornen wilh MI or

vascular events in children.

rftecsptor Polymorphisms

nein llb/llla

itein llb/llla (integrin «nißi) is the plalelei surlace receplor

in. von \Villebrand facior (vWK). and scvcral other

adhe-ands. The glvcoprolein lila subunil consisis of iwo common

ns,PI.

A:

( I I P A - l a ) a n d ΡΙΛ' (IIPA-lb) . Thisdimorphism isdu e

C niicleoiide .siibMilution involving niicleoiide 1565 witliin

l2 of ihc glvcoprotein l i l a gene. and rcsiilh in a I.cu33/Pro33

substilution thal is associaied wilh a confonnational chan.ue in die

N-ienninal disulfide loop öl 'glwoprolcin lila relatixe to ihe llbrinotien

binding sile (98). The allele l'reqiiency ol'ihe ΙΊ.

Λ:

variani ranges l'roin

~l() 10 18 perceni in (..'auca^ian populatioib. ~8'·ί in African

po|)i:la-lions. and is viriually abscnl in A>ian popnlaiions.

In 1996. V\'eiss et al. rcponcd lhat ihe fre(|iiency of carr\ing al Ica

1

·!

one copy ol'ihe ΙΊ.

Λ:

allele was iwice äs hish in 7l hospiuili/.ed paiienis

wiih MI or unsiablc anüina 1 39.4'. r l compared wiih 68 lios|)iiali/ed

conirol subjecls 1 1 9. l '"t ) (OR = 2.8: 95 '·ί (Ί = l .1 6.4) (99i. \Vhen the

analysis was resiricied lo ihe subgronp of paiienis under Ihe agc of 60.

ihe relaiive risk of acnto coronarj disease associaied \vilh Ihe I'I..

N:

allele increased 10 6.2 (95'. ί (Ί = 1.8-22.4). Since ihis initial repon.

liiere have been over 20 addilional observaiional siudies ihat have

c.xamincd ihc relaiionship bciwecn ihe ΡΙ.

Λ

'/ΙΊ.

Χ

·' polymorphi^ni of

cKcoproiein l i l a and risk öl acnie or Mahle coronary arlerv disease.

\\hile- a leu ofihese siudies have confinned ihe a>Miciation beiween

Ihe ΙΊΛ! allele and increased risk of early-iMixei coronury hcan disease

(73. l (K)- 1 02). ihe majoriiv have nol. The noüaii\e \iudie> include ihe

5 iinoKing al leasi 300 cases ( 1 03- 1 07 1. Iknvever. cornparison öl ihe

resulis among studies is complicaied by differences in definilion öl

coronary disease. selection of conirol siibjecis. and study de>ij;n. Thu.v

neüalive reMilts obserxed in some siudies mi»lii he e.xpected if ihe

;ΙΛ·>Ο-eiaiion is confined 10 younger paiients \\illi MI and/or to specific sub

jiroups. such äs womcn l l()2i. eiuarcnc smoker«. (73 1. individuals wiih

pre-cxisiing coronary atherosclerosis (10h. or indi\iduals wiih

addi-lional geneiic prcdisposiiioris ί 108 1.

Of ihe published siudies ihai have asse.ssed Ihe rclalionship beiween

ΡΙ.

Λ

- and ischemic siroke. one (109) observcd an associaiion only

in subgroups of paiienis under ihe age öl 50 (OR ~ 1.68: 95

r

i CI

1.00- 2^X2 1 and in non-smokers ι OK = 137: 95'·ί (Ί 1.Ι94.74.Ι. and

anoiher only in a small subgronp of womcn (n = 18) who wcre whiie

and had an identillable ciiology ( l 10). In contra.·.!. IWO other siudies

involving a broaderage ränge ol'both men and womcn ( I I I . 112). and

a siudj of male physicians ( 1 06) found no a>sociation beiween ihe l'l .

Λ

"

variant and risk of ischemic siroke.

The assessmem ol'ihe relaiionship between ihe l'l.·'

1

geneiic variani

ofgKcopmiein lila and risk of arlerial ihronibolic disease is aNo

ham-pcred by the lack of a clearlv del'ined el'feci on plaielet fuiiciioii. \\ hile

some studies ha\e deinonsiraicd Ihai Pl/^-posilivc plaielcls displav

in-creased in \iiro platclcl reaciiviiy compared to ΡΙΛ'-negaiive plaielets

(1 13. 1 14). oihers have not ( 1 15. 1 16). The associaiion beiween Pl..'

:

and an increased ihronibolic icndcncy is also tupponcd by experiments

usin» stähle eell lines overexpressin« ihe ΙΊ.

υ

and Ρ Ι Λ · polxmorphic

fornis ol'glvcoproicin llb/llla ( 1 1 7i. äs well äs a recenl auiopsy sludy in

which the presencc ol'ihe ΡΙ.

Λ

· allek· was associaied wilh an increased

l'rec|ueucy (>f aeuie coronary Ihrombo.iis and complicaied

aiherosclerot-ic plaques in men wilh fatal M i d i 8).

In summary. both ihe clinical and experimcnial data assessing ihe

role ol'ihe PL

C

variani of ghcoprotein lila in arterial Ihronibolic

dis-ease have yiclded conlliciing resulis. Addilional moleculargcneiic and

l'unciional analysis of ihe plalelei glycoproiein llb/llla recepior. äs well

äs \iudics iuvolving larger nnnibcr> of subjeeis that allow straiilicalion

by 01 her cardiovascular risk l'aclors. w i l l be recjuired lo resolve ihese

issues.

Iw'llu

Thromb Haernost 2001: 85: 584-95

glycoprotem la (a, integrin) subunit of the glycoprotein ia/IIa receptor have been described. T wo silent single nucleotide polymorphisms. C807T and G873A are in complete linkage disequilibrium with each other, äs well äs with several other single nucleotide polymorphisms located in the adjoining introns ( l 19. 120). Among normal individuals. there is a 5-10 fold Variation in the surface level of glycoprotein Ia/IIa that correlates with collagen-induced platelet adhesion and aggregation in vitro. The nucleotide 807T variant of glycoprotein Ja is associated with increased platelet glycoprotein Ia/IIa receptor levels and in-creased collagen-induced platelet adhesion compared to the 807C allele (l 19-121). The mechanism of the association between the 807T allele and increased receptor density is unknown. The 807T allele may be linked to another polymorphism within the glycoprotein la gene in-volving a regulatory element that controls gene transcription levels or mRNA stability.

The prothrombotic tendency of the glycoprotein la 807T allele is supported by four recent case-control studies. In a study of 2,237 con-secutive German men undergoing coronary angiography, Santoso et al. (122) reported a higher frequency of the 807T allele in individuals who had suffered an MI compared to those without MI (OR 1.57; 95% CI 1.14-2.13) that was most pronounced in obese men under the age of 49 (OR 4.92; 95% CI 1.71-14.2). In a smaller case-control study, Moshfegh et al. reported a 3-fold increased risk of MI in individuals who carried two copies of the 807T allele compared to individuals who possessed the CC or CT genotype (OR 3.3; 95% CI 1.23-8.83) (123). Similarly, the 807T allele was associated with a 2-3 fold increased risk of ischemic stroke in men under the age of 50 (124) and women under the age of 45 (125). In contrast to these 4 positive studies, Croft et al. reported no association between glycoprotein C807T and risk of MI. either overall (OR 0.88. 95% CI 0.74-1.05). in younger individuals, or in subgroups defined by other risk factors (126). Corral et al. also re-ported no association between the 807T allele and risk of either acute coronary heart disease or ischemic stroke in a middle-aged to elderly study population (121). Furthermore, it should be noted that the fre-quency of 807TT homozygotes among the control group of one of the positive studies (5.6%) was considerably lower than other published population studies (123). In summary, preliminary results suggest that the 807T variant of glycoprotein la may be a genetic risk factor for early-onset arterial thrombotic disease, but further studies involving larger numbers of subjects, äs well äs further characterization of the mechanism responsible for increased receptor levels, are required to settle this issue.

Glycoprotein Ib/IX

Glycoprotein Ib/IX receptor is the major platelet receptor for von Willebrand factor. Two polymorphisms that affect the amino acid se-quence of the glycoprotein Ib heavy chain (glycoprotein Iba) have been described. The first, an ACG to ATG Substitution at codon 145, results in a Thr/Met dimorphism that is responsible for the human platelet anti-gen (HPA)-2 System. The second is a size polymorphism that results from a variable number of tandem repeats (VNTR) of a 13-amino-acid sequence (Ser399-Thr411) that is present in either one (D), two (C), three (B), or four (A) copies (127). The HPA-2 and VNTR polymor-phisms are in strong linkage disequilibrium with each other. The HPA-2 Thrl45Met dimorphism results in a protein conformational change in a region adjacent to the vWF binding region of glycopro-tein Iba, but an effect on in vitro platelet function or von Willbebrand factor binding has not been demonstrated (128, 129). The VNTR poly-morphism involves the macroglycopeptide region of glycoprotein Iba, 588

with the addition of each repeat resulting in an increased distan^· between the ligand binding region and the platelet surface. While it hj. been hypothesized that this distance Variation may affect platelet \IK ceptibility to shear-induced activation (127), this has yet to be demon-strated experimentally.

Despite the lack of an associated prothrombotic phenotype, se\erai case-control studies have noted a 2-3 fold increased risk of coro-nary heart disease or stroke associated with the Met 145 (HPA-2bi or VNTR-B allele. particularly in younger patients (130-132). In contra^ Ardissino et al. did not find an association between the HPA-2 pol\-morphism and premature MI in a case-control study involvins pre-dominantly men (73). Two other studies did not observe an association between HPA-2 or VNTR genotypes and risk of ischemic stroke in older patient populations (109, 112).

Other Hemostatic Factors Fibrinohtic Svstein

The fibrinolytic System consists of the circulating proen/\mc plasminogen, which is converted to plasmin by the plasminogen acuu tors, tPA and urokinase. Plasminogen activator inhibitor-1 (PA1-111> j major inhibitor of the fibrinolytic system, and is synthesi/ed h\ j variety of cell types, including platelets, endothelial cells. and \avcuL· smooth muscle cells. Data from several prospective studies indicaie r association between plasma levels of various fibrinolytic markers 11 c tPA antigen, PAI-1 activity and antigen levels, and fibrin degradamM· products) and risk of arterial thrombotic disease (133-137). Thi· pre* ence of increased levels of PAI-1 mRNA in atherosclerotic plaqui· (138) and the association of elevated plasma PAI-1 levels uith ^ clinical atherosclerosis (139) also suggest a role for impaired fihnno!» sis in the occurrence of athero-thrombotic disease. However. ihh ro; is complex; Inhibition of fibrinolysis may promote thrombosi·,. h.< also may inhibit matrix metalloendopeptidase activation and xascui;· smooth muscle proliferation. which may alter atherosderolic plaqu: stability (140).

The Interpretation of cardiovascular epidemiologic Studie1· <>t fit".' olytic markers is complicated by several other factors. Plasma ιΡ·\ jn1

gen and PAI-1 activity levels are highly correlated. m pari hecaux.· '* tPA antigen assay measures both free tPA and tPA complcvii · PAI-1. which increases in patients with high PAI-1 le\els du.· i··* layed clearance (141). Measurement and Interpretation öl pb"··

PAI-1 activity levels are particularly problematic due to dmmai >-·'» tion, spontaneous transformation of PAI-1 to an inaclhe or 'sj'Z' form at neutral pH, and the potential for release of PAI-1 from pbicsf· these necessitate special measures during blood collection and Μ"Ϊ'

processing (142). PAI-1 is also an acute phase reactant. and cirniU'·?·· levels are influenced by a number of hormones and c>tokinev 114"· Λ creased PAI-1 levels are also correlated with obesit\. hNpenrwfe-"--· and hypertriglyceridemia, (i.e., the "insulin resistancc s)mlf« Thus, in most epidemiologic studies, the association between Γ V tPA levels and risk of arterial thrombotic disease ma\ ho c> >«<''·-"'*'' by these other cardiovascular risk factors.

Reiner et al.: Hemostatic Risk Faclors and Aricnal Thromholic Disease

l allele (144-146), these findings have not been con- Lipoprotein (a)

fi'.*W>

' , m xjieral larger studies (147-149). A meta-analysis of nine

'". -vh:! mcluded 1.521 cases and 2,120 controls indicated an

over-<S'*-fcih increased risk of MI associated with the 40 allele (OR l .23,

1' Π |'|t4-l.45) (150). However, the increased risk was confined to *. ,-ί-wtJp of case-control studies in which both cases and controls

trom "high-risk" populations.

a sulfur-containing amino acid that is formed äs an Λ compound during methionine metabolism. Congenital >, ,·-.ν> ^timiria. a rare autosomal recessive metabolic disorder, is asso-„' min *ery high levels (>100μηιο1/ί) of fasting plasma total

Sl r. o>ieini. and is often accompanied by premature thrombotic

jiv·-^' imohing the coronary, cerebral, and peripheral arteries. Mild ι.,-rtiiniocweinemia (i.e., >15μηιο1/ί) is not uncommon in the

•,',:r.u popukition. and is associated with a variety of acquired and

:'i\ uiors. including increasing age, male sex, cigarette smoking,

r.ivaK'i'iMiniption of nutritional factors (folate, vitamin B6, vitamin

» . , rcnal failure. and heterozygous deficiencies of enzymes involved ,; ΜΙΗΚΛsteine metabolism (151, 152). The adverse effects of sfvoivteine include vascular endothelial injury, smooth muscle yiitiraiion. oxidation of low-density lipoprotein cholesterol, and in-Aition of a prothrombotic vascular endothelial microenvironment

,<l. 152).

>iincc the very high levels of plasma homocysteine that accompany iejenital homocystinuria are strongly related to risk of arterial

throm-\& disease, some degree of association with mildly elevated levels i.s tut plausible. There have been a number of case-control and

cross-ctiunal studies that indicate that mild hyperhomocysteinemia is

asso-ίαά ttith an increased risk of coronary heart disease, cerebrovascular

käse, and peripheral vascular disease (153-155). However, it should *soted that homocysteine levels may increase following acute arte-•al thrombosis (156, 157), which complicates the Interpretation of in which levels are measured retrospectively. Prospective assessmg the risk of arterial thrombotic disease associated with »oied homocysteine levels have yielded mixed results with most showing a weak or no association (154. 155). The strengest \\iaiions have tended to occur in studies involving subjects with wxisling cardiovascular disease. This raises the issue of whether the wied homocysteine levels are a consequence rather than a cause of Winical atherosclerosis.

•Mild hyperhomocysteinemia has been associated with a thermola-wriant of MTHFR that is due to a C to T transition at nucleotide Homozygosity for the C677T MTHFR variant is present in 5% fo ofCaucasians. A recent meta-analysis of 23 case-control studies ling over 12,000 subjects concluded that homozygosity for the 'labile MTHFR variant is not associated with risk of cardiovas-disease (158). This absence of an association raises doubt about •'role of mild hyperhomocysteinemia äs a cause of arterial throm-: disease. However. based on the association of MTHFR and

κΐίίι Dcysteine levels, the predicted relative risk of the thermolabile genotype is small (possibly no more than a 20-percent risk ,· pr*. öse), and even a meta-analysis may have insufficient power to

Ö a risk of this magnitude. Ongoing randomized clinical trials of and B vitamin supplementation to reduce plasma homocysteine Should provide more definitive answers to the public health (ns surroundins homocvsteine and risk of athero-thrombotic

Lp(a) is a complex serum lipoprotein composed of an LDL particle linked by its surface apolipoprotein (apo) B-100 via a disulfide bond to a unique and highly polymorphic glycoprotein, apo(a) (159). Apo(a) shares extensive structural homology with plasminogen, and consists of a ηοπ-functional serine protease domain, a variable number of multiple

repeats of kringle IV, and a single copy of kringle V. The variable number of kringle IV repeats is due to varying number of copies within the apo(a) gene. and results in at least 35 different molecular weight isoforms.

Plasma Lp(a) levels are largely genetically determined (160). and vary inversely with apo(a) isoform size. Levels increase in patients with renal disease, and are lower m white populations compared to African Americans (161). In contrast to plasma LDL levels, Lp(a) levels are unaffected by diet. physical activity, and most lipid-lowenng agents. On the other band, estrogen replacement therapy and high-dose niacin can lower plasma Lp(a) levels.

Experimental evidence suggests that Lp(a) may contribute to the occurrence of arterial thrombotic disease through effects on both atherosclerosis and thrombosis (159). Oxidized Lp(a) can be internal-ized by macrophages and contribute to delivery of cholesterol to sites of vessel injury. Because of its structural similarity to plasminogen. Lp(a) inhibits plasminogen binding to fibrin and endothelial cells (162). and thereby inteiferes with fibrinolysis and promotes thrombosis (163). Lp(a) accumulates in atherosclerotic lesions, and has been impli-cated in the progression of coronary atherosclerosis (164). Lp(a) may also impair endothelial function and induce smooth muscle prolifera-tion.

Despite these experimental data. clinical data supporting the role of Lp(a) äs a cardiovascular risk factor are controversial.

A number of studies have shown an association between elevated Lp(a) levels and mcreased risk of coronary. cerebrovascular, and peripheral arterial disease (161, 165, 166). However, like homo-cysteine, Lp(a) levels increase following an acute thrombotic event. Thus. retrospective .studies may not be able to discern whether elevated Lp(a) levels are the cause or consequence of cardiovascular disease, and prospective studies have yielded conflicting results (167-174). Based on the cumulative epidemiologic data. if an associa-tion between Lp(a) levels and cardiovascular exists, the effect is likely to be small and confined to a small proportion of the popula-tion with the very highest serum levels (175), onset at a young age (170) or those with additional dyslipidemias (167, 174, 176. 177). Further evaluation of the significance of apo(a) isoform size on the occurrence of athero-thrombotic disease (171. 177) äs well äs other genetic mechanisms that underlie Lp(a) heterogeneity (178) may lead to improvements in the use of Lp(a) äs a determinant of cardiovascular risk.

Thrombomodulin

Thrombomodulin is an endothelial cell surface receptor for thrombin that functions äs an anticoagulant by greatly accelerating thrombin-induced activation of protein C. A soluble, truncated form of thrombo-modulin circulates in plasma, but its significance is unknown. In a large prospective case-cohort study. decreased plasma thrombomodulin levels were associated with an increased risk of MI (179). Some reports have suggested that several rare polymorphisms within the thrombo-modulin promoter and coding sequence may be related to risk of MI (180-182).

inioinn Macniosi ζυυι. fo. j

Recommendations for Screening

Despite the proliferation of epidemiologic studies. increased fibrino-gen level remains the only hemostatic biomarker that has been clearly

identified äs a risk factor for arterial thrombotic disease. And even for

fibrinogen one may question the causality of the association. The deci-sion whether to perform screening for a hemostatic marker, however, should be based on whether screening offers a cost-effective way of identifying individuals who would benefit from treatment. Therefore, screening of plasma fibrinogen is generally not advocated because levels are favorably affected by modification of other risk factors (e. g., smoking cessation, exercise, and post-menopausal hormone replace-ment therapy) and because a beneficial effect of lowering fibrinogen level on cardiovascular risk has yet to be demonstrated. There is some epidemiologic and biologic evidence that elevated plasma levels of factor VIII. homocysteine, and Lp(a). äs well äs several genetic hemo-static variants (factor V Leiden, factor XIII Leu34, prothrombin G20210A, the PLA: variant of glycoprotein lila, and glycoprotein la 807T) are related to the occurrence of arterial thrombosis. However, there is currently no evidence that screening for any of the above hemo-static markers either within the general population or among subgroups, such äs individuals who develop arterial thrombotic disease at a young age, would have any prognostic or therapeutic consequences. Several inherited conditions associated with venous thrombosis (deficiencies of protein C. protein S or antithrombin, factor V Leiden, and prothrombin G20210A) are not äs important in the overall occurrence of arterial thrombosis. except possibly in subgroups such äs the very young or pre-menopausal women. Thus, evaluation of these disorders in the setting of arterial thrombosis may be considered in young patients once more likely candidates (lipid disorders, hyperhomocystenemia, lupus anti-coagulant/antiphospholipid antibody syndrome) have been excluded, or in MI patients without significant coronary atherosclerosis (183). In addition, a thrombin time and fibrinogen level should be obtained in these young patients to exclude congenital dysfibrinogenemia, another rare cause of both venous and arterial thrombophilia (184).

Conclusion

With the progression of the human genome project and the recent focus on identification of inter-individual genetic Variation, the poten-tial for evaluating the relationship between functional variants of candi-date genes, associated prothrombotic phenotypes. and risk of arterial thrombotic disease will greatly increase. Several guidelines for future studies that evaluate novel hemostatic markers should be considered. First, molecular epidemiologic studies should focus on genetic muta-tions that are associated with clearly defined effects on hemostatic function and that contribute significantly to inter-individual phenotypic Variation. Second. examination of homogenous populations and pre-cise. well-defined clinical outcomes should enhance the ability to detect associations. For example, hereditary determination by äs yet unidenti-fied genetic factors may be particularly important in MI that occurs at younger ages and in women (185). Furthermore, restriction to ethnical-ly homogeneous populations or eliciting detailed Information regarding genetic ancestry from study subjects should minimize the problem of false positive associations arising from population admixture. In addi-tion, any preliminary associations observed in population-based studies should be complemented with family-based studies that provide evi-dence that the marker of interest is genetically linked to the disease (186). Third, large sample sizes will be required to provide enough statistical power to assess the interaction of genetic susceptibility mark-590

ers with other genetic and environmental risk factors. UndoubtedK other candidate genes and associated functional polymoφhisms m volved in arterial thrombotic disease will continue to be ideniiile^ Integration of knowledge involving novel hemostatic biomarkers at iht

genetic, biochemical, and clinical-epidemiologic levels should lead i, significant developments in understanding the pathophysiology of arte rial thrombotic disease, the assessment of cardiovascular risk, and thc targeting of specific antithrombotic therapies to high-risk individuaN

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