Proc. Natl. Acad. Sci. USAVol. 88, pp. 6377-6381, July 1991Medical Sciences

Expression of von Willebrand factor "Normandy": An autosomalmutation that mimics hemophilia A

(factor VIII/hemostasis)

ELODEE A. TULEY*, CHRISTINE GAUCHERt, SYLVIE JORIEUXt, NEIL K. WORRALL*, J. EVAN SADLER*f,AND CLAUDINE MAZURIERt§*Howard Hughes Medical Institute, Departments of Medicine and Biochemistry and Molecular Biophysics, The Jewish Hospital of St. Louis, WashingtonUniversity School of Medicine, St. Louis, MO 63110; and tLaboratoire de Recherche sur L'H6mostase, Centre Regional de Transfusion Sanguine,Lille, France

Communicated by Philip W. Majerus, April 22, 1991 (received for review March 21, 1991)

ABSTRACT von Willebrand disease Normandy (vWDNormandy) is a recently described phenotype in which amutant von Willebrand factor (vWF) appears structurally andfunctionally normal except that it does not bind to bloodcoagulation factor VIII. This interaction is required for normalsurvival of factor VIII in the circulation; consequently, vWDNormandy can present as apparent hemophilia A but withautosomal recessive rather than X chromosome-linked inher-itance. A vWF missense mutation, Thr28 -* Met, was identifiedin the propositus in or near the factor VIII binding site. Thecorresponding mutant recombinant vWF(T28M) formed nor-mal multimers and had normal ristocetin cofactor activity.However, vWF(T28M) exhibited the same defect in factor VIIIbinding as natural vWF Normandy, confirming that thismutation causes the vWD Normandy phenotype. The distinc-tion between hemophilia A and vWD Normandy is clinicallyimportant and should be considered in families affected byapparent mild hemophilia A that fail to show strict X chro-mosome-linked inheritance and, particularly, in potential fe-male carriers with low factor VIII levels attributed to extremeIyonization.

Hemophilia A and von Willebrand disease (vWD) are causedby deficiency or structural defect of blood coagulation factorVIII and von Willebrand factor (vWF), respectively, andthese two diseases are the most common inherited bleedingdisorders of man. Factor VIII is encoded by a gene onchromosome Xq28, and hemophiliaA shows X chromosome-linked recessive inheritance. vWF is encoded by a gene onchromosome 12p, and most forms of vWD show autosomaldominant inheritance. Before the cloning of these genes, thedistinction between factor VIII and vWF was obscured bythe tendency of these proteins to form a noncovalent factorVIII-vWF complex in blood plasma. Factor VIII constitutes<1% (by weight) of the factor VIII-vWF complex, andplasma factor VIII levels generally parallel vWF levels (1).The binding of factor VIII to plasma vWF is necessary for

the normal survival of factor VIII in the blood circulation. Ineither normal individuals or patients with hemophilia A,transfused factor VIII is cleared from the circulation with ahalf-life of "12 hr (2-4), and the clearance of factor VIII isindistinguishable from that of either vWF or preformed factorVIII-vWF complex (4). This probably reflects the rapidbinding of exogenous factor VIII to vWF and the subsequentslow degradation of factor VIII-vWF complexes. In patientswith severe vWD, however, the half-life of transfused factorVIII is only -2.4 hr (4). The lack of stabilization offactor VIIIby binding to vWF appears to explain the low factor VIIIlevels that occur in patients with severe deficiency of vWF.

In the absence of vWF, endogenous factor VIII is clearedrapidly, causing a secondary deficiency of factor VIII.Another important role of vWF is to promote platelet

adhesion to subendothelium, and this is the initial event ofprimary hemostasis after vascular injury. Discrete domainsthat bind to platelet receptor sites and collagen have beenlocalized on vWF, and these domains apparently are notinvolved in binding to factor VIII (5). These observationssuggest that, in principle, a defect in the factor VIII bindingsite of vWF could result in isolated deficiency of factor VIIIwithout affecting other vWF functions in hemostasis, pro-ducing an apparently autosomal form of hemophilia A (1).Recently, three patients from two unrelated families fromFrance were reported with such a defect (6, 7). One patientappeared to have a normal bleeding time, normal plasmavWF level and multimer pattern, normal binding to plateletreceptors and to collagen, but complete deficiency of factorVIII-vWF binding. Her variant of vWD was tentativelynamed vWD Normandy (6).A candidate missense mutation was identified in the vWF

gene ofthe propositus with vWD Normandy (8), in or near thefactor VIII binding site. Expression and characterization ofthe corresponding mutant recombinant vWF proves that thismissense mutation abolishes the binding of factor VIII tovWF, demonstrating that a point mutation in the autosomalgene for vWF can cause a bleeding disorder that clinicallyresembles X chromosome-linked hemophilia A.

MATERIALS AND METHODSPlasmid Constructs. Plasmid pSVHVWF1 contains a full-

length cDNA insert for human vWF cloned into the expres-sion vector pSV7D (9) and was constructed as described (10).Plasmid pSVHNOR contains a C -- T transition causing aThr -. Met substitution at amino acid 28 of the mature vWFsubunit and was derived from pSVHVWF1 by mutagenesisusing the polymerase chain reaction (11). A fragment con-taining a unique HindIII site at the 5' end and the desiredsubstitution near the 3' end was amplified using the primers5'-TCCCCGGAAGCTTGCTGCCTGACGC-3' (vWF nucle-otides 2478-2502) and 5'-CAGGTCATAGTTCTGGCA-CATTTTGGTACACTCGAGCCCT-3' (vWF nucleotides2602-2641 complement, substitution is underlined) usingpSVHVWF1 as template. The 163-base-pair product frag-ment was purified by ultrafiltration (Centricon-30, Amicon)

Abbreviations: vWD, von Willebrand disease; vWF, von Willebrandfactor.tTo whom reprint requests should be addressed at: Howard HughesMedical Institute, Washington University, 660 South Euclid Ave-nue, Box 8045, St. Louis, MO 63110.§To whom reprint requests should be addressed at: Centre Regionalde Transfusion Sanguine de Lille, 21 rue Camille Guerin, BoftePostale 2018, 59012 Lille Cedex, France.

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6378 Medical Sciences: Tuley et al.

and employed with the primer 5'-TCAGGGTCACTGGGAT-TCAAGGTGA-3' (vWF nucleotides 3885-3909 complement)using pSVHVWF1 as template to amplify a 1431-base-pairproduct with a unique Nae I site near the 3' end. Thisfragment was digested with HindIII and Nae I, and theresultant 1373-base-pair fragment was cloned into the HindIIIand Nae I sites of pSVHVWFI. Clones containing thedesired mutation were identified by specific oligonucleotidehybridization, and the DNA sequence was confirmed (12) forthe segment subjected to enzymatic amplification.

Expression of Recombinant vWF. COS-7 cells were trans-fected with plasmids pSVHVWF1 (normal) and pSVHNOR[vWF(T28M)] using a DEAE-dextran method (13). After 24hr, mediumwasreplacedwith serum-freeDME(HG) (GIBCO/BRL, 430-2100) containing sodium bicarbonate at 3.7 g/literfor an additional 72 hr. Conditioned medium was concen-trated -10-fold by ultrafiltration (Centriprep-30, Amicon)and dialyzed at 40C against 50 mM Tris Cl, pH 8.0/0.15 MNaCl. CHO-DG44 dhfr- cells (14) were cotransfected usingcalcium phosphate (15) with the dihydrofolate reductaseplasmid pCVSEII (16) and either pSVHVWF1 or pSVH-NOR, and clones were selected with nucleotide-deficient aminimal essential medium (a-MEM) and dialyzed serum. Celllines expressing recombinant normal vWF or vWF(T28M)were incubated in serum-free a-MEM for 72 hr, and condi-tioned medium was concentrated 120-fold and dialyzed asabove.

Total protein was assayed using a Coomassie blue G-250dye-binding method (Bio-Rad) with bovine serum albumin asthe standard. vWF antigen was assayed by a sandwichELISA method using rabbit polyclonal anti-human vWF(Dakopatts 082, DAKO, Carpinteria, CA) at a 1:500 dilutionfor coating and a similar antibody conjugated to horseradishperoxidase (Dakopatts P226) at a 1:5000 dilution for detectionwith the substrate o-phenylenediamine. Product was assayedby absorbance at 490 nm using an EL312 Bio-Kinetics(Bio-Tek Instruments, Winooski, VT) plate reader. vWF wasquantitated in plasma antigen units using assayed referenceplasma from Helena Laboratories. One unit is approximatelyequal to 10 ,ug of vWF (17).

Plasmin Digestion of vWF. Samples of either plasma orconcentrated conditioned medium containing 0.4-0.7 unit ofvWF antigen were incubated for 2 hr at 4°C with 100 ,l ofSephacryl-S1000 beads on which 0.08 mg of anti-vWF mono-clonal antibody MAb239 was coupled. After washing, thebeads were suspended in 10 ,u of 0.02 M Tris Cl, pH 7.4/0.15M NaCl, heated for 15 min at 60°C, and cooled to roomtemperature. Plasmin digestion was then performed over-night at 37°C after adding 0.046 unit of plasmin. SDS wasadded to a final concentration of2% (wt/vol) and each samplewas heated in boiling water for 5 min. Beads Were removedby centrifugation and supernatant solutions were analyzed bySDS/polyacrylamide gel electrophoresis (18) on a 3-16%gradient gel under nonreducing or reducing conditions.

Factor VIII Binding. Factor VIII binding to vWF wasassayed as described (19). Briefly, increasing amounts ofvWF were immobilized by binding to anti-vWF monoclonalantibody-coated microplates and any endogenous factor VIIIwas removed by washing with 0.4 M CaCl2. Purified plasmafactor VIII was allowed to bind to the immobilized vWF, andthe bound factor VIII was then detected by adding thereagents for a chromogenic assay of factor VIII-dependentfactor X activation (Diagnostica Stago, Asnieres-sur-Seine,France) and measuring the hydrolysis by factor Xa of sub-strate CBS 48.03 (CH30CO-D-Leu-Gly-Arg-p-nitroanilide)as the rate of change of optical density at 450 nm. The vWFbound per well was then quantitated by addition of 125I-labeled anti-vWF monoclonal antibody and quantitation ofbound radioactivity (cpm). For this assay the background

value for immobilized vWF was 110 cpm, and the backgroundvalue for bound factor VIII was 0.27 x 10-3 unit.

RESULTSExpression and Characterization of Recombinant vWF. The

factor VIII binding site on vWF has been localized to an34-kDa trypsin or plasmin fragment containing amino acid

residues 1-272 of the vWF subunit (20, 21), suggesting thatvWD Normandy might be caused by a mutation in this region.This segment of the protein is encoded by exons 18-23 of thevWF gene (22). Exons 18-24 were amplified from the pro-positus and sequenced. A single potential missense mutationwas identified in exon 18, consisting of a CG -+ TG transition

Apro-

mature-

205-

116-

1 2 3 4 5 6

B

1 2 3

FIG. 1. Expression of recombinant normal vWF andvWF(T28M). (A) SDS/polyacrylamide gel letohrssunderreducing conditions. vWF was immunoprecipitated with rabbit poly-clonal anti-human vWF antibody (Dakopatts 082) and proteinA-Sepharose from samples of concentrated conditioned mediumcontaining -250 ng of vWF protein by ELISA, reduced with 7%(vol/vol) 2-mercaptoethanol, electrop'horesed through 5% polyacryl-amide gels (18), electroblotted onto nitroceliulose membrane (LKBNovablot), and visualized with anti-human vWF antibody, biotiny-lated goat anti-rabbit IgG, and Vectastain avidin-biotin complexhorseradish peroxidase (Vector Laboratories). The positions ofpro-vWF and mature v'WF subunits and the apparent mass (kDa) ofmarker proteins are indicated on the left. Lanes: 1, recombinantnormal vWF from COS cells; 2, recombinant vWF(T28M) from COScells; 3, recombinant normal vWF from CHO cells; 4, recombinantvWF(T28M) from CHO cells; 5, medium from CHO cells transfectedwith vector only; 6, normal human plasma containing -250 ng ofvWF. (B) SDS/agarose multimer gel electrophoresis. Samples ofnormal plasma or-concentrated COS cell conditioned medium con-taining -25 ng of vWF proteins by ELISA were electrophoresedthrough a 1.5% agarose gel for analysis of the vWF multimer pattern(24). Lanes: 1, recombinant vWF(T28M); 2, recombinant normalvWF; 3, normal human plasma.

Proc. Nad. Acad. Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 6379

that resulted in the substitution Thr28 -. Met. This patientwas born of a consanguineous marriage and was shown to behomozygous for this substitution (8). This substitution wasincorporated into an expression vector containing a full-length vWF cDNA insert. The normal vWF and mutantvWF(T28M) plasmids were used to prepare the correspond-ing recombinant proteins in transiently transfected COS cellsand stably transfected CHO cell lines.

Control experiments showed that, except for the factorVIII binding domain, recombinant normal vWF andvWF(T28M) were similar in structure and function to plasmavWF. SDS/polyacrylamide gel electrophoresis under reduc-ing conditions showed a major vWF subunit species with anapparent mass of -220 kDa and a minor pro-vWF subunitspecies with an apparent mass of -270 kDa for both recom-binant normal vWF and vWF(T28M) (Fig. lA). This isconsistent with normal partial processing of the -270-kDapro-vWF precursor to the -220-kDa mature vWF subunit(23). The extent of processing was similar for both recombi-nant proteins, although processing was more complete inCOS cells than in CHO cells. SDS/agarose gel electropho-resis under nonreducing conditions (Fig. 1B) showed multi-mers ranging from dimers to species of very large size forboth normal and mutant proteins. Differences between re-combinant normal vWF and vWF(T28M) cannot, therefore,be attributed to differences in proteolytic processing ormultimer formation.

Platelets can bind vWF through a specific receptor, theglycoprotein lb-IX complex (5). This interaction is stimu-

tc0

CAU)

EU)

._

JW

lated by addition of the antibiotic ristocetin, leading toplatelet agglutination by polyvalent vWF (25). This propertyof ristocetin has been exploited to devise clinically usefulprocedures to assay vWF activity. Recombinant normal vWFand vWF(T28M) supported ristocetin-induced vWF-dependent platelet agglutination (Fig. 2) and had similarcollagen binding activity (S.J. and C.M., unpublished obser-vations). This is consistent with the properties of naturalplasma-derived vWF Normandy, which exhibits normal ris-tocetin-dependent binding to platelets and also binds nor-mally to collagen (6).Recombinant vWF(T28M) Reproduces the Structural and

Functional Defects in the Factor VIII Binding Domain ofPlasma-Derived vWF Normandy. Normal plasma vWF andvWF Normandy show a clear structural difference afterdigestion with plasmin (Fig. 3). Upon SDS/polyacrylamidegel electrophoresis, the amino-terminal plasmin fragment ofvWF Normandy has slightly increased mobility (lane 2, -29kDa) compared to the corresponding plasmin fragment (lane1, =31 kDa) of normal vWF, when analyzed without priorreduction. These electrophoretic mobilities are reproducedby the amino-terminal plasmin fragments of recombinantnormal vWF and vWF(T28M) (Fig. 3, lanes 4 and 3, respec-tively). After reduction this difference disappears, and thesefragments migrate with an apparent mass of 34 kDa for theplasma-derived (lanes 5 and 6) (ref. 8; C.G., B. Mercier, S.J.,D. Oufkir, and C.M., unpublished observations) or recom-binant proteins (lanes 7 and 8). Factor VIII binding toamino-terminal fragments of plasma-derived vWF is abol-ished by reduction (20, 26). These observations suggest thatthe conformation of the amino-terminal part of vWF isimportant for factor VIII binding, and this conformation isdisrupted either by reduction or by alterations in the second-ary structure of plasma vWF Normandy (8) and vWF(T28M)(Fig. 3).

nonreduced reduced

200-

97-

1min

N T28M p

FIG. 2. Ristocetin cofactor activity of recombinant normal vWFand vWF(T28M). Lyophilized platelets were reconstituted to 200,000platelets per ,ul in 60 mM Tris Cl, pH 7.5/0.15 M NaCl (Biodata,Hatboro, PA). Ristocetin (15 mg/ml; 50 ul; Sigma) was added toplatelet suspension (400 /l) and stirred for 3 min at 37°C. A sampleof normal human plasma or of recombinant vWF diluted in 50 ,.u ofbuffer was added and light transmission was monitored continuouslywith a Payton Associates (Buffalo, NY) aggregation module. Thehorizontal bar represents 1 min. The ordinate is in arbitrary lighttransmission units. The aggregation traces shown were obtained withthe following final concentrations of vWF: N, normal recombinantvWF (0.18 unit/ml); T28M, recombinant vWF(T28M) (0.18 unit/ml);P, normal human plasma vWF (0.12 unit/ml).

68-

43-

29- *4__4I&

18- __

1 2 3 4 5 6 7 8

FIG. 3. Electrophoretic mobility of the plasmin fragments ofeither plasma or recombinant normal vWF and vWF(T28M). Theincreased electrophoretic mobility of the factor VIII-binding amino-terminal unreduced plasmin fragment of plasma-derived vWF Nor-mandy is reproduced by recombinant vWF(T28M). After electro-phoretic transfer to nitrocellulose, the vWF peptide fragments wereidentified using 1251-labeled antibodies. Rabbit anti-human vWFpolyclonal antibodies were used for lanes 1-4. Monoclonal antibodyMAb184-14A12, which recognizes both the unreduced and reducedamino-terminal trypsin and plasmin fragments of vWF (C.G., B.Mercier, S.J., D. Oufkir, and C.M., unpublished observations), wasused for lanes 5-8. Lanes: 1 and 5, a pool of normal plasmas; 2 and6, vWF Normandy patient plasma; 3 and 7, recombinant vWF-(T28M); 4 and 8, normal recombinant vWF.

Medical Sciences: Tuley et al.

6380 Medical Sciences: Tuley et al.

E4

5.~~~~~~~~C

U-3

V

2

0

0 500 1000Immobilized vWF (cpm)

FIG. 4. Factor VIII binding activity of recombinant normal vWFand vWF(T28M). Recombinant normal vWF binds factor VIII in amanner similar to normal plasma vWF, whereas recombinantvWF(T28M) does not bind factor VIII and reproduces the phenotypeof plasma vWF Normandy. o, Normal plasma; A, recombinantnormal vWF; *, vWD Normandy patient plasma; A, recombinantvWF(T28M). mU, milliunit(s).

The defect in factor VIII binding of plasma-derived vWFNormandy (6) is reproduced with recombinant vWF(T28M)(Fig. 4). Recombinant normal vWF binds factor VIII in amanner similar to normal plasma vWF, whereas recombinantvWF(T28M) and plasma-derived vWF Normandy have nofactor VIII binding activity. The entire coding sequence ofthe vWF Normandy allele was not examined, so additionalamino acid substitutions between residues 311 and 2050cannot be excluded. However, the defective factor VIIIbinding of vWF(T28M) indicates that this single amino acidsubstitution is sufficient to account for the vWD Normandyphenotype (6).

DISCUSSIONThe mechanism by which the Thr28 -. Met substitution in

vWF Normandy abolishes factor VIII binding is not known,but studies with monoclonal antibodies suggest several pos-sibilities. Monoclonal antibody MAb418 recognizes the unre-duced, but not the reduced, amino-terminal plasmin fragmentof vWF, and this antibody also inhibits factor VIII binding tovWF. The epitope of MAb418 lies within the amino-terminal106 amino acids of the mature subunit (21, 27). Thus Thr28could be a critical amino acid within the factor VIII bindingsite. The epitope of MAbW5-6A, another monoclonal anti-body that inhibits factor VIII binding to vWF, has beenlocalized to a segment ofvWF that includes ThrW-Thr" (26),and the Thr28 Met substitution in vWF Normandy is 50

amino acids away from this segment. Both Thr28 and residuesbetween Thr78 and Thr9 could participate directly in factorVIII binding if these regions were held in proximity bydisulfide bonds in normal vWF. Alternatively, Thr28 may notbe part of the factor VIII binding site, but the Thr28 Met

substitution may indirectly alter the conformation of this site.Thr28 is adjacent to Cys29, and the Thr28 -) Met substitution

may prevent the normal disulfide pairing of this residue. Suchan effect on secondary structure would be consistent with themobilities on SDS/polyacrylamide gel electrophoresis of theamino-terminal plasmin fragments of normal vWF and vWFNormandy or vWF(T28M), which are different before reduc-tion but become indistinguishable after reduction (Fig. 3).

Since the first propositus was reported, three additionalunrelated families with the vWD Normandy phenotype havebeen identified (ref. 19; C.G., B. Mercier, S.J., D. Oufkir,and C.M., unpublished observations). In all cases the disor-der appears to be recessive, since obligate heterozygotes

have intermediate values for factor VIII-vWF binding andare clinically unaffected (6, 19). Aside from the first patientidentified there is no evidence for consanguinity of theparents, and these additional patients may be compoundheterozygous. Individuals with one vWF Normandy alleleand one vWF null allele should exhibit the vWD Normandyphenotype. The distribution of genotypes associated withvWD Normandy will depend on the frequencies of suchalleles, which have yet to be determined.One family affected with the vWD Normandy phenotype

was misdiagnosed as affected with mild hemophilia A, andcorrect diagnosis led to important changes in genetic coun-seling (19). Such families frequently contain affected females,whose low plasma factor VIII levels were attributed toheterozygosity for hemophilia A with extreme lyonization.Sporadic new mutations account for approximately one-thirdof all cases ofhemophilia A, so that even genetic studies usingintragenic factor VIII DNA markers may not reliably identifyfemale carriers. Misdiagnosis of hemophilia A carrier statuscan delay or prevent recognition ofvWD Normandy, and thiserror can be avoided by direct evaluation of factor VIII-vWFbinding.

Recognition of the vWD Normandy phenotype has impli-cations for the treatment ofbleeding episodes in patients withfactor VIII deficiency. In vWD Normandy, transfused puri-fied factor VIII has a markedly shortened survival, whereaspreparations containing some vWF show normal or pro-longed factor VIII survival (6, 19). In fact, sustained normal-ization of plasma factor VIII levels can be achieved byinfusion of purified vWF alone (19). As highly purifiedtherapeutic products become more widely used, recognitionof this variant may become especially important becausesome factor VIII concentrates do not contain sufficient vWFto promote normal factor VIII survival. Furthermore, incontrast to patients with reduced factor VIII levels becauseof mild hemophilia A, treatment of patients having the vWDNormandy phenotype with vasopressin analogues will beineffective.

Characterization of recombinant vWF(T28M) confirms thegenetic basis of vWD Normandy, a congenital factor VIIIdeficiency unlinked to the factor VIII locus that can mimichemophilia A. Characterization of mutations in other patientsshowing the same factor VIII binding defect will extend ourunderstanding of the structure-function relationships andphysiological importance of the factor VIII-vWF interaction.

We thank Dr. Anna M. Randi for many helpful discussions andLisa A. Westfield for the synthesis of oligonucleotides. We alsoacknowledge Dr. Hervd Broly for help with the preparation ofmonoclonal antibodies, Bernard Mercier for primer design and PCRanalysis, Driss Oufkir for performing the analysis of plasmin frag-ments, Denis Hoguet for nucleotide sequencing, and VdroniqueDuretz and Sabine Belmont for technical assistance. This work wassupported by the Howard Hughes Medical Institute (E.A.T. andJ.E.S.) and by an American Heart Association Medical studentresearch fellowship (N.K.W.).

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