von Willebrand factor mutation enhancing interaction with platelets in patients with normal multimeric structure

von Willebrand factor mutation enhancing

interaction with platelets in patients with normal

multimeric structure.

L Holmberg, … , J Ware, Z M Ruggeri

J Clin Invest. 1993;91(5):2169-2177. https://doi.org/10.1172/JCI116443.

Variant von Willebrand disease designated as type I New York or type Malmö is characterized by enhanced ristocetin-induced platelet agglutination with normal von Willebrand factor multimeric distribution in plasma. We have studied four such patients belonging to three unrelated families and found in all of them a unique cytosine-to-thymine transition changing the codon for Pro503 (CCG) to Leu (CTG). In three patients the mutant allele also had a silent mutation in the codon for Ser500 (TCG-->TCA). Both nucleotide changes are present in the von Willebrand factor pseudogene; however, the

characterization of distinctive markers where the gene and pseudogene differ, as well as the examination of amplified cDNA derived from platelet mRNA, confirmed that the abnormality occurs in the von Willebrand factor gene of the patients. Moreover, recombinant expression of the isolated glycoprotein Ib-binding domain of von Willebrand factor provided direct evidence that the Pro503-->Leu mutation is responsible for enhanced platelet reactivity to lower ristocetin concentrations. These results define a new structural element affecting the affinity of von Willebrand factor for glycoprotein Ib and establish the molecular basis of a variant form of von Willebrand disease.

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von

Willebrand Factor Mutation Enhancing Interaction

with

Platelets in

Patients with Normal Multimeric Structure

LarsHolmberg,*JudithA.Dent,* ReinhardSchneppenheim,t UlrichBudde,IJerry Ware,*and ZaverioM. Ruggeri * *Roon Research Laboratoryfor Arteriosclerosis and Thrombosis,DivisionofExperimental HemostasisandThrombosis,Department of Molecular and Experimental Medicine and CommitteeonVascular Biology, The Scripps ResearchInstitute, LaJolla,California 92037;

tKinderklinikder Christian-Albrechts-Universitdt zuKiel, 2300 Kiel 1, Germany; and §Blutspendedienst desAKHarburg, D2100

Hamburg, Germany

Abstract

VariantvonWillebrand disease designated as type I New York ortypeMalmo ischaracterizedbyenhancedristocetin-induced

plateletagglutination withnormalvonWillebrand factor multi-meric distribution in plasma. We have studied four such

pa-tientsbelongingtothree unrelatedfamilies and found in all of

them auniquecytosine-to-thymine transition changing the

co-don for

Pro"

(CCG) to Leu (CIG). In three patients the

mutant allelealso had asilent mutationin the codonfor

Ser"W

(TCG-- _{TCA).Bothnucleotidechanges are presentin thevon}

Willebrand factor pseudogene;however, thecharacterization of distinctive markers where thegeneandpseudogene differ, as well as theexamination ofamplifiedcDNAderived from plate-letmRNA, confirmed thattheabnormalityoccurs inthe von

Willebrand factorgeneofthepatients.Moreover,recombinant expression of the isolated glycoprotein lb-binding domain of von Willebrand factor provided direct evidence that the

Pro"

-- Leu mutation is responsible for enhanced platelet

reactivitytolowerristocetin concentrations. These results

de-finea newstructural elementaffectingtheaffinityof von Wille-brand factor for glycoprotein lb and establish the molecular basis ofa variant form ofvon Willebrand disease. (J. Clin.

Invest.1993.91:2169-2177.)Key words:bleeding-mutation. pseudogene*thrombosis* von Willebranddisease

Introduction

vonWillebrand disease(1)is a common inheritable bleeding

disordercharacterized by heterogeneous phenotypic manifes-tations and caused by quantitative or qualitative defects of vWF, aglycoproteinwith typicalmultimeric structure (2). By

convention, the disease isclassifiedinto three categories:typeI, with moderately decreased plasma concentration of a

seem-inglynormalvWF; typeII,withqualitativelyabnormal vWF at

normal or near normal plasma concentration; and type III,

withvery low or undetectable vWF and, unlike typesIand II,

recessiveinheritance. A numberofsubtypes existwithinthese

Dr.Holmberg'spresentaddressisDepartmentofPaediatrics, Univer-sityof Lund,University Hospital,S-221 85 Lund,Sweden.

Addressreprintrequests to Dr.ZaverioM.Ruggeri, TheScripps ResearchInstitute,SBR-8, 10666 NorthTorrey Pines Road, LaJolla, CA 92037.

Receivedfor publication24July1992and in revisedform9

De-cember 1992.

categories (1). TypeII variantshave attractedspecialinterest

because elucidation of the underlying molecular abnormalities

may yield insights into vWFstructure-function relationships

(3, 4).

vWFinteracts withcomponentsof the vessel wall and with the platelet GP complexes Ib-IX-V and IIb-IIIa to promote

plateletadhesion and thrombusformationatsites of vascular injury (2).Inthat normalvWFinsolution hasnomeasurable interaction with platelets, the initiation of vWF-dependent platelet thrombus formation is thoughtto require

conforma-tionalchanges in vWF boundtotheexposed subendothelium and/orsubjectedtothe effect of shear forces, resulting in high-affinitybindingto GP lb. Thus, different conformations ofa

specific functional domain may regulate the interaction of vWFwith GP lbandits role inhemostasis (2).

Inthetype IIBvariant ofvonWillebrand disease, vWF in solution displays enhanced affinity for GP Ib,asshowneither

directly with the purified protein (5) or by the induction of

aggregation in platelet-rich plasmaatristocetin concentrations lower than thoseeffective in normalsamples (6).The affected patientsalsoexhibit absence of the largestvWFmultimers in plasma,probablyowingtotheirspontaneousbindingto circu-lating platelets (7).In some cases,this interactionmayinduce in vivo platelet aggregation and thrombocytopenia (8, 9), which always occur in type IIB von Willebrand disease after administration of 1-deamino-8-D-arginine vasopressin (DDAVP)' (10). Differentgenemutations have been identi-fiedinanumberofthesepatients (3, 11-13)and allhavebeen foundtobe localized inadomain ofvWF, the disulfide loop createdbythelinkageof

Cys5"

andCys695,thathas been indi-catedas animportantelement intheregulationofvWF bind-ingtoGP lb(2,3).

BesidestypeIIB-andplatelet-type( 14, 15 )orpseudo-von Willebrand disease (16) which are, however, GP lb defects (17)-there is another form of von Willebrand disease in which platelet aggregationand vWFbindingto platelets occur atlowerristocetin concentrations than in normalsubjects.In contrast to typeIIB,however, there isnospontaneous

aggrega-tion nor thrombocytopenia even after DDAVP administra-tion.Furthermore,thefullrangeofvWFmultimers ispresent in theplasma ofthepatientsasischaracteristic oftype I von

Willebranddisease.Thelatterobservationsuggested the desig-nation "typeI New York"forthisvariant (18), butpatients withthe samephenotypewereconsideredby others as belong-ingtotype II(typeMalmo)becauseof their functional vWF

abnormality (19). We have now investigated four patients

fromthree unrelated families withthis form ofthe diseaseand

1.Abbreviations usedinthispaper:DDAVP,deamino-8-D-arginine

vasopressin;DEPC,diethylpyrocarbonate.

J.Clin. Invest.

©DTheAmerican Society for Clinical Investigation, Inc.

heredemonstrateinall of them thesamegene mutation lead-ingto anamino acid substitutionatposition503 ofthemature vWF subunit. This abnormality has been reproduced by in vitro mutagenesis and the properties of the expressed protein confirm the relationshipbetween the mutation andenhanced interaction with plateletsinthepresence ofristocetin.

Methods

Blood collectionandfunctionalstudiesofhemostasis. Bloodwas col-lectedwith trisodiumcitrateasanticoagulant (0.012 Mfinal

concen-tration); platelet-rich plasmaandplatelet-poorplasmawereprepared asdescribed previously ( 19). Factor VIIIprocoagulantactivity, von Willebrand factor antigen, andristocetin-cofactor activitywere

mea-suredusing methodspreviouslydescribed (19).Themultimeric struc-tureofplasmavWFwasevaluated in agarosegelscontaining SDS,as

reported(20).Bleeding timewasmeasuredusingatemplatemethod (19). Ristocetin-induced platelet aggregationwasevaluatedby

mea-suringeither the extentorthe maximalinitialvelocity of aggregation uponaddition of varying final concentrationsofristocetin(6, 7,21 ). Normal referencevaluesfor each assay were obtained in the normal populationofthe two centers where theclinicalstudies were performed (Malmo and Hamburg). Control subjects were from the laboratory staff. Allindividualsthatparticipated in the study were aware of its experimentalnatureand gave theirinformedconsent,according to the Declaration ofHelsinki.

Nucleic acid analyses. DNA was extracted from whole blood or buffycoatusingstandard procedures (22). Platelet RNA was prepared fromwashed plateletsobtained from50 mlofblood(23). The platelets werelysedin asolutioncomposedof4Mguanidinium isothiocyanate

(Gibco-BRL,Gaithersburg, MD), 5 mMsodiumcitrate, 100 mM 2-mercaptoethanol, 0.5% N-laurylsarcosine, and 0.1% antifoam A in diethylpyrocarbonate (DEPC)-treated water. 3 ml of platelet lysate werecentrifugedthrough aCsClcushion for20 h at 35,000 rpm, 20'C, in a model SW 50.1 rotor(150,000g; Beckman Instruments, Inc., Fullerton, CA). The RNA pelletwasdissolved in 20,lof DEPC-treatedwaterand 1Mlofribonucleaseinhibitor(Promega Corp., Mad-ison,WI). The RNA was stored at-70°Cas anethanolprecipitate.

TheportionofvWF gene(24) coding for residues463-736 was amplified from genomicDNAusingthe PCR(25)and thepreviously described oligonucleotide primers226 and 227A(26).Theseprimers selectively amplifyaportion ofexon28 withoutamplifyingthe homolo-gous sequence within the vWFpseudogene due to 3'basepair mis-matches between theoligonucleotidesand thepseudogene (26).After

amplificationthefragmentswereclonedin thefilamentous phageM13 for DNA sequenceanalysis.

Singlestranded vWF cDNAfragmentsweregenerated from platelet RNA using Moloney murine leukemia virus reverse transcriptase primedbyavWF-specificoligonucleotide (complementarytothe vWF mRNAcodingforresidues 899-908). Aliquotsof thisfirst reaction

were subjectedto aPCRusingoligonucleotides thatamplifyavWF fragment representingthecodingsequence forresidues441-681. The oligonucleotidesusedinthe PCRcorrespondtocodingsequence from nucleotide 23/196to 23/220 withinexon 27 and tononcoding

se-quencecomplementarytonucleotides24/841 to24/864withinexon

28(nucleotidenumbersare from reference 24). Restriction enzyme digestionwith DdeIwasperformedontheamplifiedfragments using thesupplier'srecommendeddigestionconditions.

In vitro mutagenesis and expression of recombinantvWF

frag-ments.ThePro"3-- _{Leuamino acid substitution was expressed}

utiliz-ingapreviouslydescribed construct, pAD5/WT (27), that directs the synthesisinChinesehamster ovary(CHO)cells ofa1 16-kD homodi-mericfragmentof vWF, referredto as r 116. The expressedfragment contains residues 441-733 ofmature vWFand has been shown to

recapitulatethe properties of nativevWFwith regard to its interaction with platelet GPlb(27). The normal vWFcoding sequence within pAD5/WTwasclonedinto pBS/KS-(Stratagene,Inc.,LaJolla)and

auracil-containing singlestrandedtemplatewasgeneratedusingthe

duC ung-Escherichia colistrain CJ236(28). After mutagenesisand verification of theentirevWFcodingsequence, the vWF insert was subcloned into theXhoI/NotIsite oftheexpression plasmid pCDMn`

aspreviouslydescribed(27).Themutantplasmid containsthesame

codingsequenceaspAD5/WTwithtwodifferences:the Pro13-* Leu codonsubstitution,asseenin thepatients,andachangein the codon forAsp498 (GAC-- _{GAT), insertinganewEcoRVsite used for the}

screeningofmutantplasmids.

Cellculture, DNA transfection, and selection ofstable

transfor-mants. CHO cellsweremaintainedin 5%CO2 and grownin DME

supplementedwith 10% fetal calf serum, 0.5 mMnonessential amino

acids,and 2 mML-glutamine (Whittaker Bioproducts, Walkersville, MD).Cellswereculturedatadensityof 1.5X 105in 60-mm dishes and transfectionmediatedbycalcium phosphatewasachieved with 10 Mg ofDNA perdish, as_{described previously (27, 29).}Stable

transfor-mants wereestablishedbygrowingcellsfor 10-14 din the presence of 0.8 mg/ml of the antibioticG418 (Sigma Chemical Co., St.Louis,

MO)andisolatedcolonieswereexpandedin 12-wellplates. The

cul-turesupernatantswereexamined for the presence of vWFantigen by

spotting 200-MAlaliquotsontonitrocellulose membranes and then

incu-batingthe membranessequentiallywith vWF monoclonal anti-bodiesand'251I-labeledrabbitanti-mouse IgG, followed by

autoradio-graphicdetection(3).

Characterizationofexpressed vWFfragments. Serum-freeculture mediawereconcentratedbycentrifugation using a Centricon 30 (Ami-con,Beverly, MA). Quantitative determinationof vWF antigen in the concentrated mediawasperformedwith Laurellelectroimmunoassay (30,31), usingapoolof monoclonal antibodiesrecognizingepitopes

within vWF residues 449-728. Affinity-purified r116 vWFfragment

wasused as areference standard for this assay.

Western blot analysis ofthe r 1 16fragmentwasperformed withtwo anti-vWF monoclonalantibodies: NMC-4,recognizing a disulfide-de-pendentconformation of vWF conferred by theCys509-Cys695bond and retained in the isolatedGP lb-bindingdomain (32, 33), and U-RG46, reactingwith aconformation-independent linear epitope lo-cated within residues474-488ofthe mature vWF subunit(32). Immu-noreactiveproteinswerevisualized by subsequentincubation with12511 labeled rabbit anti-mouseIgGfollowedby autoradiography.

Immunopurificationof normal and mutant r 116 vWFfragments wasperformed byapplying 500 ml ofculture supernatant onto a 20-ml column prepared by coupling the monoclonal antibody NMC-4 to CNBr-activated Sepharose aspreviouslydescribed(27).Protein deter-mination wasperformedwith bicinchonic acid assay (Pierce Chemical Co., Rockford, IL).

Foraggregation studies,eitherimmunopurifiedfragments or

con-centrated culture media were used. The latter were obtainedby filtering 100 ml of serum-free culture supernatantthrough 0.45-MmNalgene filters,then dialyzing extensively against a buffercomposedof 20 mM Hepesand0. 15 MNaCI,pH 7.4, andconcentratingto 1 ml by centrifu-gation using a Centricon 30 filter. Aggregation was evaluatedusing human washed platelets prepared by the method of Walsh et al.(34) withmodifications described elsewhere(35);thefinalplateletcountin the assay was 1.5X 108/ml.A siliconizedglasscuvettecontainingthe experimental mixture was placed in an aggregometer(Chrono-Log Corp., Havertown, PA)at 37°C with constantstirring(1,200rpm); aggregation was induced by the addition of ristocetin and monitored throughthe increaseinlighttransmission.

Results

Characterization ofthe patientsstudied. The fourpatients

de-scribed in thisstudyarefrom threefamilies,onefromSweden, onefrom Germany, and one from the UK (Bermuda). The Swedishfamilyistheoriginaloneinwhich theoccurrenceof

this variant form ofvon Willebrand disease wasdescribed in

TableI.LaboratoryDataofthe Four PatientsStudied

Bleeding

Sex Age time FVIII:C vWF:Ag RiCof RIPA

yr min Uldl mg/mi

Patient 1 M 20 14 57 55 46 0.90

Normal <12 60-160 50-175 50-160 >1.0

Patient 2 F 54 11 50 87 64 0.4

Patient 3 F 27 - 41 110 78 0.5

Patient4 F 38 5 73 68 84 0.5

Normal <9 60-160 50-160 50-160 >0.6

Factor VIII procoagulant activity (FVIII:C), von Willebrand factorantigen(vWF:Ag),andristocetin cofactoractivity(RiCof)areexpressedas arbitrary units perdeciliter;average normal plasma contains 100 U/dl.Note that thebleedingtime was not tested inpatient3.Ristocetin-induced plateletaggregation(RIPA) was evaluated with two different methods:inpatient 1, bydeterminingtheconcentration ofristocetininducing aggregationwith an initial velocity of at least 20mm/min;inpatients 2, 3,and4, by determiningtheconcentrationof ristocetininducing ag-gregation with at least 50% maximal response. Different normal referencevalues arereported forthepatientsstudied intwodifferentcenters

(Malmofor patient 1;Hamburgforpatients 2, 3, and 4).

reportedpreviouslyas III: 1 (19),isthe sonoftheoriginal

pro-positus (deceased) andwasdiagnosed in thecourseof family studies;hehadnoobviousbleedinghistory.The German fam-ily hasfive affected membersand theinheritance is autosomal dominantasin the Swedishfamily;the mother and daughter, patients 2 and 3,arecharacterized inthisreport.Patient2 had bleeding considered abnormal aftertwotoothextractions and suffered from cutaneous hematoma and menorrhagia; patient 3 hadno obvious bleeding history. The female patient from Bermuda,patient4, wastheonlymemberof her family withan

increasedbleeding tendency;sheexperiencedabnormalblood lossaftertoothextractions,as well ascutaneoushematomaand

increased bloodlossafteran abortion.Altogether, the symp-toms in all affected patients, when present, were considered mild.

Therelevantlaboratoryfindingsaresummarizedin Table I;ofnote,thebleeding timerangedfromnormal(patient4) to

only moderately prolonged.Inallthepatients, platelets in

plate-let-rich plasmawere aggregated atconcentrationsof ristocetin

lower than thosenecessary to causeaggregationin normal

sam-ples, afinding representingthedistinctive phenotypic feature oftheseindividuals (18, 19). PlasmavWF:Aglevelswere

nor-mal in allcases, andristocetin cofactor activity (which is

mea-suredatnonlimitingristocetinconcentrations)wasmoderately decreased only in patient 1. FVIII:Cwasnormalinpatient4

butwasmoderately decreased in the others; moreover, the ratio between plasma FVIII:C andvWF:Agwaslow (<0.6) in

pa-tients2 and3,belongingto the samefamily.Forpractical rea-sons, theseassays of FVIII:Cwereperformed on frozen

sam-ples; thismayaccountfor thediscrepanciesobserved dueto the

labile natureoffactorVIIIactivity. AllvWF multimerswere presentin plasma, withanormalrelativeproportionof species ofdifferentmolecular mass;thus, themultimericstructure of patientvWFwasindistinguishable from that ofnormal con-trols(Fig. 1). Nopatient had thrombocytopenia; moreover, DDAVP administration to patient 2 shortened the bleeding

time,raisedfactorVIIIand vWFlevels but, unlikein type IIB von Willebrand disease (10), did not induce thrombocyto-penia.

Geneticstudies. DNAsequence analysis was performed on

the vWF geneofthefour patientsunderconsideration.Because

their phenotypic abnormalitywascharacterized by enhanced

vWFinteraction with plateletsinthepresence ofristocetin,we focused ourattention on the GPlb-binding domainofvWF

(32, 35).The sequence encoding residues 449-673 of the ma-turevWF subunit wasdeterminedaftercloningPCR-generated fragments into the filamentous phage Ml13. The analysis of

eighttotwelveclones per DNAsample demonstratedthat each

patient had, in addition to a normal allele, a mutant allele in

which aC -* T transition (confirmedin atleasttwo clones) changedthe codon for amino acid residue 503 from the normal

proline (CCG)to leucine (CTG) (Fig. 2). Furthermore, pa-tients2,3,and4,butnotpatient 1,hadaG -* A silent muta-tion in the codon for

Ser5"

(TCG-- TCA) that was linked_to

the allele

containing

the Pro503 -- Leu substitution

(Fig.

_2).

The basechange in the codon for

Ser5"

creates a new DdeI

restriction site, the presence of which on a single allele was confirmed in the three patients(Fig. 3A).

Sequence analysis ofthe vWF gene based on

PCR-gener-ated fragments can be affected by interference from a highly

homologous pseudogene possessing a structural organization

similar to that of the "true" gene in the region containing the

codingsequence ofinterest for these studies. Thepseudogene

has both TCA and CTG at positionshomologoustothose

cod-ing for amino acids 500 and 503 in the vWF gene, thus a se-quence identical to that seen in the cloned DNA fragments

from threepatients.That thepseudogenedidnotinfluenceour

results,

however,

was _clearly demonstrated bythe

identifica-P4

N

I.

a

to

--Figure1. SDS-agarose gelelectrophoresis of plasmavWF inpatient4(P4) anda normal

control(N). Note that the distributionand

the relative proportion of the different vWF multimers is identical in the patient sample

A.

500 Ser

Glu

Pro

503 Leu

lie

Ser 500

Glu

Pro

Pro 503

Leu

B.

(5')(3') Mutant Normal (3')(5)

A AT

IGATCGAT

C T Al

lie

T - W A T

Ile

C G C

[TA ~ upATi

Ser

_[C

G _{G Ser}

G C _CG;

-G

C !C

G-Glu A T T A Glu

LA

T t $TA

-CG

GC-Pro [G b CJI Pro

-C G _OP_ ₄₀ G C_

503 Leu T A* |

_log|

CG Pro503

[T A A Ti

Leu T A A TI Leu

LGC C GJ

(3') (5')

(5') (3')

Figure 2. DNA sequence of normal and mutatedvWFalleles. The lettering indicates the base sequencecorrespondingtothe codonsof amino acid residues 499-504 of themature vWFsubunit,asshown

ontheleft andright.Thefigurepresents the sequence of the noncod-ing strands with thecodingstrandsaligned;therespective3' and 5' endsareindicated. (A) Patient2,showingthe mutation in the codon for residue 500aswellasthe Pro13-- Leusubstitution; (B) patient 1, showingthe normal codon for

Ser5",

but thesamemutation of residue 503as seeninpatient2.

tionof theexpectedgene sequence in themutantclonesatall the 16 otherpositionswhere geneandpseudogene have been

showntodiverge(26). Moreover, additional evidencethat the observedmutationsreflectvWFgene sequence in thepatients

was obtainedbycharacterizing theplatelet mRNA prepared

from patient 2. A 746-bp fragment was amplified from the

correspondingcDNAwithtwoprimers representingsequences in exons 27 and 28 separated by a 2-kb intron;thus, the ab-senceofanylargerfragmentin thePCR product demonstrated

the lackof genomicDNAcontaminationin theplateletcDNA

preparation. The cDNA fragment was foundto contain the

DdeI restriction site correspondingto thesilent mutationin the codonfor

Ser5"

(Fig. 3B),providingevidencethat the allele

containing the linked Pro503to Leusubstitutionis transcribed;

themutations, therefore,are truly presentinthe vWF gene.

Polymorphic markersofthe vWF gene were then

charac-terized toreveal the existence of possible unknown

relation-ships between the three apparently unrelated families. Patient

1,who lacked thesilent mutationatposition 500, had the

co-donfor aspartic acid 709 in themutant allele,whilethe others hadhistidine,areported vWF polymorphism (24). Patient 4

andpatients2 and3 (mother and daughter) hadconcordant sequence in the mutant clones at the polymorphic positions 618 (36) and709; however, further analysis ofthe vWF gene

for restriction fragment length polymorphisms and variable

number tandem repeats showed that both mutant and normal

alleles frompatient4 differedfrom those in patients 2 and 3 (TableII).

Studieswithexpressed recombinant fragments.The muta-tion detected in thepatientsatposition 503 was expressedin the homodimeric 116-kDfragment ofvWFknownto possess

the GPlb-binding properties of the intactmolecule; the mu-tantfragmentwasdesignatedr116 ( P503L). Thedialyzedand

concentrated supernatants from r116 and r 116(P503L)

cul-turescontained 57and 65

_,g

ofvWFfragmentpermilligram protein, respectively,asestimated from protein determination andelectroimmunoassay ofvWFwith purified r116 as

stan-dard. The immunoaffinity purified species had protein con-tentsof 150

_,gg/ml

forrl16and 208 ,ug/mlforrl 16(P503L) after 20-fold concentration of column eluates. Western blot demonstrated that the concentratedtissue culturesupernatants

and thepurified preparationscontained similar 116-kDspecies reactive with the monoclonalantibody NMC-4 (Fig. 4). Thus, the Pro503-- Leu substitution didnotalter the

ability

of the expressedfragmenttoassembleintoadimeric species like the normalfragment. By using the conformation-independent

an-tibody, LJ-RG46 (not shown), bothr116 and r116(P503L)

gave asimilar immunoreactive pattern,exhibitingthedimerof 116kDandsomemonomericfragmentof 52/48 kD (3). This further demonstrated that the normal andmutantfragments

wereexpressed inacomparablemanner.

Theconcentratedtissue culturesupernatants at afinalvWF

fragment concentration of 2.8 ,g/ml (24 nM calculated as

116-kDdimer) supported the aggregation of platelets induced by ristocetinat 1.25mg/ml final concentration. Dose-response

curves with decreasing concentrations of ristocetin demon-strated that thefragmentwith normalsequencesupported

ag-gregation withaslittleas 0.4mg/ml ofristocetin,albeit with diminished response, while r116(P503L) supported

aggrega-tion even at 0.15 mg/ml ristocetin (Fig. 5). Similar results

were obtained with the immunoaffinity-purified fragments

used atafinal concentration of60 nM(calculatedas 116-kD dimer);r116 supportedaggregationat0.4butnot0.3mg/ml ofristocetin, whereasr116(P503L) supportedaggregationat 0.2mg/mlof ristocetin(Fig. 6).Plateletaggregationcouldnot beelicitedin the absenceof ristocetinevenwhen the

concen-tration ofr116(P503L)wasraised 10-fold.

Discussion

These studies demonstrate that the same missense mutation identifiedin threeunrelatedfamiliesin the sequenceofmature

vWF, Pro503 -* Leu,isthecauseof thevonWillebranddisease

Patient

2

3

4

1

M

-872 -603 310

=281

-234 194

|-118

4,I

l

256

426

212

Normal

I

--allele

170

86

426

212

Mutant

allele

-1353

-1078

-872 -603

310

=2381

-234

194 -118 72

E!

1111!,,

C

4,

746

Normal

216

426

__

allele

13086

426

.

Mutant

I~,,

r.

_allele

Exon

27

.

Exon

28

Genomic

*s * I

1

1

DNA

Intron 27

-2 kb

Figure 3. DdeIcleavageofPCR-amplified fragments demonstratingthe TCG-. TCA silent mutationinthe codon forSer5" of thematurevWF subunit. A continuous linerepresentsthe uncleavedfragments;arrowsindicatecleavagesites and the asterisk indicates the site of mutation.

Marker lanes (M) containaHaeIIIdigestof 4X 174RF DNA;thesize of thecorresponding fragments (bp)is shownontheright.(A) Cleavage

ofa958bpfragment amplifiedfromgenomicDNAwitholigonucleotideprimersdescribedbyMancusoetal.(26).Unique fragmentsof 170 and86bparepresentinpatients 2, 3,and4,butnotinpatient 1,inadditiontothe normalfragmentsof426, 256,and212bp.Two small frag-mentsof20and46bp migratewith theprimersandarenotvisualized.(B) Cleavageofa746-bp fragment amplifiedfrom vWF cDNA ofpatient

2asdescribedinMethods. U,uncleaved;D, DdeI-cleavedfragment.Inthe normal allele(notshown in thisexperiment)therearefivecleavage sites,twocorrespondingtoexon27and threetoexon28,asshowninthe schematic representation of genomic DNA. Theextracleavagesiteat

the codon forseine 500 in themutantallelecreatesunique fragmentsof 130 and 86bp,seenintheheterozygouspatient in additiontothe normalfragmentsof426and216bp. Foursmallfragmentsof20-35bp migratewiththe oligonucleotideprimersandarenotvisualized.

Themutationwasdetectedinonlyone of the alleles, in agree-mentwith the autosomal dominant mode of transmission of thebleedingdisorder; moreover, intwo of thethreefamilies (threeoutof four patients) thesamealleleexhibiteda

nucleo-tide substitution in the codon for serine 500. Thetwoobserved mutationscreate a sequenceidenticaltothatofthevWF

pseu-Table II.PolymorphismsDetectedinthevWFGene

ofPatients2, 3, and 4

Patient Taql(A) Xbal VNTR

2 -/- -/- 10/13

3 -/- -/- 12/13

4 +/+ +/+ 7/7

Polymorphisms were detected byrestrictionfragment length analysis afterdigestionofgenomicDNAwith TaqI(52) andXbaI (53),as

indicated,andby determination ofthevariable number of tandem repeats(VNTR) (54).Patients2and3aremother anddaughter. +, allele with polymorphic cleavage site; -, allele without it.

dogene(26),thusraising theconcernthat the latter might have interfered in the cloning and sequencing procedures adoptedto

characterize the patients. That thiswasnotthecase,however, is

demonstrated by the fact that the sequence of the mutant cloneswasfoundtobe identical tothat reported for the vWF

geneat 16 otherpositions thatareknowntodiverge from the pseudogene(26); and by the identification ofatranscriptfrom themutantallele,asevidenced byanalysisof cDNA generated

fromplatelet mRNA.

Themostcharacteristic feature of thevonWillebrand dis-ease variant described here is that aggregation is induced in platelet-rich plasma by lower ristocetin concentrations than

necessaryin normalsubjects. Ristocetin-induced aggregation is

knowntoinvolve the binding ofvWFtoplatelet GP lb(35) and, thus, is thoughttomirroroneof the crucial mechanisms

supporting platelet adhesion and aggregation at high shear forces(37, 38). The GP Ib-binding siteinvWFis located

be-tweenresidues 449and 728of thematuresubunit(32, 39, 40) and canbe expressed in a nativedimeric conformation as a

fragment of 116 kD secreted from CHO cells (3, 27). This fragment reproduces the properties of full-length vWF with regardtoristocetin-induced interaction with GP Ib, including

A

1

2

3

4

Figure4. Western blot analysis of expressed re-combinant fragments

200kD

of vWF. Concentrated

tissueculture superna-tantsand immunoaffin-ity-purified vWF

frag--

_

- 97.4kD

ments were

electropho-resedin an

SDS-polyacrylamide

gel

69kD

and reacted with the

-46kD anti-vWF monoclonal

antibodyNMC-4 after transfer to a nitrocellu-lose membrane. The electrophoretic mobilityof known molecular mass markers is shown ontheright. Lane1,purified ri16; lane 2, purifiedrl16(P503L); lane 3,rl16 culture supernatant; lane 4, rl 16(P503L) culture super-natant.

ristocetin-induced platelet aggregation. Although one cannot

exclude apriori that the consequencesofasingle amino acid substitution, orother mutations, may bedifferent in an iso-latedvWFfragmentasopposedto theintact multimeric

mole-cule, results documented previously with regardto atype IIB

mutation (3) have shown thatexpression of the isolated 116-kDfragmentcanreproducetypical phenotypic abnormalities oftype IIB vWF.Thus,expressionof thisfragmentwasusedto

evaluatethe functional consequences of the Pro503 -> Leu

mu-tation detected in thepatientsdescribed here. Indeed, the

re-sults obtained with thepresentstudies lend furthersupport to

thevalidity of the approach basedonisolated domain

expres-siontostudy the functionalconsequences ofmutationsinthe complex vWFmolecule.

Plateletaggregation studieswereperformed usingboth

tis-sueculturesupernatantsandimmunopurifiedvWFfragments derived from them. With the formerwecould excludethat the

results obtainedweresolely caused by effects of thepurification procedureonthefunctionalpropertiesof thevWFfragments;

A

Ristocetin (mg/ml)

OD 0.3 0.4 1.25

E = 2 L Tranmitanc

LightTransmittance

B

4 Ristocetin

3jL (mg/ml)

E I 0.1jU0.15 0.33 1.25n

E~2F

L

I

LightTransmittance

Figure 5. Plateletaggregation elicited by ristocetin inthepresence of concentratedtissue culture supernatants. The numbers indicated for eachtracerepresent the final ristocetin concentration(mg/ ml) added

totheplatelet suspensionsatthe time indicatedbyanarrow.(A) Normalr116 fragment;(B)mutantr116(P503L)fragment.

A Ristocetin (mg/ml)

4

-0.3 0.4 1.25

'm

B3

--12

-E -~1

LightTransmittance

B Ristocetin

(mg/ml)

4 0.15 0.2 0.3 0.4 1.25

'E

1t3'

0

- 'I

)i

I

Light Transmittance

Figure 6. Plateletaggregation elicited by ristocetin in the presence of immunoaffinity purified vWF fragments. The presentation of results is the same as in Fig. 5. (A) Normal r 1 16 fragment; (B) mutant r116 (P503L)fragment.

withthe latter we could rule out thepossibilitythat other

se-creted moleculespresent in the culturemedia interfered with

thefunctionalassay.Goodaggregationresponse at1.25mg/ml ristocetin concentration requiredmorepurified than nonpuri-fiedvWFfragment, suggestingthatthepurification procedure

maynegatively affect the functional properties ofthe molecule. With eithertypeofmaterial, however,r116(P503L) supported platelet aggregation at lower ristocetin concentrations than

r 116withnormal sequence,thusreproducingthetypical phe-notypic abnormality of the von Willebrand disease variant

underconsideration. Thedifferences between themutantand

normalfragmentswere morepronounced thanexpectedonthe basis of the moderatelyincreased ristocetin sensitivity inthe patientplatelet-rich plasma(18, 19).Thismaybeexplained by the factthat thepatientshaveamixture of normal and abnor-mal vWF subunits while the expressed mutantprotein

con-tained onlyabnormalspecies.In ourexperimentalsystem uti-lizing washed platelets,aggregationwasinduced bylow ristoce-tin concentrationsthatdonotelicitaggregationinplasma;this

reflectsthefactthatinthe latter caseristocetin bindstoother

plasma proteins, thus decreasing its effective

concentra-tion (41 ).

Plateletaggregationcould notbe inducedbyr116(P503L)

in the absenceofristocetin,evenwhen used at a10-foldhigher concentrationthan thateffectivein the presenceof0.15mg/

mlristocetin. This isin apparent agreementwiththe

observa-tionthatplatelet aggregation doesnotoccur inpatients with

the Malmo variant neither spontaneously norafterDDAVP

administration ( 19). However,it has beenreportedin

prelimi-naryformthatpurified plasmavWFfromoneindividualwith

Pro503 -> Leu mutation results in increased vWFaffinityfor platelets even in the absence of ristocetin, although such an

increase isclearlyless pronounced than that caused bytypeTIB

mutations. The latter statement is justified by the lower amountsofpurifiedtype IIB vWF necessary toinduce

aggrega-tion(43, 44) as compared to typeMalmovWF(42). Thus, the phenotypeof thevonWillebrand disease variantstype IIBand

typeMalmo (and presumablytype I NewYork) is similar with

regard toincreased responsivenesstoristocetin andtothe abil-ityofvWF toinduce plateletaggregation directly;rather,the differenceappears tobe in theextent towhich theaffinityfor GPlb is changed by different mutations in the GPlb-binding domain of the molecule. Thismayberesponsibleforthe other distinctive feature oftype IIBpatients,i.e. the disappearance of

larger vWFmultimersfromthecirculation(7, 43, 44), due to

theirgreater bindingtoplatelet membranesat the vWF

con-centrationspresentin blood.

Thefailuretodemonstrateadirecteffect oftheisolated GP lb-binding domainonplateletsmaybe duetoinherent

differ-ences between multimeric vWF and dimeric r116 fragment withregard totheirrespectiveabilityto supportplatelet

aggre-gation in the absence ofristocetin (27). Indeed, the dimeric fragment has only twoGP lb-binding sitesper molecule, as

opposedtoseveralinalargervWFmultimer,andthismay not

be sufficient to support theaggregation (oragglutination) of platelets.Ristocetinmaychange thisbecauseitcandimerize in solution, itflocculates proteins andcan bindto both the GP lb-binding domain ofvWF and to the platelet membrane

(41),possibly creating functional "aggregates" of the dimeric fragment. Itshould be noted that, in unpublishedstudies,we

found thatar116fragment containingatype IIBmutation also failedto induce platelet aggregation directly,eventhough the

same fragmentcould clearlybindto GPlb in the absence of

anymodulator(3). Inthis regard, then,expression of the iso-lated GP Ib-binding domain cannot reproduce afunctional characteristic of themutantmultimericvWF.

Theexact structure andfunctionalconformationoftheGP lb-binding site in the vWF subunit have yet to be clarified, although candidateregionshavebeenidentifiedaspossible re-ceptorrecognitionsites (32,45). Moreover, evidence from

sev-eralstudiessuggeststhat thedisulfide loop betweenCys509and

Cys691

canmodulate theaffinityofvWFfor GP lb. For

exam-ple, botrocetin, asnake venom protein, forms abimolecular complex withvWFthat thenbindstoGPlb withhighaffinity; botrocetin hasbeen showntobindtothreedifferentsegments

ofsequencewithin the above mentioned disulfide loop (46).In type IIB von Willebrand disease, characterized by increased

vWFaffinity forGPlb(5, 6), all the reportedmutations

consid-eredtoberesponsible for the functional abnormalityarewithin the samedisulfide loop; moreover, six of them areinthefirst botrocetin-bindingsegmentandtwoin the secondone(3, 1

1-13, 46). Porcine vWF, which spontaneously aggregateshuman platelets, displays interesting differences as compared to hu-manvWFin theregion correspondingtothe first botrocetin-bindingsegment: infact,it containsthreeamino acid substitu-tions (47) corresponding to those found in human typeIIB vWF, which alsoaggregatesnormal humanplatelets(43,44). All these results support thecontention that conformational transitions withinthedisulfide loopbetweenCys509and

Cys691

regulate theaffinity ofvWFforGPlb.Wehave now found that

anaminoacidchangeoutsideof the 509-695 disulfide loop in

theAl domainofvWFcanalsoleadtoheightenedinteraction

with GPIb, suggestingthatthe lattereventisregulated byan extendedregionin the GPTb-bindingdomain of vWF with the

contribution ofpreviously unidentified residues. The fact that some ofthe patients reported here had negligible bleeding symptoms(patients I and 3)raises the intriguing possibility

that other vWF mutationsaffectingtheaffinityforGP lbmay

be found in the normalpopulation.

The origin ofthe mutationsdescribed here ispresently a matterof speculation, butit is clear from thecharacterization

ofdifferent polymorphicmarkers of thevWF genethat they

have occurred independently in the three families studied.

Since the base

changes

found in the

patients

areC T

transi-tions atCpG dinucleotidesineither the codingornon-coding strands,the codonsforresidues 500 and 503 in mature vWF

may representpreferentialsites ofpoint mutationsthattendto occurtogetherfor reasons that remain to beclarified. Neverthe-less, since the single point mutation inthecodon for residue 503hasarisenasanisolated event in thefamilyofpatient 1,

thisexplanation is not ofgeneral validity. Alternatively, if the

mutation inthe codon for residue 500 wereacommon poly-morphism, it wouldbepossibletoargueforthe fortuitous asso-ciation between three independent mutational events in the codon for residue 503 and one ofthe two polymorphic

se-quencesatposition500.Againstthispossibilityisthe factthat, inapreliminarysurvey (53individuals) ofthenormal popula-tion,the mutation atposition 500 was neveridentified, thus

making itscasualassociation withthePro503 -- Leumutation

unlikely. Anotherintriguing possibilityissuggested bythe ob-servation that,in two of the families studied(patients2and 3 in onefamilyandpatient4in the other),the twolinked muta-tions create a gene sequence identical to that found in the

correspondingsegmentofthevWFpseudogene;thus, they may havebeen the result ofgeneconversion, i.e.,a nonreciprocal recombinationeventinwhicha segmentofone genereplaces

thecorrespondingsegmentofarelatedgene(48). Sucha

mech-anismhasbeenimplicatedasthe causeofhuman steroid 21 -hy-droxylase deficiency (49, 50). Although in the lattersituation thegene and pseudogene involved are located on the same

chromosome, the occurrence of heterochromosomalgene

con-versionhas been clearlydocumented inlowerorganisms(51 ) and cannotbe excluded in humans (48).Inthe caseconsidered here, thesequenceofvWFpseudogeneimplicated in the

con-versionevent would be no more than 99 basepairs, represent-ingthe size ofDNA segment thatincludesthe twomutations

presentin the patientsandis delimitedbytwomutations pres-entin the pseudogene (26) butnonein themutantalleles.Itis interesting to speculate that, ifthe pseudogene hasdiverged from the vWF geneespecially at mutational hot spots, there maybe othervonWillebrand disease variantscausedby amino

acidsubstitutions coded for by sequences identicaltothose in the vWFpseudogene. Suchapossibility is presentlyunder in-vestigation.

Acknowledgments

WethankProf. IngaMarie Nilssonand Dr.Erik Berntorp for helpin obtaining samples from patient 1; and Dr. AkiraYoshiokaof Nara

Medical College,Nara,Japan, for providingthemonoclonal antibody

NMC-4.

This work was supportedinpartby grant HL-48728fromthe

Medi-cine at The Scripps Research Institute for financialsupport to theDNA core facility. Additional support for the General Clinical Research Center (National Institutes ofHealthgrant RR0833)and from the Swedish Medical Research Council (grants 4997 and 9627) is also ac-knowledged.

This is publication 7499-MEM/CVB from The Scripps Research Institute.

Noteadded,inproof Aftersubmission of the manuscript for this article, Dr. Harvey Weissprovided us with blood from a member ofthe original familyinwhich the type I New York variant ofvon Willebrand diseasewasidentified(patient 3inreference 18). We found that this individual was heterozygousfor the same mutations of vWF codons 500and 503reportedinthispaper. Thus, "typeINew York"and "type MalmV"vonWillebrand diseaseareidentical variants.

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