or thromboplastin

THROMBOSIS RESEARCH Printed in the United

BRIEF

vol. 6, pp. 273-279, 1975 Pergamon

States

Press,

Inc.

COMMUNICATION

EFFECT OF INTERMEDIATESOF RXTRINSIC CLfWTIIGONRlRIFIRDFACTORXI: FACTORVII AND/OR!CHROMt3WLASTIN

Sandra Schiffman and Francis S. Markland, Jr. Departmentsof Medicine and Biochemistry University of Southern CaliforniaMedical School Los Angeles, California 90033

(Received 4.11.1975; in revised form 3.2.1975. Accepted by Editor K.M. Brinkhous)

Activation of factor XT in vitro requires factor XII, but other mechanisms of activation of factor XI must exist in viva because patients with hereditary deficienciesof factor XII do not bleed abnormally. One possible source of activators is the extrinsicclottingsystemwhichproceedsindependently of both factor XII and factor XI. During extrinsic clotting sever-

al potentialactivatorsarise includingtissue thmmboplastin; modified factor VII (1); a complex of factor VII-thromboplastin-calcium; activated factor X; and thmmbin.

Thrombin has been shown not to activate factor XI (2). In

this report, we present results designed to determine whether factor VII or thromboplastinalone, or a combinationof the two in the presence of ionic calcium can activate factor XI.' r4ATRRIAxSAIQME!lwDS Factor XI was a fraction from normal humanplasma purifiedabout 6000 fold by a slight nwkificationof a previouslydescribedmethod (2). Plasma was chromatographedon DEAR-cellulose;adsorbed to and eluted from BaS04; chmmatographed on SP-Sephadex at pH 5.2 using a startingbufferof 0.05 M

273

ACTIVATION

274

OF FACTOR

XI

Vo1.6,No.3

sodium acetatecontaining0.15 M NaCl and0.0001HEDTAahdagradientof

450 ml startingbufferand 450 ml of the same buffer containinginstead0.s NeCl; and chxwatographedon Mcalite 4200 in the presenceof human serum albumin(Calbiochem, crystalline, B-grade). FactorXI concentration at time ofuaew88

&O$.

When dilutedto about 20% factorXI activity,this fraction

had much less then 0.15 apparentfactorIII, Fletcherfactor,or pmthroubin; aud IY)detectablefactorVII or contactactivationcofactor(2). ~~~tin~asallneattrsctofhrmYabrainprsparadaccording to Owren (3). FactorVIIpurified

fmmcohn

FractionIII. About100 grms of Cohn

FractionIII (kindlyprovidedby AbbottScientificProductsMvision, Los Angeles,Californiaand storedat -8&J) was extractedin a Waring Blender with 400 ml of 0.05 M Tris, O.OOlM EIEA,pH 7.25 (bufferl), containingO.lM f-aminocapmicacid, 5Ckallikreininhibitorunitsperml of Trasylol(FBA Pharmaceutical, Mvision of BaychemCorp.)and 0.25 mg per ul of soybean tryprininhibitor(Worthington BiochemicalCorp.). All operationswere perfomed at PC.

The mirtureuas homgenizedattop speedforlminute and

then centrifuged.The sedimeutwasextractedtwm.wretiass with

identical

were pooled, one-fifthvolumeof volums of Trir buffer. The supernatants

O.lM sodiumcitratewas added,and factorVII wasisolatedby the barium citrateadsorptiontechniqueof Lewis and Ware (h), ea@@.ng Dmmx 50-m (sodiumions) to remve bariumion as describedby Tishkoffet al (5).

Fac-

tor VII so obtainedwas concentrated to a volumeof 50 ml by dialysisagain& lO$ polyethyleneglycol in bufferlwhich contained25 inhibitorunitsof Trasylolperml.aad0.01mgpermlof (he=dimethx!.ne b-de,

soybeantrypsininhibitor. Polybrene

AldrichChemicelCo., Inc.)was addedto the concen-

trate (finalconcentration 200 ug per ml), and it was appliedto DEAE-cellulore (D&52, 2.5 x 5Ocm)thathadpreviouslybecn equilibratedandpackedin buffer1 containing200 Irgper ml polybrene. Peak fractionsof factorVII

ACTIVATION OF FACTOR XI

275

activitywere pooled (volume184 ml) and concentrated by dialysisagainst lO$ polyethyleneglycolin 0.02 M sodiumacetate,0.5 M sodiumchloride, fraction(26 ml) was ap0.00114EDTA,pH 6.0 (buffer2). The concentrated plied to a 5.5 x 100 cm columnof SephadexG-100 in buffer2 and elutedwith this buffer. Peak tubes of activitywere pooled;soybeantrypsininhibitor and Trasylolwere added to final concentrations of 0.5 mg per ml and 10 kallikreininhibitorunitsper ml, respectively; and the fractionwas concentratedfrom 200 ml to 10 ml by dialysisagainstlO$ polyethylene glycolin was rechromatographed on DEAE-ceUuJose(1.5x buffer1. The concentrate 30 cm) in buffer1, but this time in the absenceof Folybrene. An exponentialgradientemployingbuffer1 and buffer1 plus 0.45 M sodiumchloride was utilizedto elute factorVII. Peak tubesof factorVII activitywere againpooled and concentrated by dialysisagainstpolyethyleneglycolin buffer 2. After concentration, the fractionwas dialyzedagainst0.2 M potassiux phosphatebuffer,pR 6.8, and appliedto a hydxoxylspatite column equilibrated with the same buffer. A lineargradientto 0.6 M potassium phosphatepH 6.8, was employedto elute factorVII. Tubes of peak activity were pooledand concentrated by dialysisagainstpolyethyleneglycolin buffer 2. The concentrated fractionwas then appliedto SephadexG-100 (1.0x 100 cm) in buffer2. Peak tubeswere pooledand concentrated, again using polyethyleneglycolin buffer 2, and the concentrated fractionwas storedin small aliquotsat -8oOC. FactorVII preparedin this mannerwas purified between5,OOOmd 10,000fold as comparedto the level in Cohn FractionIII, with a yield of aboct 5$. When assayedat lO$ of normalplasma concentration, factorVII was found to be essentiallyfree of factorII, V, VIII, IX, X, Xa, XI, XII, andthrombin. Preliminarystudiesindicatethat the apparentmolecularweightof factor VII as determinedby gel filtrationon a calibratedSephadexG-100 column (6) was in the range of 45-55,OOO,whereasfactorVII partiallywfied

276

ACTIVATION

OF FACTOR

XI

Vo1.6,No.3

fromplasmrhad a molecularweightin the rangeof 66-73,000. Hence,in this property,

factorVIIisolatedfromCohuFractionIII (theformusedinthc

experimentsreportedbslow)appearedtobe similar

to serumfactorvII stud-

ied by Gladhaugand Prydz (1). FactorVII assay (7). Plasmacohgenitally deficientin factor VII was used in a one stageassay in which 0.05 ml of the deficientplasma,0.05 ml

of barium sulfateadsorbedbeef plasma,0.1 ml human brain thromboplastin, and 0.1 ml of test materialwera,preincubated at 37oc for 4 minutes. At zero time O.ld. of 40 d4 calciumchloridewas added and the clottingtime uoted. FactorVII activitywasdetenainedbymmpuing clottingtimesof test materialsto a stahdardnommlcurvemadewithdilutions of a normalplasma. FactorVII - thmmboplrrstin complexwas preparedby ihcubatihgCohn Fraction-factorVII,hummbraiu thro&oplastin,audcalciumchlorideinTris buffer,pH 7.4. Mixture1contairmd ~factorVII, thmmboplastindiluted l/200,0.5 mM CaC12, O.lM Tris pH 7.4, aud 0.05 H NaCl. Mixture2 contained 12.5% factorVII,

thmnboplastin

diluted1/400,0.25 mWCaC12, 0.03Mms

pH 7.4, adl 0.12 M NaCl.0These mixtureswere testedin the factorVII assay at 0, 10, and 60 Bsinutes:mixtwre1 clottedat 63.5",62.5",and 63"; mirture 2 clottedat 48", 5l.5",and 53.5",respectively(blanktime-l&"). After 35 minutesincubation,0.2 ml of mixture1~s

combihedwith factorXI;

mixture2 was similarlycombinedwith factorXI aiter42 minuteaincubation. !rrypsin (sigmaChemicalco., Type III, twice recrystallizedfrombovine gmcreas)was dilutedto 4OOj&U.n

O.OO1RRClandProzen in smallaU-

quots for up to a ye8r at -2ooC. At time of use it was dilutedl/5 in m8 bufferpH 7.4. Factor XI

activationreactions. Reactionmixturesof 0.2 ml factorXI

reagentdilutedl/l0 (34$);0.2 ml O.lM Tris bufferpii7.4, containingBCl to producean overallionic strengthof 0.15; and 0.2 ml potentialactivating substancewere incubatedat room teqmature.

At intervalsshown,0.05 ml

v01.6,~0.3

ACTIVATION OF FACTOR XI

aliquotswere rem~vdland testedfor

fadZbr

Xi

277

activationin the assay deS-

cribedbelow. Zero time of the reactionwas the time at which

test substance

was added to the mixture. !Cwenty minutesafter the test substancewas added to the reactionB&Xtrypsiu(80 ug/ti). ture, o.3mllms remved andto itwas added 0.01 nit. Then 0.05 ml aliquotsof this new reactionudxturewere remved at intervals and testedfor factorIU activationas describedbelow. Assay of factorXI activation.The followingreagentswere added in rapid successionto a plastictube containing0.05 ml cephalin(8) diluted l/30 in veroualbufYerpH 7.35: 0.05 ml test substauce,0.05 ml hereditary factorXI deficientplasma,and 0.05 ml 40 st4CaC12. The time from addition of the calciumto formationof the clot was determined.Sequentialsamples

rmth a reactionmixturewere tested. The fonuationof activatedfactorXI, which correctsthe defectin factorXI deficientplasma Fn the absenceof an activatingsurface,was indicatedby progressively decreasingclottingtimes.

Each potentialactivatorof factorXI was testedfor 1) the abiUty to increasethe factorXI actitityas detectedin the factorXI activationassay, and 2) the abilityto modifyfactorXI in such a Gay as to mske it no longer activatableby trypsin. Thrmboplastin(Table1, line 2) snd Cohn Fraction-factor VII (Table1, line 3) both shortenedthe initial(15")clottingtime in the factorxi:activationassay. This effectwas to be eqected sinceboth reagentsbypassthe intrinsicclottingsystem. However,on further

incubation, no additional

shorteningof the clottingtime was noted,indicatingthat factorXI was not undergoingadded activationin theirpresence. Fwthezmore,

the factor XI

in

these mixtureswas readilyactivatedby trypsinafter 20 minutesexposureto the test material. Therefore,it appearedthat neitherweak thromboplastin nor Cohn Fraction-factor VII activatedfactorXI.

278

ACTIVATION

OF FACTOR

XI

v01.6,~0.3

!EAELEI. The Effect of Thromboplastin,

VII, or a

Cohn Fraction-Factor

Combinationof

VII-ThmcWplastin-CalciumonFactorXI FactorXI Activation Assay ClottingTime (sac) ReactionMixture

Inc. Time

1. XI+ buffer + trypsin

VII W.59)

4.

XI+ VII (5$)-QmW0pUstin

(l@OO)-Ca++

l26

-

05

-

114 114

117

03

80

02

1!57 151 90

90

9290

05

73

80

1% 73 90

70

70

(1/4OO)4a* 125127122124

+ txypsin 6.

255

130

+ trypsin 5. x1:+ VII (l2.5$)-t-tin

240

160

+ trypein

20'

-

103

+ trypsin

10’

245

114

2. XI+ thzwkmplastin(1/2OO)-Ca++

3. Q+

,

15"

hffer

IlO

05

00

70

405

of each reactionmixtuxewere remmd at intervalashownsod terted imediately in the fact0rXI activrtionaway over a 20 mihuteperiod, The0 tryprinwas added t0 a mabaMd fractionof the first reactionmixture,and m&samples of this secondreactionmixturewere removedand testedin the factorXI activationassay (seetext for details).

Aliqwtr

If Cohn Fractio+faclxxVII aud thrombcqlastin were combinedat the con-

centrations used above in the presenceof calcium,the initialclottingtime w&d

be too shortto petit detectionof

activation

of

factorII. Therefore,

tu0 differant~~tioamirturaawarcauds,one with weakerfactmVII andthe same throlnbaplastin concentration (Table1, line 4), the otherwith the same factorVII concentration and weakerthxwkmplastin(Table1, line 5). The results show that in neithercase did factor XI undergo activation.Further-

more, in both ca8e1,the factorXI previouslyexposedto the VII-thruuboplastin-calciumcomplexwas rea&lyactivatedbytrypsin. Hence,thepmduct

v01.6,~0.3

ACTIVATION OF FACTOR XI

279

of the incubationof factorVII with thmmboplastinand calcirrm did not -tobe

an activatoroffact.orXI.

These resultsstronglysupportthe conclusionthat intrinsicclottingis not initiatedthmugh the activationof factorXI by eitherfactorVII, weak thmmboplastin,or a combination of the two.

This workwas supportedbygrantsBL-13641-03andHL-&28-13 andwas completedduringthe tenureof an EstablishedInvestigatorship of the AmericanHeart Associationto Dr. Schiffmanand aCareerDevelopmentAward of the U.S. PublicHealth

Service

to Dr. Markland. We wish to thankDr.

samuelI.Rapaport for his helpfulsuggestions.The technicalassistanceof Mrs. PearlLeeand Mrs.LauraLeeis gratefullysckmmledged.

H. Purification of the coagulationfactorsVII 1. GLADEAtG,A. and m, and X fxm human serum.Some propertiesof factorVII. Biochim.Biophys. Acta. Ug, 105, 1970. characterization, and activation 2. SCHIRMM, S. and LEE, P. Preparation, of ahi@& puriiiedfactorM:Evidence thatahitherto unrecognized plasm activityparticipatesin the interaction of factorsXI and XII. Brit. J. Haemat. 27, 101, 1974. --one-stagen&hod for the assayof prothrombin. 3. OWBEN,P.A. A quantitative Stand. J. Clin. Lab. Invest. 1, 81, 199. -_--4. LEWIS,M.L. and m, A&. A siqle procedurefor separationof prothrombin and acceleratorglobulinfrom titratedhumanplasma.E. fi Exper. Biol. Med. 84, 636, 1953. -5. !fISHKmT,G.R., WILLIAm, L.C. and BBUWN,D.M. Preparationof highly purifiedprothrombincomplexI. Crystslllzation, bioLogicalactivity, and orolecukrproperties.2. mBiol. Chew 2, 4151, 1968, 6. IUSDREWS, P. The gel filtrationbehaviorof proteinsrelatedto their mlecular weightsover a tide range.Biochem.g. 96, 595, 1965. 7. BALL, C.A., RAPAFQRT, S.I.,AMES, S.B. and DDZBOUT,J.A. A clinicaland faaIily studyof hereditarypzmonvertin (factorVII) deficiency. Am. L. Mad. 37, 172, 1964. 8. HJOIEP, P., R4PAFOB!l!, S.I. and OWFTEK, P.A. A s-e, specificone-stage pmthrombin assay using Russell’s viper venomin cephalinsuspension. g. E. & 46, 89, 19% _- cun. wed. --