ROBERTR. MONTGOMERYand THEODORES. ZIMMERMAN,Departmentof Molecular Immunology, Scripps Clinic and ResearchFoundation, La Jolla, California 92037
A B ST RACT Factor VIII-related antigen (Vlllag) isdeficient in plasma and platelets of patients with severevon Willebrand's disease. This study reports a secondvon Willebrand's disease antigen (vWagII), distinct fromVIIIag, that is also deficient in the platelets and plasmaof patients with severe von Willebrand's disease. Vlllagand vWagII are separable by molecular exclusion chro-matography, sucrose density gradient ultracentrifuga-tion, and crossed immunoelectrophoresis. They showreactions of immunologic nonidentity with each other,and thus, do not share a precursor-product relation-ship. vWagII is released from normal platelets duringblood clotting, accounting for a fourfold higher con-centration of vWagII in serum over plasma.
INTRODUCTION
Classical von Willebrand's disease (vWD)1 is char-acterized by a deficiency of Factor VIII procoagulantactivity, ristocetin cofactor activity and Factor VIII-related antigen (VIIIag) (1). These entities may resideon one molecule or on more than one molecule which
This is publication no. 1234 from the Scripps Clinic andResearch Foundation.
This work was presented in part at the 6th InternationalCongress on Thrombosis and Haemostasis in Philadelphia(Montgomery, R. R., and T. S. Zimmerman. 1977. Thromb.Haemostasis. 38: 70), and the 20th Annual Meeting of theAmerican Society of Hematology: 1977. Blood. 50: 277.
Dr. Montgomery is the recipient of a National Institute ofHealth Fellowship (N.R.S.A.). His current address is theDepartment of Pediatrics, University of Colorado MedicalCenter, Denver, Colo. 80262.
Received for publication 13 June 1977 and in revised form23 January 1978.
'Abbreviations used in this paper: /3-TG, 3-thrombo-globulin; CPD, citrate-phosphate dextrose; PPP, platelet-poorplasma, PRP, platelet-rich plasma; Vo, void volume; vWagII,von Willebrand's disease antigen II; vWD, von Willebrand'sdisease; VIIIag, Factor VIII-related antigen; VIII,, factor VIIIprocoagulant activity; VIIIr, ristocetin cofactor activity.
co-purify under a variety of conditions (1-3). Vlllagis that antigen which reacts with heterologous antibodyproduced in rabbits to purified Factor VIII. Vlllag, alsoknown as von Willebrand's disease antigen (vWagl), ispresent in normal or increased amounts in patients withhemophilia but absent or reduced in most patients withclassical vWD. This report describes a second antigen,von Willebrand's disease antigen II (vWagII), which isdeficient in the plasma and platelets of patients withsevere vWD.
METHODS
Antisera to vWagII. The antisera used to identify vWagIIwere produced by two different methods in New Zealandwhite rabbits. In the initial method, lyophilized Factor VIIIconcentrate (lot B-17, American Red Cross Bleed ResearchLaboratory, Bethesda, Md.) was subjected to molecular ex-clusion chromatography through Bio-Gel A-15M (Bio-RadLaboratories, Richmond, Calif.). Concentration immunizationof rabbits, and antiserum absorption were performed as pre-viously described (4). After absorption with cryosupernatantplasma and IgM paraproteins, this antiserum contained an anti-body to Vlllag, vWagII, and one additional platelet protein.The studies reported in this paper utilized this antiserum(antiserum I) because of its higher titer to vWagII.
Another antiserum (antiserum II) previously prepared byone of the authors (R.R.M.) at the University of Colorado Med-ical Center, Denver, Colo., was also studied and containedantibody to vWagII. This antiserum was produced as follows.Commercial Factor VIII concentrate (Profilate, Abbott Labo-ratories, Chemical Div., North Chicago, Ill.) was reconstitutedwith column buffer to a final concentration of 40 U VIIIag/ml.A 6-ml sample was subjected to gel filtration through Sepharose4B (Pharmacia Fine Chemicals, Div. of Pharmacia Inc. Piscat-away, N. J.) equilibrated with imidazole-saline buffer (0.02Minidazole, 0.14 MNaCl, pH 6.5). The bed dimensions were2.5 x 40 cm. The void volume (Vo) fractions were pooled, con-centrated by Amicon filtration (PM 30 Amicon Corp., Lexing-ton, Mass.), and injected into three NewZealand white rabbits.After boosting at 3-wk intervals, the rabbits were bled and theantiserum was obtained. This antiserum contained antibodiesto VIllag, vWagII, and six other nonidentified plasma pro-teins. This antiserum was then absorbed with equal volumes
of plasma from a patieint with severe vWtD (Vlllag <0.01U/mil, Factor VIII procoagulent activity VIII <0.05 U/ml,VIIIr Ristocetin coftactor activity <0.05 U/ml). After this absorp-tion, only antibodies to VIllag and vWagII were detected.
Antisera to marker proteins. Various plasma protein mark-ers were identified by quantitative immunoelectrophoresisand crossed immntnoelectrophoresis utilizing monospecificantisera. Antisera to C4, IgG, IgNI, and fibrinogen were ob-tained through the Scripps Immunology Reference Labo-ratory, La Jolla, Calif: Other antisera were obtained com-mercially and include prothrombin, a1-antitrypsin, FactorXIII, and a2-macroglobulin (Behring Diagnostics, Sommer-ville, N. J.).
Patient population. Plasma was obtained from 19 normalindividuals and 24 patients with v\N'D. 8 of the 24 patientswere classified as severe on the basis of no detectable Vlllag(<0.01 U/ml), VIII. (<0.05 U/ml), and VIII, (<0.05 U/ml).16 of those with vWDhad moderate vWD(VIllag 0.080-0.34U/ml). 13 of these 16 patients had VIIIag characterized by thepresence of only those forms with more anodic migration oncrossed immunoelectrophoresis. Two of the severe vWDpa-tients were also studied after being transfused at a time whentheir VIII was 40% and their Vlllag was 20%.
Plasma p)re paration for VIllag and uXVagII analysis. Bloodwas drawn into citrate-phosphate dextrose (CPD) by adding1.3 ml CPDto 8.7 ml of blood. Blood was centrifuged at 2,000g for 30 min at 4°C. "Platelet-poor" plasma (PPP) was ob-tained by centrifuging CPDplasma at 5,000 g for 30 min at40C, and platelet-rich plasma (PRP) was obtained by centrifug-ing CPDblood at 200 g for 10 min at 4°C. CPDplasma plusinhibitors was obtained by immediately mixing fresh CPDblood with 10 U/ml heparin, 10 ,ug/ml soybean trypsininhibitor, 10 U/ml Trasylol (FBA Pharmaceuticals, NewYork),and 5 mMbenzamidine.
Washed platelets for vcVagII analysis. PRP from 25 ml ofCPDblood was obtained as above. The PRPwas centrifugedat 2,000 g for 15 min at 20°C and the supernate was dis-carded. The platelets were resuspended in 25 ml of Krebs-Ringer buffer (5 mMKCl, 0.107 MNaCl, 0.02 MNaHCO3, 9mMNa2 EDTA, 2 mMNa2SO4, pH 7.4) and centrifuged at2,000 g f'or 14 min at 20(C. This procedure was repeated threeadditional times. After the final centrifugation, all the bufferwas decanted and the platelets were resuspended in 0.1 mlbarbital saline buffer (0.01 M barbital sodium, 0.015 M bar-bituric acid, 0.125 MNaCl, 0.1% NaN3, pH 7.4). This plateletconcentrate was subjected to lysis by freezing (liquid nitrogen)and thawing (four times). Platelet fragments were removed bycentrif'ugation at 11,000 g for 15 min at 20°C. An alternativemethod used modified Tyrode's buffer (0.0026 MKCl, 0.137MNaCl, 0.001 MMgCl2, 0.012 MNaHCO3, 0.03 Madenosine;Sigma Chemical Co., St. Louis, Mo.), 0.1% dextrose, and 2%bovine serum albumin (Fraction V, Sigma Chemical Co.), pH7.4, to wash the platelets. Lysis was performed as describedabove.
Crossed iminunoelectrophoresis. Crossed immunoelectro-phoresis was performed as previously described (5) exceptthat the sample well was made larger to acccept a 150-,ulsample. Since the antiserum concentration was very high (upto 26%), the slides needed to be washed in normal saline for48 h before drying and staining.
A calibration curve for quantitating vWagII was constructedby plotting the area under the crossed immunoelectro-phoresis precipitin peak on the arithmetic scale against serialdilutions of a plasma pool on the logarithmic scale. The plasmapool vas obtained from 20 normal individuals and stored at-70°C. This served as the standard for vWagII, and was as-signed the value of 1 U/ml. The area under the precipitin peakwas obtained by multiplying the height of the peak times
the width at a point which bisects the height as previouslydescribed (6).
Quantitative immunoelectrophoresis of vWagII and VIlIag.Since the amount of protein applied to a slide was very largedue to the high concentration of antibody and the large sampleutilized, the standard quantitative immunoelectrophoresis ofvWagII in plasma proved impossible. However, quantitativeimmunoelectrophoresis could be performed on vWagII thatwas partially purified by column chromatography or sucrosedensity gradient ultracentrifugation. This method could there-fore be used to localize vWagII in these experiments. Theantiserum concentration was the same as that used for quanti-tative crossed immunoelectrophoresis. The procedure forquantitative immunoelectrophoresis of Vlllag has been pre-viously described (4).
Purification of VIlIlag and separation from vWagII. Bloodwas drawn into CPD (13 ml/87 ml blood), 10 U/ml heparin,10 U/mI Trasylol and 10 jig/ml soybean trypsin inhibitor. Theblood was then centrifuged at 3,000 g for 15 min at 20°C and theplasma was separated. Diisopropyl fluorophosphate wasadded to this plasma at a 0.002-M concentration. Aluminum hy-droxide absorption was performed four times. In the firstabsorption, 5 ml A1(OH)3 gel (Rehsorptar, Reheis Co., Inc.,Phoenix, Ariz.) was added to 100 ml plasma and stirred gentlyat ambient temperature for 15 min. The A1(OH)3 was removedby centrifugation at 25,000 g for 10 min at 20°C. The second,third, and fourth absorptions were performed in the samemanner except the concentration of A1(OH)3 was reduced to1.5 ml/100 ml plasma. After the final absorption, the plasmawas centrifuged at 90,000 g for 60 min. The supernatant plasmawas flash frozen in liquid nitrogen and stored at -70°C untilused. The cryoprecipitate was produced by thawing the plasmafor 4-6 h in a stirred melting ice bath and the cryoprecipitatesedimented by centrifugation at 90,000 g for 20 min at 5°C.This precipitate was dissolved at 370C, diluted to 5 ml withbarbital saline buffer (pH 7.4), and centrifuged at 200,000 g for30 min at 20°C. The sample below the lipid layer was carefullyremoved, taking care not to disturb any of the precipitate onthe bottom of the tube, and subjected to agarose gel chro-matography. Bio-Gel A-15M was packed into two columnsconnected in series with total dimensions of 2.5 x 94 cm. Thesample was eluted with Tris-saline buffer (0.05 MTris, 0.15 MNaCl, and 0.02% NaN3, pH 7.4). Fractions were collected in2.6-ml aliquots and the optical density at 280 nmwas recorded.Highly purified VIIIag eluted in the VO with semipurifiedvWagII eluting 2.5 x VOalong with other plasma proteins. Thefractions containing the vWagII were concentrated usingAquacide II (Calbiochem, San Diego, Calif.) to 1/10th theoriginal volume and are referred to as semipurified plasmavWagII.
Semipurification of platelet vWagII. Semipurified plateletvWagII was prepared by separating PRP from fresh CPDplasma at 200 g for 10 min at room temperature. The plateletswere then sedimented at 2,000 g for 15 min and the super-natant plasma removed. These platelets were then washed sixtimes using Krebs-Ringer buffer (4 mMKCI, 0.107 M NaCl,0.02 MNaHCO3, 2 mMNa2SO4, pH 7.4). The platelets wereresuspended in a 5-ml barbital saline buffer (pH 7.4) andsubjected to freeze-thaw lysis (four times). The particulatematter was removed by centrifugation at 90,000 g and thesupernate was subjected to agarose gel chromatography asdescribed above. The f'ractions containing vWagII were con-centrated in the same fashion and are referred to as semipuri-fied vWagII.
Electrophoretic mobility of vWagII. The relative electro-phoretic mobility of vWagII was determined by crossed im-munoelectrophoresis as described above except, the firstdimension was run at a pH of 8.6 (rather than pH of 9.5) using
von Willebrand's Disease Antigen II 1499
the same buffer. Other plasma proteins were run as externalcontrols (i.e., parallel runs) and internal controls (using severalantibodies in the second dimension and running normalplasma in the first dimension). Antiserum against fibrinogen,C4, prothrombin, and a,-antitrypsin were utilized and all runswere standardized to an albumin marker that was migrated6.0 cm.
Sucrose density gradient ultracentrifugation. Sucrosedensity gradient ultracentrifugation was performed using alinear gradient (10-40% wt/vol sucrose). A 0.5-mol samplewas applied to the top of a 10-ml gradient in a cel-lulose nitrate centrifuge tube (model 331370, BeckmanInstruments, Inc., Palo Alto, Calif'.). The gradients werecentrifuged at 35,000 rpm using a SW-41 rotor in aBeckman L3-50 centrifuge (Beckman Instruments Inc.) f'or18 h at 4°C. The gradients were eluted from the bottom in 0.33-ml aliquots. The sedimentation velocity of vWagII wasobtained by running normal plasma that had been enriched invWagII by the addition of either semipurified plasma vWagIIor semipurified platelet vWagII. Sedimentation velocity ofvWagII was determined by comparing it to other plasmaproteins present in the sample (IgM, IgG, fibrinogen,prothrombin, and a1-antitrypsin). The location of the internalmarkers and vWagII was determined by (quantitativeelectrophoresis.
Apparent molecular weight estimation by gel filtration.Sephadex G-200 (Pharmacia Div. of Diagnostics, PharmaciaInc.) was equilibrated with Tris-saline buffer in a column withbed dimensions of 1.5 x 65 cm. The column was standardizedusing a calibration kit with molecular weight markers rangingfrom 13,700 to 158,000 daltons (Pharmacia Diagnostics, Div. ofPharmacia Inc.). Normal plasma was mixed with semipurifiedplasma vWagII or semipurified platelet vWagII and a 1-mlsample applied to the column. Fractions were collected in 2.5-ml increments. The elution volumes of vWagII, Vlllag, IgM,IgG, C4, prothrombin, a,-antitrypsin, Factor XIII, and a2-macroglobulin were determined by quantitative immunoelec-trophoresis. The partition coefficient (Kay) was determinedaccording to the formula Kav = (elution volume - V0)/bedvolume - V0. A standard curve was constructed relating Kav tomolecular weight.
Antibody against highly purified VIllag. Highly purifiedVlllag was prepared as described above. The fractions con-taining the middle portion of' the VIllag peak (295 U Vlllag/OD U) were used to immunize rabbits. Freund's completeadjuvant (Grand Island Biological Co., Grand Island, N. Y.)was emulsified with an equal volume of' 80-150 ,ug of' highlypurified Vlllag and injected in multiple subcutaneous sitesalong the back of New Zealand white albino rabbits. Rabbitswere boosted twice, using the same techni(lue, at 3-wk in-tervals and the antiserum obtained by bleeding the rabbit fromthe ear artery.
Proteolytic degradation of highly purified VIllag. Highlypurified Vlllag was subjected to digestion with various proteo-lytic enzymes. These included streptokinase (500 U/mg plas-minogen, Calbiochem), activated human plasminogen (150,g/ml courtesy of Dr. E. F. Plow), trypsin (bovine pancreastrypsin, 220 U/mg, Worthington Biochemical Corp., Freehold,N. J., lot TRTPCK-35K940), and porcine elastase (60 U/mg,Worthington Biochemical Corp., lot ESFF-56P343). Vlllag(80 ,ug/ml) was incubated at 37°C for 21/2 h and for 18 h withplasmin (0.009 ,tg/,ug Vlllag), porcine elastase (0.1 elastaseU/,ug Vlllag), and trypsin (0.016 U/,ug Vlllag). Thesecoinditionis gave Vlllag digests containing either threeimmunologically distinct Vlllag fragments (elastase), twoimmunologically distinct fragments (trypsin digest), or onecomponent with accelerated anodic mobility (plasmin digest)
when these digests were subjected to crossed immunoelectro-phoresis. These digests were used to immunize rabbits.
Cellular lysates of nonplatelet blood cells. Erythrocytes,granulocytes, and lymphocytes were obtained in the follow-ing manner. Heparinized blood was obtained and diluted 1:2in RPMI-1640 (Grand Island Biological Co.) and layered ontoa barrier of Ficoll-Hypa(lue (P 1.074 g/ml) (7). The tubes werecentrifuged at 2,260 g for 5 min at 20°C. The top plasma seg-ment contained predominantly platelets. The cells at the in-terface were lymphocytes together with a few adherent cellsand platelets. These cells were placed in culture (7). After4 days, the nionadherenit cells were 95% lymphocytes with noplatelets preseint. This was the source of lymphocytes. Granu-locytes were obtained from the buffy coat on top of the eryth-rocytes in the original Ficoll-Hypa(lue centrifugation step.There were 90% granulocytes with a few mononuclear cells.The erythrocytes were obtained from the bottom of the tubeand were free of platelets and leukocytes. These erythrocytes,lymphocytes, and granulocytes were washed four times withKrebs-Ringer buffer (pH 7.4), subjected to freeze-thaw lysis,and centrifuged at 11,000 g for 15 min at room temperatureto remove the particulate matter.
RESULTS
Detection of vWagII. The antisera used to detectvWagII also detected Vlllag. When antiserum I wasused at a concentration of O.6% in the second dimensionof a crossed immunoelectrophoresis, only the Vlllagpeak was produced (Fig. 1). No other precipitin peakswere detected. If, however, the sample applied to thewell in the first dimension was increased sevenfold inthe same sample volume and the antibody concentra-tion in the second dimension increased 26-fold, theVlllag precipitin peak was drastically reduced inheight and another, more anodic, precipitin peak wasdetected (Fig. 1). This peak was called vWagII forreasons to be described. Antiserum II (produced at adifferent institution) behaved in a similar manner.
Both of these antisera clearly identified vWagII andits deficiency in patients with severe vWD.
Deficiency of vWagIl in severe vWD. When plasmafrom patients with severe vWDwas subjected to identi-cal crossed immunoelectrophoresis, neither the Vlllagnor the vWagIl peaks were detected (Fig. 2). Whenplasma from patients with moderate vWD(i.e., 8-34%)were tested, the vWagII peak was present. Patients,with a f'orm of vWD characterized by a more anodicmigration of' the Vlllag peak, were tested and hadeasily detectable vWagII (Fig. 2). Quantitation of'vWagII was accomplished by measuring the area underthe peak as described in Methods. Levels of 6%vWagII or greater produced identifiable precipitinpeaks. Table I gives the VIllag and vWagII results in19 normal individuals and the patients with vWD.None of' the severe vWD patients had detectablevWagII (i.e., <0.06 U/ml plasma vWagII). All but one ofthe moderate vWDpatients had vWagII detected. Themoderate vWDpatient who had <0.06 U/ml vWagII
1500 R. R. Montgomery and T. S. Zimmerman
Anti seru.." l"IN
6
ViiiagNormal Plasma
Antiserum ConcnA6 0/t
vWag I i
aVllg
[- ) NormaI Plasma
FIGURE 1 Crossed immunoelectrophoretic analysis of nor-mal plasma using two concentrations of antibody. At the lowerantiserum concentration only the Vlllag precipitin peak isseen. When the antiserum concentration is increased, theVIIIag peak is drastically decreased and the vWagII peak isclearly identified.
had two different plasma samples tested and neitherhad detectable vWagII levels. This patient was other-wise not distinguishable from other patients withmoderate vWD. Two patients with severe vWDwerestudied at 60 min, 12, 24, and 48 h after they receivedtransfusions with cryoprecipitate (1 bag/5 kg). NovWagII could be detected in their plasma even whentheir VIllag was >30% and their VIII, was >40%.Fig. 3 shows the relationship between vWagII andVIIIag in normal individuals and patients. In the in-dividuals studied, a weak but significant linear rela-tionship exists for the normal individuals with a cor-relation coefficient of 0.71 (P < 0.01). There was nolinear relationship for the vWagII levels of the pa-tients with moderate vWD (correlation coefficient0.034; not significant).
Effect of clotting on vWagII. When normal serumwas compared to normal plasma from the same in-dividual, vWagII levels were fourfold greater. Toevaluate the source of this rise, experiments weredesigned to evaluate the effect of clotting. PRP, PPP,CPDplasma, and CPDwhole blood were recalcified inglass tubes with 30 ,ul of 1 M CaClJml plasma andpermitted to clot at 37°C for 30 min before serum
Normal
ilas:
VilGlag
Moderate vWd
)
Severe vWdve,r.wd:
Severe vWdx.]FIGURE 2 Crossed immunoelectrophoretic analysis of plasmafrom a normal individual, an individual with moderate vWD,and two individuals with severe vWD. The vWagII peak isundetectable in the severe vWDplasmas. The less anodicforms of Vlllag are absent from the moderate vWDplasma. NoVIIIag is detectable in the severe vWDplasmas.
von Willebrand's Disease Antigen II 1501
v-1 .01!21,jmp.w*!.Il J!, 11 11
TABLE IPlasma vWagII and VIllag
vWagII VIIIag
U/mi U/mi
Normal individuals (19) 0.87±0.33* 0.98±0.27(0.46- 1.44)1 (0.58-1.58)
* Mean±SD.t Range.§ If the patient with <0.06 U/ml vWagII was eliminated, themean would be 0.52±0.21 for the remaining 15 patients."Only 1 of 16 was <0.29 U/ml.
separation. Table II shows the results of these ex-periments. The vWagII level of normal CPDplasmawas unaffected by immediately adding proteolyticinhibitors (soybean trypsin inhibitor, Trasylol, benz-amidine, and heparin). Recalcification of CPDbloodcaused a fourfold increase in vWagII. Recalcificationof PPP, however, did not change the level of vWagIIcompared to the starting plasma. Recalcification ofPRP, on the other hand, produced a fourfold rise invWagII suggesting this to be a platelet effect. To testthis hypothesis, PRPwas subjected to four times freeze-
thaw lysis and a similar fourfold rise in vWagII wasdetected.
vWagII in washed normal and severe vWDplatelets.Normal platelets were obtained from 50 ml of CPDblood and washed six times in Krebs-Ringer buffer be-fore resuspension in a 100-,1u barbital saline buffer.After lysis by freezing and thawing, crossed immuno-electrophoresis showed this lysate to contain vWagIIat a concentration of 20 U/ml (see Fig. 4). When
VVv I,
) Norm iF Viflag
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0
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* 0-10
760
0
lo
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* X. 0 0
* .0
0 0.4 0.8 1.2Plasma vWagil U /ml)
1.6
FIGURE 3 The comparison of vWagII concentrations withthose of VIIIag in normal plasmas (0), individuals withmoderate vWD (-), and severe vWD (C)). The hexagonrepresents eight individuals who had undetectable levels ofboth antigens.
Severe YVD'.''
FIGURE 4 Crossed immunoelectrophoretic analysis of' plate-let lysate from a normal individual and a patient with severevWD. A third antigen "X" is identified in both the normals andsevere vWD(see text).
1502 R. R. Montgomery and T. S. Zimmerman
platelets were obtained from four patients with severevWDin the same fashion (Fig. 4), there was no vWA'agIldetected (i.e., <0.06 U/ml). Single samples of plateletlysate in three of these four patients were studiedwith three or more separate determinations of plateletvWagII. The fourth individual had platelets sampledand vWaglI studied on four separate occasions. None ofthese studies revealed any detectable vWagII in theplatelet lysate of individuals with severe vWD. Inaddition, a concentrate of vWDplatelet membranes(after freeze-thaw lysis) also did not contain vWagIIwhen tested on crossed immunoelectrophoresis. Pa-tients with severe vWDtherefore have <0.3% of nor-mal platelet vWaglI. In the process of evaluating plate-lets, it was found that antibody to a third antigen(present in both normal and severe vWD plateletsand labeled "X" in Fig. 4) was present in antiserumI. This antigen showed no identity with either Vlllag orvWagII. Further characterization of this unknown anti-gen (X) has not been undertaken since it is presentin both normal and severe vWDplatelets.
Preparation of highly purified VIllag and itsphysical separation from vWagII. Highly purifiedVlllag was obtained by the method described. Theelution pattern of the cryoprecipitate is shown inFig. 5. Since the starting plasma contains 1 U VIIIag/30ODU and the Vo peak is found at 8 U VIIIag/-0.027 ODU, this represents >8,500-fold purification.An 80-,ug sample of this material was reduced andexamined by 5% sodiumdodecyl sulfate-polyacryl-amide gel electrophoresis. A representative gel isshown in Fig. 5. Five rabbits were immunized threetimes with 150-200 ug of highly purified Vlllaggiven at 3-wk intervals. The antiserum produced in allfive individual rabbits was monospecific for VIIIagwithout absorption as determined by Ouchterlonyanalysis and crossed immunoelectrophoresis usinghigh antiserum _oncentration (18%) in the seconddimension. The titer of anti-VIIIag was fourfold greaterthan the anti-VIIIag titer of the antiserum used to
identify vWagII yet no vWVagII or other antigenswere detected.
Semipurification of plasma and platelet vWagII.Fig. 5. shows the clear physical separation of plasmavWagII from Vlllag by 4% agarose gel chromatog-raphy. The vWagIl was included in 4% agarose andwas eluted at 2.5 times the V0 and at a concentra-tion of 4.25 U vWagII/0.13 ODU (i.e. 1,200-foldpurification). Semipurified platelet vWagII was ob-tained as described in Methods from a washed plate-let lysate. The vWagII eluted in the same positionof 4%agarose and was -600-fold purified. To evaluatethe immunologic relationship of platelet and plasmavWagII, a tandem crossed immunoelectrophoresis wasperformed using the method described by Kroll (8).Fig. 6 shows the results when semipurified plasmavWagII was run in tandem with a platelet vWagII con-centrate. A reaction of immunologic identity, as de-scribed by Axelsen et al. (9) was seen between plateletand plasma vWagII.
Electrophoretic mobility of vWagII. Platelet andplasma vWagIl were found to have identical electro-phoretic mobility. To compare their mobility to themobility of other known plasma proteins, crossed im-munoelectrophoresis was performed with the pH of thefirst dimension changed to the more commonly usedpH of 8.6. Table III shows the relative mobility at pH8.6 in 0.9% agarose at ambient temperature. Themobility is between that of prothrombin (an a2-globulin) and al-antitrypsin (an a,-globulin) underthese conditions the Rf (migration distance of antigen/migration distance of Evans blue bovine serum al-bumin marker) of vWagII was 0.68 and the Rf and ofVIllag 0.285.
Sedimentation velocity of vWagII. Sucrose densitygradient ultracentrifugation was performed on semipuri-fied platelet vWagII and semipurified plasma vWagll andcompared to the sedimentation velocity of various knownplasma proteins. The sedimentation velocity of semipu-rified vWagII run in parallel with markers was identical to
I
4:i.
w11Slt IWe e vWagil
\If)
.1I
0.3
- 0.2 E
-01el
150 200 250 300 350 400
Elutlon Volume lml)
FIGURE 5 Elution profile of agarose gel filtration step ofVlllag purification. The inset is a 5%sodium dodecyl sulfate-polyacrylamide gel of 80 ,ug of eluting in the V0. This materialwas reduced in sodium dodecyl sulfate-urea before gelelectrophoresis. Vlllag elutes in the V0 and vWagII at2.5 x V0.
Platelet vWaglI
Plasma vWagil
-~~~~~~~~~~~~~~~~~~~~~~~~..I I4 4
FIGURE 6 Tandem crossed immunoelectrophoretic analysisof plasma and platelet vWagII with a reaction of immunologicidentity between platelet vWagII and plasma vWagII.
von Willebrand's Disease Antigen 11 1503
E_ 8-, 6,_ 4
q
TABLE IIIElectrophoretic Mobility at pH 8.6 in 0.9% Agarose
* Rf = (migration distance of unknown)/(migration distance ofalbumin marker).
its sedimentation velocity when mixed with normalplasma to provide internal markers. Fig. 7 shows theresults for platelet and plasma vWagII with the in-ternal markers of IgM, fibrinogen, IgG, prothrombin,and a,-trypsin. The distribution of vWagII was identi-cal for platelet and plasma vWagII and correspondedto an average sedimentation velocity of 4.8S. Theaverage S rate for VIIIag in plasma was 21S.
Behavior of vWagII in Sephadex G-200. Althoughthe determination of the true molecular weight ofvWagII awaits its further purification, gel filtration inSephadex G-200 was carried out to compare its be-havior to the partition coefficients (Kay) and molecularweights of known globular plasma proteins. Fig. 8 il-lustrates the elution profile of vWagII. The elution ofplatelets and plasma vWagII was almost identical.Whencompared to the internal plasma protein markers,the behavior was similar to that expected for a globularprotein of 135,000 daltons. The Ka, for vWagII was
.otZ
M._
xi
24 ,
116M. -116
8 e -OmU-0
Fraction Number
FIGURE 7 Sucrose density gradient analysis of vWagII andVIIIag and comparison with the sedimentation velocity ofvarious plasma proteins.
FIGURE 8 Elution profile of VIIIag, plasma vWagII, andplatelet vWagII on Sephadex G-200 and comparison withthe molecular weight of various plasma proteins.
0.0375. VIIIag was present in the Vo as would beexpected.
Immunologic relationship between vWagII andVIlIag. As noted above, antibody produced to highlypurified VIIIag does not react with vWagII, indicatingthe immunologic distinctness of these two entities. Tofurther investigate this, the antiserum containing bothanti-VIIIag in high titer and anti-vWagII was absorbedwith purified VIIIag to reduce the anti-VIllag titer.Fig. 9 shows the crossed immunoelectrophoresis ofnormal plasma. VIIIag and vWagII precipitin peakscross giving a reaction of immunologic nonidentity.Evaluation of immunologic relationships on crossedimmunoelectrophoresis has been reviewed (9, 10). In
vWgil
FIGURE 9 Crossed immunoelectrophoretic analysis of normnalplasma after partial absorption of the antiserum with purifiedVIIIag. Electrophoresis time for the first dimension wasreduced so vWagII would not migrate as far as usual and thusallow easier comparison with VIIIag. The crossing of theVIIIag and vWagII precipitin lines denotes a reaction ofimmunologic nonidentity.
1504 R. R. Montgomery and T. S. Zimmerman
fact, further absorption with purified Vlllag showedcomplete elimination of the Vlllag peak with nochange in the anti-vWagII titer (see Fig. 10).
Highly purified Factor VIII was digested withplasmin, elastase, and trypsin. None of the Vlllagfragments produced by these proteolytic enzymesshowed immunologic identity with vWagII. In ad-dition, antisera to these digests were produced in rab-bits. None of the antisera produced a vWagII precipi-tin peak when tested against normal plasma utilizingcrossed immunoelectrophoresis.
Absence of vWagII in erythrocytes, granulocytes,and lymphocytes. Erythrocyte, granulocyte, andlymphocyte suspensions were washed and subjectedto lysis by freezing and thawing. None of thesepreparations contained detectable amounts of vWagII.
Effect of temperature and anticoagulant. Since thetemperature of blood after the collection and type ofanticoagulant used have been shown to markedly af-fect the amount of platelet Factor IV (11, 12), or 8-thromboglobulin (13, 14) released from platelets,various anticoagulants, and temperatures were used inthe collection of specimens for vWagII analysis. WithEDTA, CPD, citrate, and heparin as anticoagulants,identical amounts of vWagII were present in plasmaand this was unaffected by incubation of the samplesat 40, 37, or 22°C for 30 min before centrifugationof the whole blood.
DISCUSSION
This study reports the presence of a newly recognizedplasma and platelet antigen, vWagII. This antigen isnot detected in either the platelets or plasma fromindividuals with severe WD(eight individuals). It ispresent in all but one of the patients with moderateor variant forms of vWD tested and all normal in-dividuals tested (34 individuals).
The antisera used to recognize this antigen has beenproduced using two different methods and utilizingFactor VIII concentrates from two different sources asstarting material. Neither of these two methods in-cluded a step to render the starting sample free ofplatelets or platelet fragments. Thus platelet fragmentswould elute in the Vo of both procedures. Other in-vestigators have found antisera, produced in a similarfashion, to contain antibodies to platelet proteins (1,15, 16). We feel the presence of the vWagII antibodyin our antiserum is due to the presence of plateletfragments in the immunizing materials. In fact, whenVIllag was purified as described in Methods ("purifica-tion of Vlllag and separation from vWagII") no anti-body to vWagll was produced in any of the five rabbitsimmunized with V0 material. The probable reason forthis was the inclusion of a high-speed ultracentrifugationstep before applying the sample to the column. Thus,
A
Ab+ Buffer
B
t. .It.
Ab + VOM
FIGURE 10 Crossed immunoelectrophoretic analysis ofplasma before (A) and after (B) complete absorption of anti-Vlllag antibody with purified Vlllag. The vWagII precipitinarc (A and B) was unaffected by prior absorption with Vlllag,though the heavy Vlllag precipitin arc (A) is now completelyabsent (B).
platelet fragments were removed and there was novWagII in the region of the Vo, though it did elute inthe region of 2.5 x V0 as shown in Fig. 5.
There are a number of abnormalities that have beendescribed in vWD. These abnormalities include lowVill, (17-19), low Vlllag (20-22), low VIII, (23-25),long bleeding time (14, 26-28), and decreased plate-let adhesiveness (29, 30). In Erik von Willebrand's in-itial description of vWDand in follow-up studies onthese patients, the bleeding diathesis in vWDwas feltto be due to a platelet abnormality (26, 27). Sincethe advent of platelet aggregation studies, numerousauthors have attempted to define a platelet aggrega-tion defect in vWD. Aggregation to epinephrine,ADP, collagen, and thrombin have been normal(31, 32). The abnormal tests that were felt toreflect a platelet abnormality-such as the bleedingtime, platelet adhesiveness, and aggregation inducedby ristocetin-have subse(luently been felt to besecondary to a plasma defect. Normal plasma, cryo-precipitate, and purified Factor VIII have been shownto correct or partially correct these abnormalities(33-25). It has been felt, therefore, that intrinsicplatelet function is normal (31-33). A study of plateletmembrane glycoproteins did not detect an abnormalityin the platelets of patients with vWD(36). However,both Vlllag (15, 16, 37) and vWagII are deficient inthe platelets of individuals with severe vWD, thoughno functional abnormalities can, at present, be ascribedto this lack.
von Willebrand's Disease Antigen II 1505
I
VIIIag and vWagII are physically separable fromeach other by electrophoresis in agarose, molecularexclusion chromatography, and sucrose density gradi-ent ultracentrifugation. They are also antigenicallydistinct. Antisera from rabbits immunized with highlypurified VIIIag fail to recognize vWagII. In addition,highly purified VIIIag does not absorb any of the anti-body to vWagII (Fig. 10). Of major sighificance is theobservation that VIllag and vWagII show a reactionof nonidentity on crossed immunoelectrophoresis ofnormal plasma (Fig. 9). These latter observations pro-vide direct evidence that vWag II is not a proteolyticdigestion product of VIIIag (or vice versa). If vWagIIwere a fragment of VIIIag it might lack some of theantigenic determinants of native VIIIag and (or) mightexpress neoantigens. However, it would also retainsome of the antigens expressed by native VIllag.Therefore, the two would show a reaction of partialidentity and antisera raised against one would recog-nize the other. Not only did native Vlllag fail to showany antigenic relationship to vWagII, neither didits proteolytic digestion products. In addition, anti-sera raised to Factor VIII proteolytic digestionproducts (see Methods) have likewise failed toidentify vWagII. A variety of proteolytic inhibitorsadded to blood immediately after venipuncture failedto affect vWagII levels and clotting of PPP failed toincrease vWagII content. Thus, it is unlikely thatvWagII is generated in vitro or in vivo as the resultof VIllag proteolysis. However, the absence of anti-genic determinants shared by vWagII and VIIIag doesnot exclude the possibility that both are products ofa heretofore unidentified common precursor.
Like vWagII, VIllag is present in plasma and plate-lets of normal individuals and absent from those withsevere vWD(15, 16, 37). Recent studies of Nachmanet al. (38) provide evidence that VIIIag is synthesizedby the megakaryocyte. Whether this is true for vWagIIremains to be determined.
Although vWagII is released from platelets duringclotting, it is unlikely that the presence of vWagIIin plasma is the result of in vitro platelet leakageduring the drawing of the blood and subsequentplasma separation. Incubation of the blood at 40, 200,or 37°C for 30 min before separation of plasma did notcause an increase of vWagII content. One would expectsuch a leakage to be time- and temperature-dependentas it is for ,8-thromboglobulin (,B-TG) (see below). It ispossible that the presence of vWagII in normal plasmais secondary to platelet breakdown or release in vivo.vWagII is not present in erythrocytes, lymphocytes, orgranulocytes. Study of other cells is in progress.
There appears to be a weak correlation betweenvWagII and VIIIag concentration in normal individualsand in patients with severe vWD(Fig. 4). Althoughpatients with moderate forms of vWDas a group have
decreased levels of Vlllag and vWagIII, there is nodemonstrable correlation between the two in the groupstudied.
Although vWagII is released from platelets duringclotting as are /8-TG (13, 14) and platelet Factor IV (11,12), it appears to be distinct from them. Platelet FactorIV concentration in serum is 1,000-fold greater thanthat in plasma, whereas vWagII is only fourfoldgreater. f8-TG levels are increased up to 80-fold inplasma from PRP prepared in EDTAat 20° comparedto 4°C. Use of heparin as anticoagulant results in asixfold rise in f8-TG in plasma from PRP prepared at4°C compared to PRP from EDTA-anticoagulatedblood. Plasma vWagII concentration, however, is unaf-fected by the anticoagulant used (CPD, citrate, EDTA,or heparin) and is unaffected by incubation of wholeblood at 40, 200, or 37°C for 30 min with any ofthese anticoagulants. The electrophoretic mobility ofvWagII is more anodic (a-mobility) than either, 3-TG(,8-mobility) or platelet Factor IV (y-mobility).
The function of vWagII is unknown at present.Nevertheless, the description of its deficiency in severevWD imposes a new level of complexity on thegenetics and pathobiology of this disorder.
ACKNOWLEDGMENTSMany of the plasma and platelet samples of the patients withvWDwere kindly provided by Doctors C. F. Abildgaard,C. K. Kasper, and W. E. Hathaway. We wish to thankDiana Patterson, Mary Gortmaker, and Sharon Dinwiddie forpreparation of the manuscript.
This work was supported by grants HL-15491, HL-20517,HL-05195, HL-21730, RR-05514, RR-05357, and HL-16411from the National Institutes of Health.
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