Characterization of recombinant von Willebrand factor corresponding ...

Vol. 267, No. 32, Issue of November 15, pp. 23209-23215,1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of Recombinant von Willebrand Factor Corresponding to Mutations in Type IIA and Type IIB von Willebrand Disease* (Received for publication, June 19, 1992)

Anne-Sophie Ribba, J a n VoorbergS, Dominique Meyer, Hans PannekoekS, and Genevieve Pietug From the Znstitut National de la Sante et de la Recherche Medicale U. 143, H6pital de Bicttre, Paris,France and the fDepartment of Molecular Biology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Seruice, Amsterdam, the Netherlands ~~

Type IIA andIIB von Willebrand disease (vWD) lium components which induces a conformational change in result from defects in von Willebrand factor (vWF). vWF (2). This interaction is reproduced in vitro by action of Although both type IIA and IIBvWD are characterized nonphysiologic modulators such as ristocetin (3) or botrocetin by the absence of high molecular weight multimers in (4). Subsequently vWF interacts with the platelet membrane plasma, vWF from patients with type IIA vWD dem- receptor glycoprotein Ib (GPIb) (5). onstrates a decreased affinity for the platelet receptor The vWF gene which spans 178 kb and contains 52 exons glycoprotein Ib (GPIb), whereas vWF from patients ( 6 ) has been localized to chromosome 12 (7,8). A pseudogene with type IIB vWD show an increased affinity for GPIb. To investigate how structural alterations in vWF has been identified on chromosome 22 and corresponds to the affect its interaction with GPIb, we reproduced the central partof the gene (exons 23-34), its sequence presenting reported potential mutations in type IIA and IIB vWD 97% of homology with that of the gene (9). The primary in vWF cDNAand expressed the recombinant proteins transcript is approximately 9 kb in length (7-8, 10-12) and in COS-1 cells. The type IIA vWF potential mutation encodes a 2,318 amino acids (aa) prepro-vWF composed of a was represented by a G +A transversion which results 22-aa signal peptide, a 741-aa propolypeptide (von Willebrand in the substitutionof Lys for Glu at position 875 in the antigen 11), and the mature vWF subunit (2,050 aa). mature vWF subunit (rvWFLyss7'). The type IIBvWF Among patients with von Willebrand disease (vWD), type mutation corresponds to a duplicated ATG codon, re- I1 variants are characterized by an abnormal interaction of sulting in three contiguous methionines starting at po- vWF with platelets and an absence of high molecular weight sition 640-541 inthenormal vWF sequence (rv- multimers in plasma (13). A decreased reactivity of vWF W F d ~ p l M e t ~ ~ ~The - ' ~ subunit ~). composition and mul- toward platelet GPIb is observed in type IIA vWD, whereas timeric structureof both mutant proteins were similar it is increased in type IIB vWD (14). In type IIA vWD, the to the wild-type rvWF. The ~ v W F L ~ Sbound " ~ to fixed largest multimers may be more sensitive to proteolysis than platelets in thepresence of ristocetin similar to wildthose in normal plasma (15, 16).IntypeIIB vWD, vWF type rvWF. The r v W F d ~ p l M e t ~ ~ bound ~ - ' ~ to ~ fixed shows increased affinity for platelet GPIb, and the largest platelets in the absence of agonist. The rvWFLysS7' appears to interact normally with GPIb, and the de- multimers are removed from the circulation by platelet bindcreased affinity for the platelet receptor observed in ing (17-19). The molecular variants of vWD represent amodel plasma isprobably a consequence of prior reduction in for the study of the mechanism of the binding of vWF to multimeric size resulting from the defect. In contrast, platelet GPIb. the duplication of Met640-541 increases the reactivityof Recently, molecular defects have been identified in exon 28 vWF for its platelet receptor. of the vWF gene in these two types of variants of vWD. In patients with type IIB vWD, aa substitutions and duplication of a single residue have been found between aa 540 and 578 within a loop formed by an intramolecular disulfide bond von Willebrand Factor(vWF)' is a multimeric plasma between Cys509and Cysfg5 (20-27).The conformation of this glycoprotein with two essential functions, asacarrier of loop may be important to juxtapose the two noncontiguous Factor VIII, stabilizing its activity, andasa mediator of sequences (aa 474-488 and 694-708) (28) and the recently platelet adhesion to the subendothelium following damage of described third domain (aa 514-542) (29) involved in the the vascular endothelium (1).At high shear rates, platelet interaction of vWF with GPIb. Furthermore, binding sites of adhesion depends first on the binding of vWF to subendothe- vWF tocollagen (30), heparin (31), sulfatides (32), and botrocetin (33) have been localized within this loop. The type IIB * This work was supported by the Ministkre de la Recherche et de mutations are thought to induce a conformational modificala Technologie andthe Fondation pour la Recherche MBdicale tion of this part of vWF which may result in an increase of (France). The costs of publication of this article were defrayed in part by the payment of page charges. This article musttherefore be hereby affinity of vWF for GPIb; a similar conformational change of marked "aduertisement" in accordance with 18 U.S.C. Section 1734 vWF would result from botrocetin or subendothelium binding. solely to indicate this fact. Potential mutationsdetected in type IIA variants have been I To whom correspondence should be addressed INSERM U.143, located between aa 742 and 865 (34-37) in an area which is Hbpital de Bicitre, 94275le Kremlin-Bicitre Cedex, France. Tel.: carboxyl-terminal to the GPIb binding site of vWF where a 33-1-46-58-15-44;Fax: 33-1-46-70-64-46. has been identified The abbreviations used are: vWF, von Willebrand factor; vWD, proteolytic cleavage site at TyrS42-Met843 von Willebrand disease; GPIb, glycoprotein Ib; mAb, monoclonal (38). The type IIA mutations may change the conformation antibody. of the molecule around this site and increase the sensitivity 23209

23210

Expression and Characterization

of vWF toplasma proteolysis, resulting inthe loss of the high molecular weight multimers (35,38) and thedecreased affinity for GPIb. In previous studies, we have identified two potential molecular abnormalities in 'type IIA and type ZIB vWD patients and excluded a polymorphism by studying 100 normal alleles (27,39). In thisstudy, we have reproduced in the vWFcDNA a G +A transversion, substituting the aa residue lysine (Lys) for glutamic acid (Glu) 875 observed in a patient with type IIA vWD (39) and a duplicated ATG codon for methionine (Met) detected in a patient with type IIB vWD (27).We have expressed the full-length cDNAs and compared the functional properties of the resulting mutant proteins with those of the wild-type recombinant vWF (rvWF). The rvWF containing the mutation Glu 4 Lyss75and the wild-type rvWF bind to platelet GPIb only in the presence of ristocetin. The rvWF containing the duplication of Met640-541 has an increased affinity for GPIb which is consistent with the type IIB phenotype.

of Mutated uon Willebrand

Factor

firm thecorrect ligation of the fragments. Plasmids were then amplified and purified by cesium chloride-ethidium bromide equilibrium centrifugation before being used for transfection. Cell Culture and Transfection-Monkey kidney cells (COS-1) were cultured for 3 days inIscove's modified Dulbecco's medium (GIBCO) supplemented with penicillin (100 units/ml) (GIBCO), streptomycin (100 pg/ml) (GIBCO),and10%(v/v) fetal calf serum(GIBCO). When cultures were confluent, cells were harvested with 0.05% trypsin and 0.02% EDTA and cultured overnight in the samemedium as described above. Transfections were performedusing the DEAEdextran procedure (43). Cells were transfected with 20 pg of the appropriate plasmid DNA (pSVLvWF, r v w F L y ~ ~ ' ~a, n d rvW F d ~ p l M e t ~ ~ ' in - ~ 4~ ml ' ) of culture medium supplemented with 2% fetal calf serum, 51.6 pg/ml chloroquine(Sigma) and 300 pg/ml DEAE-dextran (Pharmacia Fine Chemical, Uppsala, Sweden). After 4 h at 37 "C, this incubation was followed by addition of 1 ml of 10% dimethyl sulfoxide solution to the culturesfor 1 min at room temperature. Subsequently, the cells were washed twice and maintained overnightin culture medium containing penicillin (100 units/ml), streptomycin (100 pg/ml), and 10% (v/v) fetal calf serum. The transfected cells were thencultured for 48 hinserum-free medium. Conditioned media containing the expressed rvWF was frozen at -20 "C.Conditioned medium from cells transfected withplasmid pSVL without full-length vWF cDNA was used as a control. MATERIALS ANDMETHODS Metabolic Labeling of Transfected Cells and ZmmunoprecipitationPatients-The two patients demonstrated all criteria for either In some experiments, expressed rvWF was metabolically labeled with type IIA or type IIB vWD. In the patientwith type IIA vWD, a G + [3sS]methionine (Amersham International,Amersham, United KingA substitution replacing the aa residue G h P 5 by Lys was observed dom) as described previously (44). %-Labeled rvWF contained in (391, and in the patient with type IIB, a duplicated ATG codon was conditioned media or in cell extracts was immunoprecipitated overidentified (27). Both patients had prolonged Ivy bleeding time and night at 4 "C with rabbit anti-vWF IgG (Dakopatts, Glostrup, Denmultimer analysis by 0.1% SDS, 1.6%agarose gel electrophoresis mark) coupled to proteinA-Sepharose (Pharmacia). Theimmunopredemonstrated the loss of high molecular weight multimers of vWF. cipitates were analyzed by 0.1% SDS, 5%polyacrylamide gel electroThey had levels of vWF antigen(57 units/dl for type IIA patient and phoresis and visualized by autoradiography (45). Molecular weigth (M,) markers used included myosin (200,000). 36 units/dl for type IIB patient)higher than those of vWF ristocetin Characterization of Antibodies-A polyclonal antiserum (Ab 44) cofactor activity (11 and 14 units/dl, respectively). In platelet-rich produced in rabbits by immunization with purified human vWF was plasma from the patient with type IIA, a lack of ristocetin-induced platelet aggregation (RIPA) was observed as a ristocetin concentra- prepared and rendered monospecific by immunoadsorption of contion of 1.3 mg/ml, whereas in the patientwith type IIB, a ristocetin- taminants with plasma from a patient with severe vWD as reported induced platelet aggregation was obtained using 0.3 mg/ml of this (46). mAbs to human vWF were produced and characterized as described antibiotic. Plasmid Constructs-The expression vector pSVLand theplasmid previously (47). mAb 324 is directed toward the part of vWF compSVLvWF containing the wild-type full-length cDNA of vWF have prised between aa 449 and 728 and known to inhibit the vWF binding been described previously (40). The G + Amissense mutation, to GPIb(48). mAb 9 specifially inhibits thebinding of vWF to GPIIb/ substituting the aa residueGlu to Lys at position 875 (39), was IIIa (46) and is directedagainst an epitope located betweenaa residues introduced into theplasmid pSVLvWF in a five-fragmentligation. A 1698 and 1773 of the mature vWF subunit (49). A pool of mAbs to single-stranded M13 clone was obtained after subcloning and se- vWF thatrecognize epitopes distributed across the NH2- and COOHquencing of an amplified DNA fragment from patient genomic DNA terminal parts of the mature vWF subunitwas prepared (mAbs 424, by polymerase chain reaction (39). This clone, containing an insert 439, 456, 532, and 543 to the NH2-terminal part, mAbs 8,4, and433 of genomic DNA, possessed the G + A mutation at position 5142 to the COOH-terminal part). No mAb from the pool inhibited the binding of vWF to GPIb. and was digested usinga standard procedurewith the restriction binding of vWF mAb 6D1 is directed toward GPIb and inhibits the enzymesAcc1 and BspEI (Boehringer Mannheim, Mannheim,Federal to GPIb (50). mAb 7E3 is directed toward GPIIb/IIIa and inhibits Republic of Germany) to obtain a 205 base pair fragment (vWF nucleotides 5039-5244). Four restriction fragments of 8.3, 0.5, 1.9, the binding of vWF to thatreceptor (51). Radiolabeling of the Antibodies-Rabbit anti-vWF polyclonal Ab and 2.5 kb in length were isolated from the plasmid pSVLvWF after double digestion with the restriction enzymesBspEI-XhoI, XhoI- 44 and thepool of mAbs to vWFwere labeled with Na1251(Amersham) KpnI, KpnI-HindIII, and HindIII-AccI (Boehringer),respectively, and using the Iodo-Gen method (52). Specific radioactivity was 10 pCi/ gg for Ab 44 and 2.5 pCi/pg for the pool of mAbs to vWF. purified on low meltingagarose gels (GIBCO-BethesdaResearch oWF AntigenDetermination-The amount of vWF antigen Laboratories, Cergy Pontoise, France). The ligation was performed usingstandard methods, andthe new construct was named rv- (vWFAg) in conditioned media after transfection was determined by enzyme-linked immunoadsorbent assay as described previously (40). WFL~S'~~. The duplication of a Met at position 540-541 was reproduced onto Conditionedmedia were concentrated by Centricon 100 filtration the vWF subunitby introducing an ATG codon at position 4139 into (Amicon Corp., Danvers, MA), and the levels of vWFAg in concenthe complete cDNA of vWF. Themutagenesis was realized using the trated media were measured by two-site immunoradiometric assay M13-gapped duplex protocol (41). 1.6-kb A SstI-KpnI fragment (vWF (53). The pool of mAbs was diluted in 0.05 M Na2C03, pH 9.6, and nucleotides 3310-4977) was subcloned into the double-strandedM13 coated at 5 pg/ml on a polystyrene plate. The samples were diluted mp18 phage DNA and annealedwith a 23-mer mutagenicoligonucle- in 0.05 M disodium tetraborate, 0.5 M NaC1, pH 8, containing 2% otide (5'AGCCGCTCCAT=TCATGTCCAC3') (inserted bases are bovine serum albumin, added to the plate, and incubated overnight underlined). A clonecontaining thedesired mutation was selected by at 37 "C. The labeled polyclonal Ab 44 (0.4 lo6 cpm/ml) was used for sequencing (42) the entire cDNA. The clone was then digested with detection. Levels of vWFAg present in the concentrated media of the restriction enzymes PstI and NcoI, and the 669-base pair PstI- transfected cells were expressed relative to a reference pool of 20 NcoI cDNA fragment (vWF nucleotides 4042-4711), containing the normal plasmas arbitrarilydefined as 100 units/dl of vWFAg. ru WF Multimer Analysis-Multimeric analysis of the rvWF was ATG insertion, was isolated. The plasmid pSVLvWF was digested with therestriction enzymes NcoI-KpnI,KpnI-Sa& SalI-BamHI, and performed by 0.1% SDS, 1.4% agarosegel electrophoresis as described BamHI-PstI (Boehringer). The resulting restriction fragments, hav- in detail by Nishino et al. (54). Approximately 20 pl of rvWF at a ing a size of 0.2, 4.5, 7.3, and 1.1 kb, respectively, were purified as concentration of vWFAg of 5 units/dl were mixed in 10 mM TrisHC1, pH 7.8, 2% SDS and layered on the gel. After electrophoresis, described above and ligated to the PstI-NcoI-mutated insert. The the gel was fixed, incubated with 1251-labeledpolyclonal Ab44 (IO6 obtained plasmid was called r v W F d ~ p l M e t ~ ~ ' - ~ ~ ' . Each DNA construct was digested by restriction enzymes to con- cpm/ml), and dried. The multimeric composition of the samples was

23211

Expression and Characterization of Mutated uon Willebrand Factor visualized after 36 h of autoradiography a t -80 "C. Normal plasma vWF was treated in the sameway and used as a control. Preparation of Formaldehyde-fixed Platelets-Platelet-rich plasma was obtained from fresh human hlood collected into 0.10 volume of 3.8% sodium citrate by centrifugation for 10 min at 190 x g a t 20 "C. Platelet-rich plasma (100 ml) was incubated with 98 ml of 150 mM NaCI, 10 mM Tris, pH7.4, and 2 ml of formaldehyde 35-40% (Merck, Darmstadt, Federal Republic of Germany) for 18 h a t room temperat,ure in the dark. The mixture was centrifuged 15 mina t 2530 X g a t 4 'C. The platelet pellet was washed three times in 150mM NaCI, 10 mM KH2P04, pH 6.5. The platelets were resuspended in 150 mM NaCI, 10 mM Tris, pH 7.4, to a final concentration of 6 X 10R/mland stored a t 4 "C before be ng used. rv WF BindingAssay to Platelet GPIb-To measure the capacityof a binding assay was performed based rvWF to bind to platelet GPIb, on described protocols (55, 56) and previously used to characterize rvWF expressed in different types of cells (57). Various amounts of rvWF (final concentration from 0 to 5 units/dl) were incubated with the '2'II-labeled pool of mAbs to vWF (lo6 cpm/ml) for 30 min a t room temperature. After incubation, 30 p1 of the mlxture was added t o 200 pl of formaldehyde-fixed platelets which were diluted in 25 mM Tris-HCI, pH 7.4, 2% bovine serum albumin (Calbiochem) to a final count of 10"/ml. Binding was performed in the absence or the presence of ristocetin a t a concentration of 1.5 mg/ml. After incubation for 1 h a t room temperature, platelets were centrifuged 3 min a t 10,000 X g through 250 pl of Ficoll solution (Pharmacia) to separate hound from free ligand. Radioactivity associated to the platelet pellet or to the supernatant was determined in a y-scintillation counter (LKB,Bromma,Sweden),andthebindingwasestimatedasthe percent of total radioactivity bound to platelets (BIT). As controls, HIT was also estimated using conditionedmedia from cells untransfected and transfected with the expression vector pSVL without the full-length vWF cDNA. In order to assess the accuracyof the method, binding of vWF to platelet GPIb was estimated using plasma from normal subjects or a concentration of vWFAg patients with type IIA or type IIB vWD. At of 20 units/dl, binding of plasma vWF to GPIb varied asa function of the concentration of ristocetin added (from 0 to 1.5 mg/ml). The plasma vWF of the type IIB patient bound to GPIb at a lower concentration of ristocetin (0.5 mg/ml) than that necessary to bind the normal plasma vWF (1 mg/ml). At high concentrations of ristocetin (1.5 mg/ml), no significant binding wasobserved with the type IIA plasma vWF. Using that concentration of ristocetin, binding of vWF from normal plasma and from type IIB plasma was a function of the amount of vWFAg (from 0 to 20 units/dl) added to fixed platelets whereas binding of vWF from type IIA plasma was similar to that obtained with vWF-depleted normal plasma. Inhibition of rv WF Bindingto Platelets by Anti-u WFor Anti-GPIb mAbs-The effect of anti-vWF mAb 324 was evaluated on the ristocetin-mediated binding of rvWF or normal plasma vWF to GPIb. T h e samples of rvWF or normal plasma vWF were first incubated with various concentrations of mAb 324 (from 0 to 20 pg/ml) for 30 min atroom temperature and thenwith the "'I-labeled pool of mAbs ( loficpm/ml) and added to 10'/ml fixed platelets as described above. mAb 9 which inhibits vWF binding to GPIIb/IIIa wasused as a negative control. The effect of mAb 6D1 to GPIbwas tested on the binding of rvWF or normal plasma vWF to GPIb induced by ristocetin. Various concentrations of mAb 6D1 (from 0 to 20 pg/ml) were incubated with fixed platelets (lOR/ml)for 30 min a t room temperature prior to adding the samples of rvWF or normal plasma vWF. Binding was performed as described above, using mAb 7E3 to GPIIb/IIIa as a negative control.

fected with the expressionvector pSVL without the fulllength vWFcDNA were used as controls. Structural Characterization of ru WF-NWF metabolically labeled with ["S]methionine was immunoprecipitated from the conditioned media of COS-1 cells transfected with the wild-type (pSVLvWF) or mutant constructs(rvWFLysR'sand rvWFd~plMet"~"-"').Following reduction and SDS-polyacrylamide gel electrophoresis, the NWF appeared as two bands with an apparent M, (360,000 and 275,000) corresponding to the pro-vWF and mature vWF subunit, respectively (Fig. U). In addition, the proportion of secreted pro-NWF and mature NWF for the two mutant proteins was approximately 50% and similar to that of wild-type NWF (Fig. lA). Only the unprocessed form was observed for wild-type or mutant NWF from cell extracts (data not shown). No band was detected in the medium of cells transfected with the expression vector pSVL without the vWFcDNA (Fig. lA). The multimeric structure of the recombinant proteinswas analyzed by SDS, 1.4% agarose gel electrophoresis. All the multimeric forms were present when compared with normal plasma vWF, but bands corresponding to smaller multimers had greater intensity in the wild-type and mutant NWF than in normal plasma vWF (Fig. 1B). No vWF multimers were detected in the medium of cells transfected with the expression vector pSVL without the vWFcDNA (Fig. 1B). Functional Churacterization of ru WF-The wild-type and mutants r v w F L y ~a n~d~ ~~W F d u p l M e t " ~ ~were " ' tested for their ability to bind to platelets in the absenceor in the presence of ristocetin. The bindingof the wild-type and both mutant NWF to fixed platelets was similar to thatobtained with the normal plasma vWF in the presence of ristocetin (1.5 mg/ml) (Fig. 2). This bindingincreased with the amount of vWFAg added (from 0 to 5 units/dl) and at the maximal concentration of vWFAg used (5 units/dl), a B I T of 20-2596 was observed for the wild-type and the two mutant NWF, E

A 1

2

3

4

MW

1

2

"

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5 1

* !

pro-vWF vWF+

F 2 0 0 000

u*

FIG.1. Structural characterization of wild-type and mutants r v W F s e c r e t e d into the conditioned medium by transfected COS-1 cells. A, subunit composition of wild-t-ype and mutants rvWF. Cells were metabolically labeled with [:"S]methionine. Labeled rvWF was immunoprecipitated with rabbit anti-vWF IgG coupled to protein A-Sepharose andanalyzed under reducing conditions by 0.1% SDS, 5% polyacrylamide gel electrophoresis. COS-1 cells transfected with the expression vector pSVL without the fulllength cDNA were treated in the same way and used as a control. The position of pro-vWF and mature vWF subunit is indicated on' the left by the arrows. The ratio of pro-vWF to mature vWF within each sample was compared by densitometry. The molecular weight RESULTS (MW) markers included (200,000). Lane I, wild-type rvWF; lane 2, Expression of ruWF-The wild-type and two mutant full- mutant rvWFLys"'; lane 3, mutant rvWFduplMet540-s4';lane 4, negativecontrol pSVL. R, multimeric composition of wild-type and length cDNAscloned in the expressionvector pSVL and mutants rvWF. Conditioned medium was concentrated and electrocalled pSVLvWF, rvWFLys875, and rvWFduplMet.540-541 phoresed onto a 0.1% SDS, 1.4% agarose gel under nonreducing were expressedunder the controlof the SV40 "late" promoter conditions for analysis of the rvWF multimeric pattern. Approxiin the COS-1 cells. Expression levels of COS-1 cells trans- mately 20 pl of normal plasma vWF or rvWF a t a concentration of fected withthe wild-type or with the mutantplasmids reached vWFAg of 5 units/dl were loaded on thegel. After electrophoresis the was fixed, incubated with 'Y51-labeledrabbit anti-vWF polyclonal about 1unit/dl of vWFAg secreted in the conditionedmedia. gel antibody (lo6 cpm/ml), and dried. The gel was exposed to autoradiAfter concentration, the maximal levels of vWFAg obtained ography for 36 h a t -80 "C. Lane I, normal plasma vWF; lune 2, wildwere approximately 5 units/dl for each recombinant protein. type rvWF; lane 3, mutant rvWFLysR'"; lane 4, mutant rvWFduplConditioned media from COS-1cells untransfected and trans- Met540-541; lane 5 , negative control pSVL.

23212

Expression and Characterization

of Mutated uon Willebrand

A

FIG. 2. Ristocetin-induced bind32 ing of wild-type andmutant rvWF to fixed platelets. Various amounts (from 0 to 5 units/dl of vWFAg) of vWF from normal plasma and from wild-type or mutant rvWF from conditioned medium were first incubated for 30 min at room temperature with the pool of '=I- t labeled mAbs to vWF (lo6cpmlml) and then for 1 h at room temperature with formaldehyde-fixed platelets (lOa/ml) in the presence of 1.5 mg/ml of ristocetin (filled symbols) or in the absence of this antibiotic (open symbols) as 5described 0 under "Material and Methods." After centrifugation, the radioactivitv associ- c atedtothe platelet pellet or to the su32 pernatant was determined and binding was expressed as the percent of the total radioactivity bound to platelets (B/T76). Nonspecific binding was estimated using the conditioned medium alone or the conditioned medium of cells transfected with the vector pSVL without the cDNA 16 of vWF (0 unit/dl of vWFAg). Results represent the mean & S.D. of three separate experiments. A , normal plasma vWF (m, 0; B , wild-type rvWF (A, A); I C, mutant ~ ~ W F (0,O); L ~ D, S mutant ~ ~ ~ rvWFd~plMet''~-~~~ (+, 0).

*

2.5

'

~

Factor

B WildType

32

Normal Plasma

4

T 16

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I

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vWFAg added (U/dl)

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rVWFLys 875

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-

vWFAg added (U/dl) rvWFduplMet 540-541

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vWFAg added (Uldl)

vWFAg added (U/dl)

comparable with the binding of the normal plasma vWF (Fig. ~ ~ W F L ~and S ' ~r v~W, F d ~ p l M e t ' ~ ' -mutants ~~~ was of 90, 98, 2). In the absence of ristocetin, the wild-type rvWF and and 75%, respectively, with 20 pg/ml ofmAb 6D1 (Fig. 3B). ~ v W F L ~ were S ' ~ unable ~ to interact with platelets at all con- The binding of wild-type rvWF, of normal plasma vWF, and S ' ~ ~ is inhibited to the same extent by centrations of vWFAg tested (Fig. 2, B and C); BIT was of ~ ~ W F L to~ platelets similar to that obtained with the conditioned media of cells this antibody as expected (Fig. 3B). The binding of rvuntransfected or transfected with the expression vector pSVL W F d ~ p l M e t ~ ~ 'however, - ~ ~ l , was less inhibited than that of without the vWF cDNA (BIT, 5.4%). In contrast, the rv- the both recombinant proteins and the normal plasma (Fig. WFd~plMet'~'-'~~ bound to platelets in the absence of risto- 3B). The anti-GPIIb/IIIa mAb 7E3 inhibiting the binding of cetin.This binding was dependent upon the amount of vWF to GPIIb/IIIa was used asa control to evaluate the vWFAg added and was similar to thatobserved in the presence nonspecific inhibition (less than 10%). of ristocetin (Fig. 2 0 ) . mAb 324 vWF to known to inhibit vWF binding to GPIb DISCUSSION was tested upon the binding of normal plasma vWF andwildDirect evidence that mutations in type IIA or type IIBvWD type and mutants rvWF to platelets in the Presence of risto- are responsible for the phenotype of the patientsrequires the &in. Different concentrations of mAb 324 (from 0 to 2 rg/ analysis of the expression products from mammalian cells ml) were incubated with normal plasma vWF or rvWF at a transfected with the full-length mutated vWF cDNA. The concentration of vWFAg of5 units/dl prior to performing the corresponding mutated recombinant proteins should mimick binding assay. The maximal inhibition (95%)of the binding the abnormal multimeric structure and platelet interactionof of the normal plasma vWF was reached for a mAb 324 the variants of VWD. concentration corresponding to 2 (Fig. 3A). Withthe In this paper, we have transfected the full-length vWF 324, the ristocetin-mediated cDNA containing mutations into COS cells. Using site-&sameconcentration ofmAb platelet binding of the wild-type rvWF and the ~ ~ W F L Y Srected ' ~ ~ mutagenesis we have reproduced the G + A mutation was inhibited to 75 and 50%, respectively (Fig. 3A). The substituting the aa residue Glu + LysS7', corresponding to a partial inhibition of the binding observed with the ~ ~ W F L ~potential S ' ~ ~ mutation observed in a type IIA patient (39), and was not explained. mAb 324 was, however,not able to interact the insertion of an ATG codon, encoding for a duplication of withthe r v W F d ~ p l M e t ' ~ ' -by ~~~ immunoradiometric assay Met at position 540-541, detected in a type IIB patient (27). (data not shown), thus it did not interfere with the binding Analysis by SDS-polyacrylamide gel electrophoresis of the of the mutant r v W F d ~ p l M e t ~ ~ ' - ~platelets ~ ' t o (Fig. 3A). The subunit composition of the wild-type and the two mutants anti-vWF mAb 9 used as acontrol, which inhibits the binding ~ ~ W F Land ~ SrvWFd~plMet'~'-~~' ' ~ ~ indicated that therecomof vWF to GPIIb/IIIa, had no significant inhibitory effect binant proteins are secreted both as nonprocessed and proc(less than 10%) on platelet interaction of the normal vWF essed forms, corresponding to the pro-vWF and mature vWF subunit, respectively. The pattern of the proteolytic processand rvWF proteins. The anti-GPIbmAb 6D1, which blocks the vWFinteraction ing of the mutants rvWF was identical to that of the wildwith GPIb, inhibited the binding of normal plasma vWF to type rvWF. It can be deduced that the processing for these platelets in a dose-dependent fashion, and 10 pg/ml of mAb mutants occurs in the same way as thatof the wild-type pro6D1 were sufficient to obtain an inhibition of 90% (Fig. 3B). rvWF and is consistent with the normal processing of the The inhibition by mAb6D1 of the binding of wild-type rvWF, plasma pro-vWF. Nevertheless, the processing of the recom-

23213

Expression and Characterizationof Mutated uon WillebrandFactor B

A

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20

MAb 6D1 added (pglml)

FIG. 3. Effect of anti-vWF and anti-GPIb mAbs on the ristocetin-induced binding of normal vWF and of wild-type and mutants rvWF. A , effect of anti-vWF mAb 324 on the ristocetin-induced binding of normal vWF and of wild-type and mutants rvWF. Normal plasma vWF or wild-type or mutants rvWF (5 units/dl of vWFAg) were incubated for 30 min a t room temperature with various concentrations of mAb 324 (from 0 to 2 pg/ml) prior to adding to thepool of lZ5I-labeledmAbs to vWF andfixed platelets with 1.5 mg/ml of ristocetin as described under "Material and Methods." B, effect of anti-GPIb mAb 6D1 on the ristocetin-induced binding of wild-type and mutants rvWF. Fixed platelets (108/ml) were incubated for 3 0 min at room temperature with different concentrations of mAb 6D1 (from 0 t o 20 pg/ml) before performing the binding assay with 5 units/dl of vWFAg of normal plasma vWF, wild-type or mutants rvWF, and 1.5 mg/ ml of ristocetin. Results are expressed as percent of inhibition of maximal binding to platelets of rvWF or normal vWF in the absence of mAb 324 or mAb 6D1. Nonspecific binding obtained with conditioned medium of cells transfected with the vector pSVL without the vWF cDNA was substracted in all experiments. Results represent the mean +. S.D. of three separate experiments. M, normal vWF; A, wild-type ~; rvWFd~plMet'~~-~~'. rvWF; 0, mutant ~ ~ W F L ~ S "mutant

4,

binant proteins occurs only partially in COS-1 cells, whereas the rvWF is completely processed in COS-7 cells (58, 59). Potential mutations clustered in a small region around the proteolytic site T ~ r ~ * - Mmay e t be ~ ~responsible for type IIA vWD expression by causing the absence of the high molecular weight multimers of vWF in plasma. They could alter the synthesis of the large vWF multimers or enhance the sensitivity of the mutant vWF to proteases in plasma. Lyons et al. (37) have shown the existence of two groups of type IIA mutants rvWF expressed in COS cells depending on the different pattern of multimers. The first group exhibited the full range of the vWF multimers, suggesting that the loss of high molecular weight multimers in patients occurs following secretion and is possibly due to proteolysis in plasma at the site aa842-843. The second group demonstrated aloss of high molecular weight multimers and appeared to be defective in intracellular processing. In our study, the multimeric structure of the mutant NWFLyP5analyzed by SDS-agarose gel electrophoresis is similar to that of the wild-type NWF and to the first group of the type IIA mutant NWF described by Lyons et al. (37) (Arg + Trp'34 and Gly + G ~ u ~The ~ ~mutation ) . L Y S ' ~does ~ not prevent the rvWF to multimerize in the COS cells and thus the mutant rvWFdoes not exhibitthe structuralabnormality of plasma vWF observed in the patient with type IIA vWD. This difference of multimeric patterns observed between plasma of type IIA patient and medium of COS cells may be explained by the existence of proteases in plasma. Calpain, a protease which is postulated to be implicated in the proteolysis of vWF at Tyra2-Meta3, could be present in plasma and responsible for the increased proteolysis of vWF in type IIA vWD, whereas it would be absent inthe medium of COS cells leading to thelack of proteolysis of the mutated NWFL~S'~'. Functional studies demonstrated that the binding of the mutant ~ ~ W F L to ~ GPIb S ' ~ is ~ similar to that of the wildtype NWF. This binding occurred only in the presence of ristocetin, was dependent upon the amount of rvWF added, and was inhibited by mAb 324 to vWF and by mAb 6D1 to GPIb which block the binding of vWF to GPIb. Thus, the presence of the mutation Glu + L y P 5 in the mature vWF

subunit of theNWF does not reproduce the absence of binding observed with the plasma of the patientwith type IIA using the same method. The characteristic loss of the multimers seen in type IIA has been postulated to affect the function of the protein, resulting in adecreased affinity of the vWF for platelet GPIb. We speculate that the normal binding of the ~ ~ W F L to~ S ' ~ ~ platelets is due to the presence of all the multimers in the recombinant protein, thus not mimicking the type IIA phenotype. Consequently, our hypothesis is that the potential mutation Glu + L y P 5 detected in type IIA (39) may predispose to enhanced proteolysis in vivo and be responsible for the altered structureof multimers in plasma and thedecreased reactivity of vWF toward GPIb. The most characteristic abnormalityof vWF from patients with type IIB is its increased affinity for GPIb. Mutations that cause this phenotype should identify a structure of vWF that can modulate the affinity of the protein for its receptor. The rvWFd~plMet'~'-~~~ shows the full range of vWF multimers, similar to that of wild-type NWF. In type IIB vWD, the loss of high molecular weight multimers is explained by their removal from the circulation by platelet binding due to an increased affinity of vWF for GPIb (17-19). In the expression system used where rvWF is not in contact with platelets, the rvWFd~plMet'~'-'~~ exhibits a multimerization identical to thatof the wild-type NWF. Direct evidence of the effect of the mutation is the enhanced binding of r v W F d ~ p l M e t ~ ~ ' - ~platelets. ' ~ t o The mutantrvWF is able to bind to GPIb in the presence and also in the absence of ristocetin. The rvWFd~plMet'~'-~~~ is not recognized by mAb 324 which blocks the binding of normal vWF to GPIb; thus the conformation of its epitope is probably modified by the presence of the duplication of Met540-541 in this region of vWF. The binding of the r v W F d ~ p l M e t ~ ~is' inhibited -~~~ by the mAb 6D1 to GPIb which blocks the binding of vWF to its platelet receptor. This mAb inhibited more strongly the binding of the wild-type rvWF than that of the rvWFd~p1Met~~'541. This could be explained by the ability of the rvW F d ~ p l M e t ~ ~ to O -bind ~ ~ l to platelets in absence of agonist, thus mAb 6D1 could not completely compete off the N-

23214

Expression and Characterization of Mutated von Willebrand Factor

State University of New WFd~plMet'~'-'~~ from platelets. The affinity of N - Dr. B. S. Coller (Division of Hematology, WFd~plMet'~'-'~~ for GPIb is increased as compared with that York at Stony Brook, Stony Brook, NY)is gratefully acknowledged. of the wild-type rvWF and similar to thatof the plasma vWF REFERENCES in type IIB vWD. The duplication of the Met540-541 recreates 1. Baruch, D., Bahnak, B.R., Girma, J. P. & Meyer, D. (1989) in Platelet Disorders (Caen, J. P., eds) Bailliere Tindall-WB, London the phenotypic abnormality identified in the type IIB patient, 2. Sakariassen, K. S., Bolhuis, P. A. & Sixma, J. J. (1979) Nature 2 7 9 , 636and we conclude that itis the molecular basis for the type IIB 638 3. Kao, K. J., Pizzo, S. V. & McKee, P. A. (1979) J. Clin. Inuest. 63,656-664 in this patient. 4. Read, M. S., Shermer, R. W. & Brinkhous, K. M. (1978) Proc. Natl Acad. Thus, the native conformation of vWF prevents itsbinding Sct. U. S. A. 75,4514-4518 5. Sakariassen, K. S., Nievelstein, P. F. E. M., Coller, B. S. & Sixma, J. J. to GPIb, and the loop located between the Cys residues 509 (1986) Br. J. Haematol. 63,681-691 and 695 may be required to maintain the appropriate confor6. Mancuso, D. J., Tuley, E., Westfield, L. A., Worrall, N. K., Shelton-Inloes, B. B., Sorace, J. M., Alevy, Y. G. & Sadler, J. E. (1989) J. Biol. Chem. mation of the three noncontiguous segments 474-488, 514264,19514-19527 542, and 694-708. All the alterations in the structure of this 7. Ginsburg, D., Handin, R. I., Bonthron, D. T., Donlon, T. A,, Bruns, G. A. P., Latt, S. A. & Orkin, S. H. (1985) Science 2 2 8 , 1401-1406 loop that were observed in type IIB patients seem to enhance 8. Verweij, C.L., De Vries, C. J. M., Distel, B., Van Zonneveld, A. J., Van the binding of vWF to GPIb. Conversely, two proposed muKessel, A. G., Van Mourik, J. A. & Pannekoek, H. (1985) Nucleic Acids Res. 13,4699-4717 tations in this loop detected in vWD type B (Gly + S e P ) 9. Mancuso, D. J., Tuley, E. A,, Westfield, M.A., Lester-Mancuso, T. L., (60) and in a putative type I variant (Phe +Ile606)(61) appear Lebeau, M. M., Sorace, J. M. & Sadler, J. E. (1991) Biochemistry 3 0 , 253-269 to decrease the binding of vWF toGPIb. The proteolytic vWF 10. Sadler, J. E., Shelton-Inloes, B. B., Sorace, J. M., Harlan, J. M., Titani, K. fragment 111-T2 (30) deleted of aa 512-673 but containing the & Davie, E. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,6394-6398 peptidic sequences 474-488 and 694-708 was able to bind to 11. Lynch, D. C., Zimmerman, T. S., Collins, C. J., Brown, M., Morin, M. J., Ling, E. H. & Livingston, D. M. (1985) Cell 41,49-56 platelets without any modulator. In porcine vWF which di- 12. Verweij, C. L., Diergaarde, P. J., Hart, M. & Pannekoek, H. (1986) EMBO J. 5 , 1839-1847 rectly aggregates platelets in uitro, the mismatches between 13. Ruggeri, 2. M. & Zimmerman, T. S. (1987) Blood 70,895-904 the porcine and the human molecules are at thesame position 14. De Marco, L., Mazzucato, M., De Roia, D., Casonato, A., Federici, A. B., Girolami, A. & Ruggeri, Z. M. (1990) J. Clin. Inuest. 8 6 , 785-792 as threepointmutations associated with type IIB vWD 15. Gralnick,H. R., Williams, S. B., McKeown, L. P., Maisonneuve, P., A r p , and Trp"') (62).As demonstrated in this study, Jeanneau, C., Sultan, Y. & Rick, M. E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,5968-5972 the insertion of an amino acid within the loop modifies the 16. Berkowitz, S. D., Dent, J., Roberts, J., Fujimara, Y., Plow, E. F., Titani, complex interaction of vWF with GPIb and theother mutaK., Ruggeri, Z. M. & Zimmerman, T. S. (1987) J. Clin. Inuest. 7 9 , 524531 tions observed in type IIB vWD in a small region of the vWF 17. Ruggeri, Z. M., Pareti, F. I., Mannucci, P. M., Ciavarella, N. & Zimmerman, subunit between aa 540 and 578 also appear to modulate the T. S. (1980) N. End. J. Med. 302. 1047-1051 18. Ruggeri,'Z. M:,-Lombkdi, R., Gatti, L., Bader, R., Valsecchi, C. & Zimmerbinding of vWF toGPIb. man, T. S. (1982) Blood 60,1453-1456 Ware et al. (22) have reported the type IIB substitutionTrp 19. De Marco. L.. Girolami. A,. Zimmerman. T. S. & Ruageri, 2. M. (1985) Proc. Natl. Acad. Sci. U . S . A. 8 2 , 742417428 + Cys"' on a recombinant fragment spanning aa 441 to 730. 20. Randi. A. M.. Rabinowitz. I.. Mancuso. D. J.. Mannucci. P. M. & Sadler. J. The mutation had no functional consequence when this fragE . (i991) j.Clin. Inuest. 87, 1220-1226 K. A,, Nichols, W. C., Bruck, M. E., Bahou, W. F., Shapiro, A. D., ment was expressed in Escherichia coli and did not exhibit 21. Cooney, Bowie. E. J. W.. Gralnick. H. R. & Ginsburg, D. (1991) J. Clin. Inuest. the native conformation of the molecule. On the contrary, the 87,1227-1233 binding of the mutated fragment expressed in Chinese ham- 22. Ware, J., Dent, J. A,, Hiroyuki, A., Sugimoto, M., Kyrle, P. A., Yoshioka, A. & Ruggeri, 2. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,2946-2950 ster ovary cells was 10-fold higher than that obtained with 23. Kroner, M. L., Kluessendorf, M.L., Scott, J. P. & Montgomery, R. R. (1992) Blood 7 9 , 2048-2055 the nonmutated fragment. Other type IIB mutations, Pro + 24. Lillicrap, D., Murray, E. W., Benford, K., Blanchette, V. S., Rivard, G. E., Val + Met653,Arg + Gln578,and Arg + were Wensley, R. & Giles, A. R. (1991) Br. J . Haematol. 79,612-617 expressed on the full-length vWF (23,63,64),and the mutant 25. Donner, M., Andersson, A. M., Kristoffersson, A. C., Nilsson, I. M., Dahlhack, B. & Holmberg, L. (1991) Eur. J. Haematol. 47,342-345 rvWF also showed an increased affinity for GPIb. 26. Holmbera. L.. Donner.. M... Dahlback, B. & Nilsson, I. M. (1991) Blood 7 8 , (suppl.j.15Oa (abstr.) The conformational change of the loop induced by the type 27. Ribba. A. S.. Laverene. J. M.. Bahnak.. B. R... Derlon,. A.,. Pietu, G. & Mever, IIB mutations could be similar to thatobtained when vWF is D. (1991)'Blood %3,'1738-'1743 H. Fujimura, Y., Shima, M., Yoshioka, A., Houghten, R. A., Ruggeri, complexed with botrocetin which promotes the association of 28. Mohri, Z. M. & Zimmerman T, S. (1988) J. Biol. Chem. 263,17901-17904 vWF with GPIb. The duplication of Met640-541 and five type 29. Berndt, M.C., Booth, W. J., Andrews, R. K. & Castaldi, P. A. (1991) Thromb. Haemost. 6 5 , 748 (abstr.) IIB mutations are located in the first sequence involved in 30. Roth, G. J., Titani, K., Hoyer, L. W. & Hickey, M. J. (1986) Biochemistry the binding of vWF tobotrocetin, defined between aa residues 25.8357-8361 H., Yoshioka, A., Zimmerman, T. S. & Ruggeri, 2. M. (1989) J. Bioi. 539 and 553 (33);two other type IIB mutations,Leu + Pro574 31. Mohri, Chem. 264,17361-17367 and Arg + Gln578,are located in the second sequence, com- 32. Ch:$?phe, O., Obert, B., Meyer, D. & Girma, J. P. (1991) Blood 78,2310La1 I prised between aa residues 569 and 583, which probably also 33. Sugimoto, M., Mohri, H., McClintock, R. A. & Ruggeri, 2. M. (1991) J . participates in the interaction with the modulator (33). Biol. Chem. 266,18172-18178 34. Chang, H. Y., Chen, Y. P., Chediak, J. R., Levene, R. B. & Lynch, D. C. Botrocetin interaction with vWF mimicks the action of (1989) Blood 7 4 , (suppl.) 131a (abstr.) potential physiologic modulators of vWF function such as 35. Ginsburg, D., Konkle, B. A., Gill, J. C., Montgomery, R. R., Bockenstedt, P. L.. Johnson. T. A. & Yane. A. Y. (1989) . , Proc. Natl. Acad. Sci. U. S. A. collagen and heparin-like proteoglycans (33). In uiuo, expo86,3723-3727 sure of the normally inaccessible GPIb binding site seems to 36. Iannuzzi, M.C., Hidaka, N., Boehnke, M., Bruck, M. E., Hanna, W. H., Collins, F. S. & Ginsburg, D. (1991) Am. J. Hum. Genet. 4 8 , 757-763 require conformational changes induced by binding of vWF 37. Lyons, S. E., Bruck, M. E., Bowie, E. J. W. & Ginsburg, D. (1992) J. Biol. to collagen. The present results suggest that a physiological Chem. 267,4424-4430 J. A,, Berkowitz, S. D., Ware, J., Kasper, C. K. & Ruggeri, Z. M. equivalent of the conformational change may be represented 38. Dent, (1990) Proc. Natl. Acad. Sci. U. S. A. 87,6306-6310 by alterations of the structure of the loop. 39. Lavergne, J. M., De Paillette, L., Bahnak, B. R., Ribba, A. S., Fressinaud, E., Meyer, D. & Pletu, G. (1992) Br. J. Haematol., in press In conclusion, this study of the expression of NWF with 40. Voorberg, J., Fontijn, R., Van Mourik, J. A. & Pannekoek, H. (1990)EMBO type IIA and type IIB mutationsis a useful approach to define J. 9 , 797-803 W., Drutsa, V., Jansen, H. W., Kramer, B., Pflugfelder, M. & regions of vWF involved in the regulation of its binding to 41. Kramer, Fritz, H. J. (1984) Nucleic Acids Res. 1 2 , 9441-9456 platelet GPIb and a better understanding of the complex 42. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467 mechanisms involved. 43. Lonata. M. A,. Cleveland. D. W. & Sollner-Webb, B. (1984) Nucleic Acids "

'

~

Y .

Acknowledgments-We thank Dr. J. P. Girma and B. Bahnak for critical revision of this manuscript and B. Obert for performing the SDS-agarose gel. The gift of monoclonalantibodies 6D1 and 7E3 from

Res. 12,5707-5717 44. Voorberg J. Fontijn, R., Calafat, J., Janssen, H., Van Mourik, J. A. & Panneioek, H. (1991) J. Cell Biol. 1 1 3 , 195-205 45. Laemmli, U. K. (1970) Nature 227,680-685 46. Girma, J. P., Kalafatis, M., Pietu, G., Lavergne, J. M., Chopek, M. W.,

Expression and Characterization of Mutated uon Willebrand Factor Edgington, T. S. & Meyer, D. (1986) Blood 67,1356-1366 47. Meyer, D., Baumgartner, H. R. & Edgington, T. S. (1984) Br. J. Haemutol. 67,609-620 48. Meyer, D., Fressinaud, E., Sakariassen,K. S., Baumgartner, H. R. & Girma, J. P. (1987) Ann. N . Y. Acad. Sci. 609,11&130 49. Pietu, G., Ribba, A. S., Cherel, G. & Meyer, D. (1992) Biochem. J. 284, 711-715 50. Coller, B. S., Peerschke, E. I., Scudder, L. E. & Sullivan, C. A. (1983) Blood 61,99-110 51. Coller, B. S. (1985) J. Clin. Inuest. 76,101-108 52. Fraker, P. J. & Speck, J. C. (1978) Biochem. Biophys. Res. Commun. 80, 849-857 53. Ardaillou, N., Girma, J. P., Meyer, D., Lavergne, J. M., Shoa'i, I. & Larrieu, M. J. (1978) Thromb. Res. 12.817-830 54. Nishino,'M., dirma, J.P., Rothschild, C., Fressinaud, E. & Meyer, D.(1989) Blood 74, 1591-1599 55. Schullek, J., Jordan, J. & Montgomery, R. R. (1984) J . Clin. Inuest. 73, 421-428

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56. Lo ez Fernandez, M. F., Lopez-Berges, C., Corral, M., Garcia-Talavera, J. Lopez-Borrasca, A. & Battle, J. (1987) Vox Sang. 62, 15-19 57. Meulien P., Nishino M., Mazurier, C., Dott, K., Pietu, G., Jorieux, S., Pavirini, A., Girmi,, J. P., Oufkir, D., Courtney, M. & Meyer, D. (1992) Thromb. Haemostasls 67,154-160 58. Tuley, E. A., Gaucher, C., Jorieux, S., Worrall N. K., Sadler, J. E. & Mazurier, C. (1991) Proc. Nutl. Acad. Sci. U. S.'A. 88,6377-6381 59. Kroner, P.A., Friedman, K. D., Fahs, S. A., Scott, P. & Montgomery, R. R. (1991) J. Biol. Chem. 266,19146-19149 60. Rabinowitz, I., Mancuso, D. J Tuley, E.A., Randi, A. M., Firkin, B. G., Boward, M. A. & Sadler, J. E . (1991) Blood 78, (sup 1.) 179a (abstr.) 61. Mancuso, D. J., Montgomery, R. R. & Adam, P. (1991PBlood 78, (suppl.) 67a (abstr.) 62. Bahnak B. R., Lavergne, J. M Ferreira, V., Kerbiriou-Nabias, D. & Meyer, D. (1892) Biochem. Biophysy Res. Commun. 182,561-568 63. Cooney K. A., Lyons S. E. & Ginsburg, D. (1992) Proc. Natl. Acad. Sci. U. S.'A. 89, 2869-2'872 , S., Rabinowitz, I. & Sadler, J. E. (1991) 64. Randi, A. M., Tule , E A Jorieux, Blood 78, (suppl) l i 9 a (abstr.)

E.,