5G POLYMORPHISM WITHIN THE PROMOTER OF THE PLASMINOGEN ACTIVATOR INHIBITOR-1 GENE IN PATIENTS WITH DEEP VEIN THROMBOSIS

ThrombosisResearch,Vol.84,No.6, pp.431P443,19% CopyrightO 1996ElsevierScience Ltd PrintedintheUSA. Allrightsresewed G049-3848/96 $12.00+.IM

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PII S0049-3848(96)00211-3

A NOVELG/AANDTHE4G/5GPOLYMORPHISM WITHINTHE PROMOTEROF THEPLASMINOGEN ACTIVATORINHIBITOR-1GENEIN PATIENTSWITHDEEP VEINTHROMBOSIS NellyGrubicl,Mojca Stegnarl,Z,PolonaPeterne12,AlexandraKaiders,Bemd R. Binderl Univ.Vienna,IDept.Vase, Biol.& Thromb.Res. and 3Dept.Med. Comp. Sci.,Vienna,Austria and Wniv. Med. Ctr.,TrnovoHosp. Intern.Med., Ljubljana,Slovenia

(Received25 September 1996by Editor1.Pabinger;revisecVaccepted24October1996)

Abstract Plasmaplasminogenactivatorinhibitor-1(PM-I) levelwas observedto be associated with sequence variationsat the PAI-1 locus. Therefore, PAI-1 gene promoter was screenedfor possiblynew polymorphismsand to investigatethe contributionof these sequencevariationsto PAI-1 levelsin patientswith deep veinthrombosis(DVT). DNA was isolated from blood of 83 consecutiveunrelated patients (42+11 years old) and from 50 apparentlyhealthysubjectsof similarage and gender distribution.Six fragmentscovering DNA sequence-1523base pairs(bp) upstreamfromthe start of PAI-1 gene transcriptionto +90 bp in the first exon, were amplifiedby polymerasechainreaction and analyzed by single-strandconformationpolymorphisms.Two polymorphismswere found: a previouslydescribed4G/5G deletion/insertionpolymorphism675 bp upstreamfromthe start of transcriptionand a novelG/A singlebase substitution polymorphismfln-therupstream at -844 bp. The two polymorphismswere in strong linkage disequilibrium.Significantdifferencesbetween patients and controls were observed neither for the frequenciesof the 4G/5G alleles(0.60/0.40 and 0.59/0.41, respectively)nor for the frequenciesof the G/A alleles (0.33/0.67 and 0.41/0.59, respectively),The distributionof both polymorphismswas similarin idiopathicand secondaryDVT as well as in first and recurrentDVT. In patientsassociationbetween the 4G/5G genotypesand PAI activitywas observed,with the highest values in the 4G/4G genotype(13.3 U/mL), medianvaluesin the 4G/5G genotype(9.8 U/mL) imd the lowest values in the 5G/5G genotype(2.0 U/mL). Despite the lack of association between the G/A genotypes and plasma PAI-1 levels, electrophoreticmobilityshift assay showed specificbindingof a nuclear protein from human vascular endothelial cellsextractsto both the G and the A variant,suggestingfictional importanceof this novelG/A polymorphismin regulatingthe expressionof PAI-1 gene. Copyright01996 ElsevierScienceLtd

Key words:PAI-I gene,polymorphism,plasmaPAI-1, deep veinthrombosis Correspondingauthor: Mojca Stegnar, UniversityMedicalCentre, Trnc-.mHospital of Inte:”;+i Medicine,Riharjeva24, 1000Ljubljana,Slovenia,Fax:+386-61-333155

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Considerableevidencesupportsthe view that elevatedplasmaplasminogenactivator inhibitor-1 (PAI-1), the principalrapid inhibitorof tissue-typeplasminogenactivator, is mainlyresponsible for the reduced fibnnolyticactivityobservedin patientswith myocardialinfarction(l-3) and in patientswith deep vein thrombosis(DVT) (4, 5), In young patientswith myocardialinfarction cause-and-effectrelation has been establishedbetween PAI-I and risk of reinfarction (6). Increasedlevelsof PAI-1 correlate also with the developmentof recurrent DVT (7), indicating that impairedfibrinolysisdue to increasedPAI-1 mighthavea role in the pathogenesisof vascular diseasepossiblyby contributingto a pro-thromboticstate. The cause of high PAI-1 in vasculardiseaseis not clear. Increased plasma levels of PM-1 are relatedto severalanthropometricand metabolicfactors of whichbody constitution,plasmalevels of triglycerides and insulin seem to be the most important (8-10). In addition to these environmentalfeatures there is data suggestingthat PAI-1 levelsmay be influencedby genetic polymorphism.Levels of PAI-1 have been found to relate to sequencevariationsat the PM-1 locus. MultiallelicCA dinucleotiderepeat polymorphismin the third intron, Hind III restriction fragment length polymorphismat the 3’ end of the PAI-1 gene and a single guanosine deletion/insertion(4G/5G polymorphism)-675 base pairs (bp) upstream from the transcription start of the PAI-1 gene were associatedwith PAI-1 levelsin myocardialinfarction(11-14). It seems that sequencevariationsin the promoter of PM-1 gene are of importancenot only in regulatingexpressionof the PM-1 gene but influencealso the relationshipbetween metabolic factors suchas triglycerideand PM-1 levels(15, 16). The aim of the present study was to screen PM-1 gene promoter sequencesfor possiblynew polymorphismsand to evaluatethe contributionof these sequencevariationsto PAI-1 levelsin patientswith DVT. SUBJECTSAND LABORATORYMETHODS Subjects All subjectsinvestigatedwere Caucasian,originatedfrom Central Europe and were unrelated. They were asked to participateafter they had given their fill informedconsent. The study was approvedby the SloveneEthicalCommittee. Eighty-threeconsecutivepatients (35 women and 48 men) aged 19 to 60 (42+11, meanMD) years, were asked to participateat least three months,but on average 18*1Omonths(meafiSD) after acute DVT. At the time of the acute event clinicaldiagnosisof DVT was confirmedby at least one of the followingmethods:ultrasoundveinimaging,impedance pletismography,isotope or contrastvenography.Locationof DVT was in 75 (90’%0) cases proximallower limb,in 3 (4’%0) distallower limband in 4 (5’XO) upper limb.One patientsufferedthrombosisof vena cava. Sixtyfive (78?40)patients suffered a single event and in 18 (22%) DVT was recurrent. Pefision/ventilation lung scanningwas petiormedin patientsin whom pulmonaryembolismwas suspected and a positive scan was establishedin 17 (20°/0)patients. In 34 (41°/0)patients no common factors predisposingto DVT could be established(idiopathicDVT). However, in 49 (59%) DVT was secondaryto common predisposingfactors such as surgery (N=17), trauma @=15), bed-rest (N=lO) and immobilization(N=lO). In women the most frequent predisposing factors were oral contraceptives(N=12), hormonal replacement therapy (N=4), puerperium

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(N=4)andpregnancy(N=3).At the time of blood sampling25 (30%) patientswere receivingoral anticoagulant treatment. None of the women was taking oral contraceptives or hormonal replacementtherapy.When includedin the study patientswere screenedfor hemostatic defects predisposingto DVT. Fiftyapparentlyhealthysubjects(23 womenand 27 men) 21 to 60 (43+10,metiSD) years old were asked to participateas controls. They were mainlystudents and membersof the hospital staffwith no DVT or other cardiovasculardisordersin theirhistory. Blood samplingand DNA isolation Blood sampleswere obtainedfrom fasting subjectsbetween 7 and 9 a.m. after a 20 min rest. Blood was sampledfrom an antecubitalvein, in most cases without applicationof a tourniquet. For measurementof hemostatic factors blood flowed directlyinto precooled siliconizedglass vacuumtubes with 0.13 mol/Ltrisodiumcitrate(1 volumeof citrateto 9 volumesof blood),was placedin ice water and then centrifugedwithinone hour for 30 min at 2000 g and 4° C. Platelet poor plasmawas transferredto smallplasticvials,frozen in liquidnitrogenand stored at -70° C until analyzed. For biochemicalassays blood was collected into vacuum tubes without an anticoagulant;afler one hour serumwas harvestedand analyzedthe sameday. For DNA analysis blood was sampled into K3-EDTA vacuum tubes (Becton Dickinson Vacutainer System Europe), thoroughly mixed with the anticoagulantby inverting the tube severaltimes and stored at -70°, DNA was isolatedusing commerciallyavailablegenomicDNA purificationkit (WizardTM, Promega,Madison,WI, USA). Screeningfor PAI-I genepromoterpolymorphisms Six fragments covering the promoter between -1523 bp upstream from the start of the transcriptionsite to +90 bp in the first PM-1 gene exon (Fig. 1) were amplifiedby polymerase chainreaction.200-500ng of genomicDNA in a 25 WLvolumewere used as a templatefor the following6 pairsof primers(ViennaBiocenter,Austria):TAA GGG CCA CGG GGC CCG TAC TGG TCC ATA G and CAA TTC ACC ATC GTG TAG AAT GAC for fragmentI, CTG ATT CTA CAC GAT GGT GAA TTG and CAC GAG CTG CCC GCT AGG ACT TGG GCC for fragmentII, CCT GCC ATG AAT TGA CAC TC and GTG TCC AG ACT CTC TGT G for fragmentIII, CAC AGA GAG AGT CTG GACAC and CTG CCA GGT TGA CTG TCT CG for fragmentIV, CAG ACA GTC AAC CTG GCA GG and GTC TGA ACA GCC AGC GGG TC for fragmentV, GAC CCG CTG GCT GTT CAG AC and GGC TGC TGA GCT GCA GGA ATT CAG CTG CTG for fragmentVI. The primerswere designedaccordingto the published sequenceof the PAI-1 gene (17). Conditionsfor polymerasechainreactionwere: 94° C: 40 see, 62° C: 40 see, 72° C: 40 sec for 30 cycles. Double strandedDNA from polymerasechainreactionwas denaturedto a singlestrandedDNA and analyzed for single-strand conformation polymorphisms (18) in a semi-automated programmableelectrophoresisinstrument(Phastsystem)usingprecastpolyacrylamidegels (Phast gels 8-25 gradient).Electrophoresiswas run under non-denaturingconditions(Phast gel native bufferstrips,all Pharmacia,Sweden).Electrophoreticconditionswere adjustedto obtain optimal separation of differentDNA entities (pre electrophoresis:400V, 5mA, IW, 200Vh; loading: 400V, 0.5mA, IW, 6Vh; separation:400V, 5mA, IW, 300Vh),Gelswere silverstained(19).

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FIG. 1 Schemeof the PM-1 gene promoter.Sixfragmentscoveringthe promoterfrom -1523bp from the start of the transcriptionto +90 bp in the firstexon,amplifiedby polymerasechainreaction are shown. Single-strandconformationpolymorphismsfound in polymerasechainreactionfragmentsIII and IV were confirmedby direct sequencingin two homozygoussubjects.Polymerasechainreaction products were first purified on purification columns (Qiaquick, Qiagen, USA) and then precipitated by ethanol. They were sequenceddirectly using the dideoxy method with DNA polymerase(AmpliTaq,Perkin Elmer, USA) and capillaryelectrophoresisequipment(Abi Prism 310, Perkin Elmer, USA), Primerspurifiedby highperformanceliquidchromatographywere the sameas those utilizedfor the polymerasechainreactions. Electrophoreticmobilityshljitassays A 28-mer oligonucleotidewas designedfor each type of the polymorphicGIA sequence:TTA GCG GGC AGC TCG AGG AAG TGA AAC T for the G variant and TTA GCG GGC AGC TCG AAG AAG TGA AAC T for the A variant. Complementaryoligonucleotidesfor each variant were synthesized by an Applied Biosystems Oligonucleotide Synthesizer (USA). Equimolar amounts of the complementaryoligonucleotideswere annealed, and radioactivity labeledby fillingin the overhangswith Klenowenzymein the presenceof [ct32P]dATP, Labeled probes were purified on 7“Apolyacrylamidegel, eluted and precipitatedwith ethanol.Nuclear extracts were prepared from humanvascularendothelialcells (HUVECS)accordingto Dignam and coworkers (20) with exception of a modifiedbuffer C, which was used during nuclear extraction(20 mM Hepes-KOH,PH 7.9, 4Z0 w Nacl, @O M ~4)2s04> 1s W M@2, O.z mM EDTA, 0.5 rnM dithiothreitol,zs~. glycerol).Cellswere eitherunstimulatedor treated with lipopolysaccharide.In electrophoretic mobility shit? assays, l-5vg of nuclear protein were incubatedwith 0.3-1.0 VLof radioactivitylabeledprobe (105cpmhg) in bindingbuffer(100 mM HEPES-KOH, pH 7.9, 5 mM EDTA, 25 mM MgC12,250 m Kcl,5mM dithiothreitol50 % glycerol).Protein-DNAcomplexeswere loaded on 5Y0polyacrylamidegel and run 2-3 hours at 150V. The gel was driedon Whatman3MMpaperand autoradiographedovernight.

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Haemostasisand biochemicalassays PM-1 antigenwas determinedby an enzyme-linkedimmunosorbentassay and PAI activityby a chromogenic substrate assay utilizing commercially available kits (Imulysew PAI-1 and Spectrolyse@fibrin,Biopool, Sweden, respectively).Resistance to activated protein C was estimatedby a modifiedactivatedpartialthromboplastintime (Coatest@,Chromogenic,Sweden). Antithrombin III, protein C and plasminogen were determined by amidolytic assays (all Berichrom, Behring, Germany) according to the instructions of the manufacturer. Glucose, triglyceride, total-cholesterol, high-density lipoprotein (HDL) -cholesterol and low-density lipoprotein(LDL) -cholesterolwere determinedby routine biochemicalmethods. Body mass indexwas expressedas: (bodyweightin kg)/(bodyheightin m)z. Statisticalmethoa% The skewnessof PAI-1 antigen,triglycerideand glucose distrubutionswas normalizedby logtransformation,while PAI activity was square-rooted, in order to permit use of parametric methods. In the tables the transformed values are presented as anti-log means with gSO/O confidenceintervals(PAI-1 antigen,triglyceride)or squaredmeanswith 95°/0confidenceintervals (PAI activity).The t-test and analysisof variancewere used to comparevaluesbetween the two groupsof subjectsand betweendifferentgenotypes.Alternatively,non-parametricKruskal-Wallis analysisof variancewas utilizedin order to establishdifferencesbetween non-transformedPM-1 antigenand PAI activitylevelsin variousgenotypes.Pearson’scorrelationcoefficientwas used to evaluate associationsbetween PAI-1 and other variables.To compare PAI-I antigen and PAI activitylevels in differentgenotypes,these values were adjustedfor the triglyceridelevels and body massindexwith the use of analysisof covariance. The XZ-testwas used to analyzedeviationof the genotypedistributionfrom the distributionthat would be expectedif the alleleswere in the Hardy-Weinbergequilibriumand to test differencesin frequenciesof genotypesbetweenpatientsand controls. Calculationswere performedusingthe GLM Procedureof the SAS/Statprogram (SAS Institute Inc., Cary, NC, USA). Linkagedisequilibriumbetween the 4G/5G and the G/A genotypeswas calculatedwith the Genepopcomputerprogram(21). P value of <0.05 was taken as statistically significant. RESULTS The patients and the controlshad similarage and gender distribution.The two groups did not differ significantlyin body mass index, fastingblood glucose,HDL-cholesteroland triglyceride levels.On the other hand, patientshad higherlevelsof total- and LDL-cholesterolthan controls (TableI). In two of the PAI-1 promoter fragmentssingle-strandconformationpolymorphismwas found. FragmentIV (Fig. 1) containedpreviouslydescribed4G/5G deletion/insertionpolymorphismat -675 bp from the transcriptionstart resultingin alleleswith either 4 or 5 guaninesin a row. Fragment III (Fig. 1) contained a new hitherto undescribedsingle substitutionof guanine to adenine(G/A polymorphism)flu-therupstreamat -844bp (Fig.2).

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TABLEI Anthropometricand metabolicdata of DVT patientsand healthycontrols(meansMD or means with 95°Aconfidenceintervals,NS: not significant).

Women/Men(N) Age (years) Body massindex(kg/cmz) Glucose(mmol/L) Total Cholesterol(mmol/L) HDL-Cholesterol(mmoVL) LDL-Cholesterol(mmol/L) Triglyceride(mmol/L)

Patients (N=83)

Controls (N=50)

P

35/48 42.2 k 11.4 26.2 k 3,8 5.2 (5.1-5.4) 6.2 t 1.2 1.3t 0.4

23/27 43.9 t 10.0

NS NS NS NS <0.001 NS <(),0()1 NS

26.5 k 3,5 5.4 (5.2-5,6) 5.3 t 1.0 1.3* 0.4 3.4 * 0.9 1.3(1,2-1.6)

4,1 + 1.1 1.5(1.3-1.7)

Frequenciesof the 4G and 5G alleleswere 0.60/0.40in patientsand 0.59/0.41in controls.Allele frequenciesin the novelG/A polymorphismwere 0.33/0.67in patientsand 0.41/0.59in controls. The distributionof the 4G/5Gand the G/A genotypesin both groups of subjectsstudiedis shown in TablesII and III. Therewere no significantdifferencesin the frequenciesof differentgenotypes betweenpatientsand controlseitherfor the 4G/5Gor for the G/A polymorphism.In patients(X2test=5.46), but not in controls(X2-test=l.97) the observedfrequenciesof the 4G/5G genotypes differed significantlyfrom the distribution expected from the Hardy-Weinberg equilibrium. Distributionof the G/A genotypesdifferedboth in patients(~2-test=l1.55) and in controls (X2test=3,95) from the expectedequilibrium.The 4G/5Gand the G/A polymorphismswere in strong linkage disequilibriumin both groups of subjects investigated (patients: p=0,001, controls: p=o.oo5).

El

4G14G 4G15G 5CV5G

G/G GIA A/A

FIG. 2 Typicalsingle-strandconformationpolymorphismpatternsof the 4G/5Gpolymorphism(A) and the G/A polymorphism(B).

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TABLEII Distributionof 4G/5Ggenotypesin DVT patientsand in healthycontrols. Genotype

4G/4G

4G15G

Patients(N=83)

25 (30.1)

50 (60.2)

8 ( 9.7)

Controls(N=50)

15(30.0)

29 (58.0)

6 (12.0)

N

5G/5G

(~0)

The distributionof the 4G/5G and the G/A genotypesdid not changesignificantlyif patientswith resistanceto activatedproteinC (N=9) and with deficienciesof antithrombinIII (N=l), proteinC (N=4) or plasminogen(N=l) were excluded.Likely,the distributionof the 4G/5G and the G/A genotypeswas not significantlydifferentbetween the subgroupsof patientswith idiopathicand secondaryDVT or with singleand recurrentDVT (datanot shown). In subjectsinvestigatedPAI-1 antigenand PAI activitysignificantlycorrelatedwith triglyceride levels(1=0.43and rO.37, respectively),body mass index (r=O.35and r=O.17,respectively)and total cholesterol(1=0.20and r=O.25,respectively).Analysisof covariancerevealed significant influenceof both triglyceridelevel and body mass index on PAI-1 antigen and PAI activity, thereforeadjustmentsfor thesetwo independentvariableswere performed(TablesIV and V). ,..

The patients and controls had similarlevels of PAI-1 antigen (12,8, 10.6-15.4ng/mL vs 13.3, 10.2-17.4ng/mL) and PAI activity(11,4, 8.8-14.3 IU/rnLvs 9.1, 6.3-12.5 IIJ/mL). For PAI-1 antigenanalysisof varianceindicatedno significantinteractionsbetween groups of subjectsand different4G/5Ggenotypes:For PAI activityinteractionswere almoststatisticallysignificantusing parametricanalysisof variance(p=O.067)and significant(p=O.034)using non-parametrictest. In patientsthe lowestPAI activitywas observedin the 5G/5Ggenotype,intermediatein the 4G15G genotypeand the highestin the 4G/4G genotype(TableIV). For the G/A genotypesneitherthe patientsnor the controlsdifferedsignificantlyin PAI-1 antigenand PAI activitylevels(TableV). TABLEIII Distributionof G/A genotypesin DVT patientsand in healthycontrols. Genotype N (%)

GIG

GIA

AIA

Patients(N=83)

2 ( 2.4)

50 (60.2)

31 (37.4)

Controls(N=50)

5 (10.0)

31 (62.0)

14 (28.0)

438

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3 4

5 6 7 8

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9 10

FIG. 3 Specific binding of human vascular endothelial cells (HUVECS) nuclear extracts to G/A polymorphism.G variant (lanes 1-4) and A variant oligonucleotide(lanes 5-8) incubatedwith non-inducedHUVECS nuclear extracts without (lanes 1 and 5) and with 100-foldexcess of unlabeledoligonucleotide(lanes2 and 6), or incubatedwith lipopolysaccharide-induced HUVECS without (lane 3 and 7) and with 100-foldexcess of unlabeledoligonucleotide(lye 4 and 8); A variant oligonucleotidecompetedwith unlabeledG oligonucleotidevariant, incubatedwith noninducedHUVECSnuclearextracts(lane9); G variantoligonucleotidecompetedwith unlabeledA oligonucleotidevariant,incubatedwithnon-inducedFIUVECSnuclearextracts(lane 10). To investigate weather the G/A polymorphic site specificallybinds to nuclear proteins electrophoreticmobilityshift assayswere performedwith two double stranded oligonuceotides correspondingto the G and the A variant, using nuclear extracts from HUVECS. With both variants specific binding was observed. Binding activity was similar for non-induced and Iipopolysaccharide-induced nuclear extracts and for the A and G variant. One major specific protein-DNAcomplexwas formed.Competitionreactionswere performedwith 100-foldexcess of the unlabeledoligonucleotidefor each type of the sequence. The strong reduction of the protein-DNAcomplexesin all competitionreactionsindicatedspecificityof binding.In addition, the formation of the complexwith the A variant oligonucleotidecould be competed with an excessof the G variantand viceversa, suggestingthat similarproteinsbindto both forms(Fig.3). DISCUSSION In this study a novel polymorphismin the promoter of the PAI-1 gene is described. This polymorphicsite is located -844 bp upstreamfrom the transcriptionstart and representsa single guanineto adeninesubstitution(G/A polymorphism)accordingto the publishedsequence(17). The distributionof the G/A genotypesboth in the patientand in the controlgroup differedfrom

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the distributionthat would be expected from the Hardy-Weinbergequilibrium,with an over dominanceof heterozygotes.The excessof heterozygotesmightbe due to the balancingselection which is one of the importantfactors for maintainingpolymorphismsin naturalpopulations.The novel G/A polymorphismwas in strong linkage disequilibriumwith the previouslydescribed 4G/5G polymorphism.Such allelic associationwas expected because of the vicinity of their chromosomal location. Presumably, the guanine to adenine substitution in the novel polymorphismhappenedin the distantpast becauseallfour possiblehaplotypeswere present. This study is to the best of our knowledgealso the first report on the 4G/5G polymorphismin patients with DVT. It shows a relationshipbetween the 4G/5G genotype and plasma PAI-1 activity levels and thus supports previous findings on such associations in patients after myocardialinfarction(11-14) and in patientswith non-insulin-dependentdiabetesmellitus(15), with the highestPAI-I levelsin the 4G/4G genotypesand the lowest in the 5G/5G genotypes.In spiteof a strong correlationbetweenPAI activityand P.M-l antigenlevelsin this study(data not shown)and approximately40°/0higherPAX-1antigenlevelsin patientswith the 4G/4G genotype compared to the 5G/5G genotype (Table IV), relationshipbetween the 4G/5G genotypes and PAI-1 antigendid not reach the levelof statisticalsignificance.Thislack of significantassociation between the 4G/5G genotypesand PAI-1 antigen levels may be explainedin part by different et%ciencieswith which variousfroms of PM-1 (active,latent, complexed)are detected with the PAI-I antigen assay and/or by contributionof latent platelet PAI-1 to PAI-1 antigen levels determinedin plasma,possiblyblurringthe differencesbetweenthe genotypes. The same studies showingthe relationshipbetween the 4G/5G genotype and PAI-1 in patients (11, 12) failedto show such a relationshipin controlsubjects.Similarly,no such associationwas establishedin healthycontrolsinvestigatedin this study.The discrepancybetween DVT patients and healthy controls could probablybe explainedeither by a relativelylow number of healthy individualsinvestigated(only 6 healthy controls had the 5G/5G genotype) and/or by different interactionsbetween determinantsof PAI-1 in plasma in patients and controls. It has been observedthat the influenceof triglycerideon PAI-1 seems to be genotype dependent(15, 16). Sincepatients studiedhad increasedtotal- and LDL-cholesterolindicatingdisturbedlipoprotein metabolism,it might be speculatedthat interactionsbetween genotype and metabolicfactors affectedplasmalevelsof PAI-1 in the patientbut not in the controlgroup. In young patients atler myocardialinfarctionthe prevalenceof the unfavorable4G allele was higher than in population-basedcontrols (13). Associationbetween the 4G/4G genotype and coronary artery disease was observed also in non-insulin-dependentdiabetes mellitus (22). However, in the present study distributionof the 4G/5G genotypesand prevalenceof the 4G allele in patients was not different from the distributionand prevalence observed in healthy controls.Differentgenotypeswere also not associatedwith the severityof the disease,sincethe distributionwas differentneither between patients with singleor recurrent DVT nor between patientswith idiopathicor secondaVDVT. The regulationof PAI-1 expressionin vivo is complex.PAI-1 behavesas an acute phasereactant, and increasing plasma level are observed in response to inflammation and trauma. The polymorphism(s)in the promoterregioncouldbe envisagedto alter eitherthe responseof PAI-1 to a specificphysiologicalregulatoror the basal levelof PM-1. For the 4G/5G polymorphismin

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vitro assaysof PAI-1 gene promoteractivitydemonstratedsignificantlyhigheractivityof the 4G than the 5G alleleunder conditionsof cytokinestimulation,such as are found in the acute phase (12). On the other hand Erikssonand coworkers(13) observedin accordancewith the previous study (12) sequence-specificbindingto the 4G and 5G allelesof two factors presentin a nuclear extractderivedfrom liver,smoothmuscleand endothelialcells.The 5G alleleboundan additional factor to an overlappingbindingsite, which acted as a transcriptionalrepressor.This sequencespecificbindingof nuclearproteinswas observedin unstimulatedcellsindicatingthat the 4G/5G polymorphisminfluencesthe basal level of transcription.Our data seem to support the latter possibilitysince DVT patients investigatedwere relativelylong afler the acute phase of the diseaseand yet, increasedPAI-1 activitywas observedwith increasingnumberof the 4G alleles. Althoughno associationwas observedbetweenthe novelG/A polymorphismand plasmaPAI-1 levelsneitherin patientsnor in healthycontrols,this findingdoes not excludea possiblerole of this novel polymorphismin regulatingplasmaPAI-I levels.An evidencesuggestingfi-mctional importancewas obtainedby electrophoreticmobilityshiftassays,demonstratingspecificbinding of a nuclearproteinto the G and the A allele.Similarbindingwas observedwith nuclearextracts from unstimulatedand stimulatedHUVECS.In spite of the latter observationimportanceof the G/A polymorphicsite in up-regulationof PAI-I levels during the acute phase can not be excluded.It was concludedthat this novelG/A polymorphicsite mightbe similarlyto the 4G/5G polymorphicsite involvedin promoteractivityof the PAI-1 gene with presumablypathogenetic importancein thrombosis. Acknowledgments The studywas conducted whileM. Stegnarwas the recipientof a SlovenianScienceFoundation Fellowship.Adviceon genetic data of W. Pinsker(Institutefor MedicalBi’ology,Universityof Vienna),adviceand help with the electrophoreticmobilityshifiassaysof E.’Hofer and technical assistanceof M. Tehovnikare gratetldlyacknowledged.The studywas supportedby a grant No. 5566from AustrianNationalBank and grantNo. J3-7822fromthe SlovenianMinistryof Science and Technology. The results of this study were presented during the XIIIth International Congresson Fibrinolysisand Thrombolysis,Barcelona,Spain,June 1996. REFERENCES 1. JOHNSON, O., MELLBRING, G., NILSSON, T. Defective fibrinolysisin survivors of myocardialinfarction.Int J Cardiol6, 380-382,1984. 2. PARAMO, J.A., COLUCCI,M., COLLEN, D., VAN DE WERF, F. Plasminogenactivator inhibitorin the blood of patientswith coronaryartery disease.Br Med J Clin Res Ed 291, 573574, 1985. 3. AZN~ J., ESTELLES, A., TORMO, G., SAPENL P., TORMO, V., BLANCH, S., ESPANA, F. Plasminogenactivatorinhibitoractivityand other fibrinolyticvariablesin patients with coronaryartery disease.Br Heart J 59, 535-541,1988.

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