Circulating heparan sulfate proteoglycan anticoagulant from a patient with a plasma cell disorder

Circulating heparan sulfate proteoglycan

anticoagulant from a patient with a plasma cell

disorder.

M S Khoory, … , E J Bowie, K G Mann

J Clin Invest. 1980;65(3):666-674. https://doi.org/10.1172/JCI109712.

A woman, aged 68, with multiple myeloma (immunoglobulin[Ig]A kappa type) developed an anticoagulant with properties suggestive of heparin. The anticoagulant prolonged the thrombin time but not the reptilase time and was resistant to boiling, proteolytic enzyme digestion, and trichloracetic acid precipitation. The thrombin time was corrected by the addition (in vitro) of protamine sulfate or the addition of purified platelet Factor 4 (PF4) to the plasma. The anticoagulant was isolated by PF4-Sepharose affinity chromatography and analyzed in terms of its molecular weight, uronic acid, and amino acid composition. The proteoglycan isolated had a mol wt of 116,000 and appears to consist of two 38,000 dalton polysaccharide units interconnected by peptide material totaling 39,000 daltons.

Electrophoretic analysis of the pronase digested peptidoglycan using the lithium acetate-agarose technique suggested the material was of the heparan sulfate type. The

peptidoglycan had about one-tenth the specific activity of commercially available heparin on a weight basis. The isolated proteoglycan was indistinguishable from commercial heparin when analyzed in terms of its ability to act as a cofactor in the antithrombin III inhibition of thrombin.

Research Article

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Circulating Heparan Sulfate Proteoglycan Anticoagulant

from

a

Patient with

a

Plasma Cell Disorder

\MAGED S. KHOORY, \MICHAEL E. NESHEINI, E. J. WALTER BOWIE, and

KENNETH G. MANN,Hematology Researchi Sectioni,

AMailo

Cliniic,

Roch5ester, MlinnICesota 55901

A BS T RAC T A womiiani, aged 68, with miiultiple imiyelomiia(im munoglobulin

[IgJA

kappa type) developed

ananticoagulanitwith properties suggestiveofheparin. Theanticoagulanit proloniged the thromiibini tiunle but not the reptilase timiie and(l was resistanit to boilinig,

proteo-lyticenizymiedligestioni, anid tricliloracetic acid precipi-tation.The thrombin timiiewascorrectedbytheadditioni

(invitro) of protaminie sulfhateortheadditioni of purified platelet Factor4(PF4) to theplasmiia. The aniticoagulanit wasisolatedbyPF4-Sepharoseaffinity chromnatography

and analyzed in terms of'itsmiiolecular weight, uroniic acid, anid aminio acid coimlpositionl. The proteoglycan isolated had amiiolwtof 116,000anidappears to consist of two 38,000 daltoln polysacchari(le unlits

initerconi-nected by peptide miiaterial totalinig 39,000 daltonis. Electrophoretic analysis of the proinase digested

pep-tidoglycanusing the lithiumii acetate-agarose techniique

suggestedthemiiaterial wasof theheparani sulfatetype. The peptidoglycan ha(l about onie-ten-thl the specific activity of commercially available heparini onlaweight basis. Theisolatedproteoglycaniwasinidistinguishable

from commercialheparinwlheni analyzedintermiis ofits

ability to act as a cof'actor in the

anitithrolmpbini

III

inhibitioni of thrombini.

INTRODUCTION

Mlultiple myeloma may interfere with hemiiostasis in

severalways (1). These incltildethe dlirect comiiplexinig

of the paraprotein with a coagtulationi f;actor (2), or

in-direct interactionby interferenicewithfibrin miioniomiier

Apreliminaryreportof thisstudvwas

presenited

ataimieetinlg

ofthe Federation for Amiierican Societies for Experimiienital

Biologv, April 1978,Atlaniitic

City,

N. J. 1978.Fed. Proc. 37: 3. (Abstr. 618)

Dr.Khoorv wvassupporte(dbh Bloo(o Banikiniganid lemiiostasis traininlg granit HL-07069 fromiithe National Heart, Ltiung, and(l BloodInstitute. Dr.Mann isani EstablishedInvestigator ofthe

Am-ierican Heart Associationi. Address reprinit re(luests to

Dr. Manni.

Receive(iforpuiblicatiom 9 Mlarch1979ani(I inirevtise(lform

9Novemnber1979.

666 J. Clitn. Inoest. © The Americani Societyfoir

polymerization (3-5). The patienit described in this study appears to be unii(que in that she developed a coagulopathy not directly related to the miyeloma protein,but clharacterized by thepresenceofa

circulat-ingproteoglycan withproperties similartoheparini in that itfunctionied as cofiictorto antithrombin III.

The acid mnucopolysaceharidesarepresenit asstirLctual miiembers of conniective aiud other supportinig tissues

of mammiiiials, anid their presenieeinavarietyof celllinles has been described(6, 7). The presenice ofheparini in blood has beeni reportedl(8) altlhouigh this observation

wasbx'iindirect proceduires anid lhas nlot beeni conifirmiied. The presenlt stu(ly clearly demiionistrattesthe presenceof ani antitlhromiibini III cofactoractive miiaterial in humilan plasmiia and shovs that it was nlotassociated withl the

plasmacytes

or the

immunoglobtlini

fractioni

of the

plasmiia. The isolationi anid partial characterizationi of tlhe material is also presen-itedl.

METHODS

Aminohexvl Sepharose wasobtained fromil Phariiaciaii Finie Chemilicals, Inic., Piscataway, N. J. 10% Agarose and(l P-150 were obtainied fromil Bio-Rad Laboratories, Richilloild, Calif.

The comimillercial heparini used for routinle assays was froml1 Upjohn Co., Kalamilazoo, Mich. (heef lunlg),wlhereasreference stanidards of the acidmucopolysaccharideswereprovidedby Dr. Martiii NMatthewvs,' Universitv of Chicago. Agarose for electrophoretic analysis was SeaKemil (ME), ob)tained firolim MIarine Colloids, Inc.' Rockland, Maine. Azoruhiin S dye,

D-glucuromo-3,6-lactone, and( carbazolq wvere fi'oiln Aldrich Chemiiical Co., Milwaukee, Wise. The water soluble r-e-agenit I-ethl\1-3-(3-d imllethvlai\tinopropvl-)carl)odiiidi(le was

fromilSigmila Chemiiical Co., St. Louiis, Mo.Crtuldethromhin foi routinie_{aniticoaggulanit}analysis wasoltainedfromiiParke,D)avis

& Co., Detroit, Mich. Proniase was obtaine(d fromim Sigmiia Chemical Co., (protease IV). Platelet Factor 4 was purifie(d bh themethlo(dof Ruiz etal.(9).Purifie(d bovinea-thrombinm use(d forexamiiniationi ofiniteractioniof the inhihitorofantithromhlln

III wasprepared1ythemiiethlodlofLunidbladetal.(10). Bovine antithrombinIIIwasprepareduisinigthemiietlho(dof Datmnsatn(d

IAcidl mucopolysaccharide referenice stanl(lar-ds, April

1977,MI. B.Mlatthews,J.A.Ciff)oielli, D)epartmnentofPediatrics,

University ofChicago, Chicago, Ill.

tCClinical Intvestig(ation), Inc. 0021-9738I80I0'3I066609) $1.(00

Rosenberg (11). Analyses of thrombin concentrations were performed by the modified National Institutes of Health (NIH) procedure (12)described byMann etal. (13).

Coagulation tests were performed by published methods (14). The appearanceof the anticoagulant materialthroughout the differentstepsoftheisolation andpurificationprocedures

were monitored by the thrombin time using crude bovine thrombin reconstituted to yield anormal thrombin time of 16-18 s in the absence of added inhibitor. As a substrate,

50,ulof a 0.15 M NaCl solution in which the unknownmaterial was dissolved was added to 150 ,ul ofnormal pool of fresh plasma collected in oxalate; as a control, 50,ulof 0.15 M NaCl was added to 150 ,ul of normal plasma. The anticoagulant activity of thematerialwasmeasuredagainst astandardcurve prepared with increasing dilutions of beef lungheparin with 0.15 M NaCl as a diluent. Because of the variation of the

thrombin solutionfrom day to day, such a standardcurve was freshly plotted every time an assay ofthe anticoagulantactivity

was tobe done.

Proteolytic digestion was accomplished byincubatingthe

thawedpatientplasma,freshly collectedinacidcitratedextrose

andstoredat-700C, orthe isolatedproteoglycan fromplatelet

Factor 4(PF4)2-Sepharosecolumn, with pronase (3 mg/100ml) for48 h at 450C. Atthe endof thisperiod, solid trichloroacetic acidwasaddedto 5%(wt/vol)and incubationcontinued for2h at40C.Thesupernate from this aswellas two washes ofthe precipitate withdeionizedwater wereexhaustivelydialyzed

against the water and lyophilized.

PF4wascoupled to aminohexyl Sepharose 4B in the follow-ing manner: to 1 mlof packed resin was added 2.5 ml human PF4(2.6mg/ml) in 0.5 M NaCl, pH 5.5, 22°C. 20 mg of solid water-soluble carbodiimide was then added with stirring and

thepH maintained at 4.5-5.5bythe addition ofdilute HCl over aperiod of 1 h. Stirring was then continued overnight

at22°C. The resin was thenfiltered, washed with 100 ml of 0.02 MTris-HCl, 1.5 M sodium chloride, pH 7.4. Resin was

then washedin 0.02 MTris-HCl,0.15 MNaCl, pH 7.4 for stor-age orfurtheruse.

Molecular weight estimations were performed with the

high speed sedimentation equilibrium technique(15) using a

Beckman modelEanalytical centrifuge (BeckmanInstruments, Inc., Fullerton, Calif.). No gross curvature was observed in

high speed sedimentation equilibrium experiments. Therefore,

weightaverage molecularweights(NMw)werecalculated from directanalysisoftheslopeofplots of log fringe displacement (lnc)vs.radial distancesquared (r2) using the equation:

2RT dlnc

Mw =

coNl - vp) dr2

The Z average molecular weights were calculatedby extrap-olation ofMwr,theweightaverage molecularweightatany pointrtothecell bottom.Avalue of 0.53cm3/g(16) wasusedl

forv,for the peptidoglycan,since this value(determined for

chondroitin4-sulfate)isappropriateforthedegreeof sulfation

reportedforheparansulfate. Avalue of0.61cm3/gwas used for theproteoglycanbased upon composition(38-41%protein)

andavalue of0.73cm3/gwasusedfor protein.

Speedswerechosen such thatdlnc/dr2wasbetween 1.5and

2.5. For allexperiments, meniscus depletion andthe attain-mentof equilibrium were verifiedexperimentally.

Aminoacidanalyseswereperformedby singlecolumn tech-niques on a Beckman model 119 amino acid analyzer. Before

analysis sampleswerehydrolyzedin 6 NHCIfor24 hat 110°C.

Uronic acid content was measured by the carbazole reaction

accordingtoBitterand Muir (17). Gel electrophoretic analyses

2Abbreviationt

used itl this paper: PF4, platelet Factor 4.

of acidmucopolysaccharidewereperformed accordingtothe

procedure of Homer(18) in lithium acetate buffer and 0.6 or 1.2% agarosegels.Stainingwasaccomplished usingtoluidine blue 0.

AntithrombinIIIcofactor activitv of the isolated anticoagulant was evaluated with purified coomponents. Solutions of the isolated inhibitor at concentrations equivalent to 0.66 U

heparin/ml(deten-nined from thrombin-timeassaysonl nlorimnal

human plasma) wereincubatedat220Cin 0.02 M Tris-HCl,

0.15 NI NaCl,pH 7.4 with bovineantithrombinIII(2.44,utM) andhuman a-thrombin(0.856KLM).Atregularintervals after theaddition ofthrombinaliquotsof thereactionmixturewere

withdrawnandassayed forresidualthrombin-catalyzed clotting

activitvaccordingtothe NIHprocedure (12)as modified by Mann et al. (13). Control experiments werealso performedl

using an eqtuivalentamount ofcommercial heparin inplace

of the isolatedanticoagulant. In some instances human PF4 at afinalconcentrationof 0.14mg/!lll wasincluded to compare theneutralization ofthe heparin-like cofactor activitv of the

patient-derived inhibitortothe neutralizationi of commercial

heparin.

Thepatient's leukocvtes,obtainedby leukophoresis,were

pelletedbycentrifugationat 800g for 8 min.Cellswerethen

preparedforeithershort-tenrmculture orhomogenization.For

short-term cultures, the cells were resuspended in RPMI 1640 medium and cultured in Marbrook vessels (19) as

described by Katzmann (20). Cellpelletswerehomogenized

in a pestle tissue grinder maintained at 0°C. Nuclei and cellular debris were removedbycentrifugationat1,000 g for 10min.The supernate oftheshort-termleukocyte culturedid

not possess anticoagulant activity, nor did the extracts of

homogenizedcells.

Case report. The patient was a 68-yr-old female with a

10-mo history ofmultiple myeloma (imunuoglobulin[Ig]A kappa)who hadbeentreatedwithL-phenvlalaninemustar(dand prednisone. She presented with generalized petechiae antd largehematomataonthe upperextremities.

Laboratorv studies: hemoglobini 10.2gIlO0 ml, leukocvtes 29,500/mm3 with 63% plasma cells; platelet count 12,000/

mnm3; calcium 15 rng/100 ml, uric acid 6.6 mg/100 ml, and creatinine 7.2 mg/100 ml. Total serum protein 7.2 g/100ml with 4.5 g/100 mlalbuminand 0.28g/100ml alpha 1, 0.92 g/ 100mlalpha2, 1.4g/100ml beta, and 0.82g/100 mlgammlla globulin. The IgA was 4.9mg/ml, IgM 0.15mg/mIl,andIgG

4.0 mg/mIl. The urine was positive for Bence Jones protein and showed a monoclonal kappa spike on immunoelectro-phoresis.Bonemarrowaspiration showed over 90%abnormal plasmacells. No mast cells were seen. Coagulation studies arepresented inTable I.

RESULTS

The chromatographic separation of the anticoagulant

material present in the patient's plasma on

PF4-Sepharose is presented in Fig. IA, whereas the chro-matographic behavior of beef lung heparinized-humani acidcitratedextrose plasma usedas acontrolispresented

in Fig. lB. In each case, 5 ml of the anticoagulated plasmawasallowed to percolate through -3mlof

PF4-Sepharose.The material notboundineach case repre-sentsmost of theplasmaprotein present in the original

samples. The plasma usedin the heparin control was

anticoagulated by the addition of 250 U ofbeef lung

heparin, and before application to the column had a

clottimeof>100 s.The initial fractions fromthiscolumn

TABLE I

Patient'sCoagtulationi Studie,s*

Prothrombin time

Partial thromboplastin time Activated partialthromboplastin

time Stypven time

Reptilase time Thrombin time

Thrombin time with equal volume ofnormalplasma Thrombin time with 10,ug/mlof

protamine sulfate

Thrombintime afterbarium

sulfate adsorption

Thrombin time with 50,g/mlof purified PF4

Antithrombin III Fibrinogen

Fibrin split products Protamine gel

12(11)s

50 (45-60) s

58 (25-40) s 14 (15)s

21 (14) s 600 (16-18) s

600 s

22 s

24 s

19s

21 (18-24),ug/ml 250 (190-300) mg/100ml 5(<5)

_t.g/ila

positive (negative)

*

_Normal

_{values in parentheses.}

had a thrombin clot time of 22.6 s. Similarly, patient

plasma, which before application to the column had a

plasmathrombin clottimeof>100 s,exhibitedathrombin clottimefor the initial fractions of25.5 s.Thus,inboth

the experimental and control PF4-Sepharose columlln, the anticoagulantactivity wasremovedonthe column, and the clottability of the plasma coming through the

colunmnwasrestored.Subsequent elution ofthe column after washing with 0.5 M sodium chloride, was

ac-complishedwith 0.75Msalt. The anticoagulantactivity

eluted from each column is expressed in terms ofthe fraction of thequantity of anticoagulant appliedtothe column based upon the heparin assay described in Methods. Under this set of conditions, anticoagulanit

activity, some protein (as exhibited by absorbanice at 280 nm), and uronic acid were elaborated from both the chromatography of thepatientplasma and

chromatog-raphy ofthe heparinized normal plasma. Subsequent elution of both columns with 1.5M sodium chloride

resulted inthe elution ofasmall amount of additional

anticoagulantactivityfrom thepatient'splasmaaindthe

largerfraction ofanticoagulantactivityfrom the column

to which the heparinized normal plasma was applied. The appearance oftwo peaks from each column is a

reproducible occurrence that does not appear to be relatedtoindividualheparincomponents ineachsample,

but rather to anomalous columiinbehavior, inthat

reap-plication of either of thecomponents eluted at 0.75 M

or 1.5 M results in a similarchromatographic pattern;

thatis, twopeaksonelution.Most likelythisanomalous behaviorisareflectionofheterogenous

bindinig

ofPF4

totheagarose supportrather than heterogeneity inthe anticoagulant activityapplied tothe column.

The amountof' anticoagulant materialpresent inthe patient's plasma was about 30

_gsg/ml,

basedupoIn

re-coveries from the PF4-Sepharose columlln.

For the columnn to which the patient's plasimia was applied, the comiiponents eluted at 0.75 -M salt coin-tained,in additiontoanticoagulantactivityand tironic acid, sonme proteinaceous comiiponients. In addition, immunodiffusioni experiments revealed the presence

of trace amnountsof human antithrombin III in the comil-ponents eluted in this fraction, whereas sodiu-mdodecyl suilfate gel electrophoretic analysis showed fainit an-iotunts of a variety of proteins associated with this peak. The components eluted in the 0.75 M fraction fromn the

pa-tient's plasmawerepooled and suibjecte(dto chromatog-raphy on Bio-Rad P-150. Anticoagulanit activity was

elutedinthe void volume of this column. The miiaterial retained itsabsorbance at 280nm, suiggestinigthe

con-tinued presence ofprotein.

To coiipare the patient-derived ainticoagulanit with commercialhepariin, the 0.75 M fraction wassubjected

toproniase digestion and trichloroacetic acid precipita-tion to removethe protein comlponenit(s). These steps were taken because commlllercial heparin is obtaine(d

onily after rigorous proteolytic and chemical digestioni

ofthe animiial tissue fronm which the polysaccharideis

derived. Afterthis treatinent all absorbance at 280nIn

was lost from the anticoagulanit preparation, whereas theanticoagulantactivity wascompletelyrecoveredl.A

comparison of thegelfiltration behavior of this material

with comnmercial heparin, usinig 10% agarose, is pre-sented in Fig. 2. In contrast withcommnercial heparin, the patient-derived aniticoagulanit peptidoglycani is excluded from a 10% agarose column and appears to be of substantially larger size than commiiiiercial beef

lulng heparin.

The isolatedpeptidoglyeanwas subjectedtoagarose

gel electrophoretic analysis in lithiumii acetate, pH 3,

tocompare its relative electrophoreticmobility to that of other known sulfate(dpolysacchari(le standards.The

electrophoretic miobilityof the acidmulcopolysaccharides

are functions oftheircharge density,andmnostofthese

canbe distinguished electrophoretically. Represented inFig. 3 is aphotographof agarosegel

electroplhoreto-gramsof referencestanidard acidmucopolysaccharides

and the anticoaguilant peptidoglycan derived from the patient's plasma. Reference standard acid miiucopoly-saccharides arepresent ingels Bthrough H. The

dye-frontmarker,amaranth,is ingelK.Thepatient-derived

anticoagulant ispresent in gelsA, I,and J; asthe iso-latedi material (gel A), as the isolated material mixed with heparin (gel I), andthe isolated material mixed

with heparan sulfate (gel J). It canl be seen that the patient-derivedpeptidoglycan ainticoagulantis

electro-phoretically homogeneousinthissystemandiseintirely distinguishedfrom hepariniand five of the other sulfated

polysaccharide reference standardsused for comparison.

A

l.5M

0

co

'

0.4

.JO

0.3--0

"I _0.2

0.11

0.75 M

1.5M

10 20 30 40

125

120

Fractions- 1.5 ml each B

a

4) 0i

(4

0.5

0.4

0.3[

0.2

0.1

0

1.5M

t f

0.75M

30 40

25

0N

>. co

4- a

30 *-20 c

10

0

.m

Fractions-1.5 ml each

FiGuRE 1 (A) Chromatography of

patienit

ACD plasma oIn humiiian PF4-aminohexyl Sepharose

and (B) of heparin anticoagulated niormal humani acid citrate dextrose plasmaon human

PF4-amninohexyl Sepharose. Absorbance at 280 nm (0), aniticoagulation activity (0), and uronic

acid contenit(x)areplotted vs.fraction member.

The material undergoes coelectrophoresis with heparan sulfate. Subsequenit to this experimient, reference

standard

heparani

sulfatewassubjectedtoPF4-Sepharose affiniity chromatography, anid was showni tobehave in similarfashion to the

anticoagu-laint

derived

fromn

the patienit's plasma.

The amino acidl composition of the proteoglycan relative to uronic acid

comnpositioni

is

presente(d

in

Table11.Thesuin foraminoacid compositionindicates about2.23aminiioacidsperhexuironiicacid

residute.

For

heparan sulfate, the hexuronicacid content

correspondls

to 44 (21)-49%' of the total polysaccharide

Unlit

mass. Ifone

presumnles

thatthe material isolatedfromii the

pa-tienitisaproteoglycanforwhich theacid polysaccharide

portion is heparan sulfate, one can estimatea weight-fractionproteiniof from 38 to41%based upon the amino

acid to hexuronic acidratio. After pronase treatment, 85%of theaminoacidsarelost from the totalcomposite toprovide a ratioof0.317aminoacidsper uronicacid

residue for thepepticloglycan.Again assumingthatthe acid polysaccharidle is heparan sulfate for which the

polvsacchari(le chaiin is 44-49% hexuronic acid, the

peptidoglycan resultinig from pronase digestion

cor-respondlsto -91-92% acidpolysaccharide with8-9%

ofresiduial(notdigestiblebypronase)peptidematerial. Frequently, gel filtration has been used to estimate

(CirculatinigProteoglycan Aniticoagulant 669

0.5

N. 1.0

S 0.8 ,i 0.6

t 0.4

*g 0.2 n

A

5 10 15 20 25 30 35 40 45 50 Elution volume,ml

FIGURE 2 Elution profiles of pronase-digested, TCA-treated anticoagulant (0) and Upjohn heparin (0) on a gel filtration

column prepared with 10% agarose. Relative anticoagulant activity is plotted vs. elution volume. The void volume for

the column is -18 ml.

the physical size of acid mucopolysaccharides. Gel filtration chromatography as atechnique for molecular weightestimation, issomewhatcomplicated bythe need to calibrate the gel filtration column in (luestion with

anappropriate setof markers thatrepresentmodel

com-pounds of known chemical structure and molecular weight, whicharethe hydrodynamic equivalents of the molecule whose molecularsize is tobeestimated. When used for the analysis of complex proteoglycans that

may have peptide backbone as well as multichain or

branch chain structures,gel filtration analysiscan lead

togross error. Forthese reasonsand the fact thatwedo

nothave detailed knowledge of thenature ofthe struc-tureof the anticoagulant,wechosetoevaluate themass

A

.If

B C D E F G H K

FIGuRE 3 Lithium acetate-agarose electrophoretic analysis

of reference standard acid mucopolysaccharides and

patient-derived peptidoglycan. Gel A contains patient-derived

pep-tidoglycan anticoagulant; gel B, referencestandard heparin; gelC,heparin obtained from Upjohn; gelD,keratan sulfate; gel E, dermatan sulfate; gelF, heparan sulfate; gel C,

hyal-uronic acid; gel H,chondroitin-6-sulfate; gel I, patient

anti-coagulant peptidoglycan mixed with reference standard

heparin; gel J, patient peptidoglycan anticoagulant mixed withreferencestandardheparan sulfate; andgelK,amaranth

dye marker.

TABLE II

AmiinoAcidComposition ofAnticoagulantl laterial

Proteoglvca,i Peptidoglyean

n1/oll/nioluronic(aci(d

Aspartic acid 0.18 0.035

Threonine 0.14 0.032

Serine 0.26 0.030

Glutamicacidl 0.32 0.049

Proline 0.16 0.031

Glycine 0.32 0.037

Alanine 0.18 0.023

Valine 0.11

Methionine 0.02

Isoleucin1e 0.05

Leucine 0.12 0.009

Tyrosine 0.05

Phenylalanine 0.08 0.014

Histidine 0.08 0.009

Lysine 0.09 0.009

Arginine 0.07 0.009

Hexuronic acidl 1.0 1.0

Amino acid 2.23 0.317

ofthe patient-derivedanticoagulantbyanalytical ultra-centrifugation.Amostpreciseanalysis ofsedimentation

equilibrium datarequires knowledge of the total

com-positionof the material forthe calculation of the partial

specific volume terms. At present, detailed total

compositions for the patient-derived anticoagulant is notavailable. However, sufficient partial composition dataisavailabletopermitestimationof the appropriate

partial specificvolume term for the peptidoglycan and

proteoglycan (see Methods).

We have performed sedimentation equilibrium ex-perimentswiththe isolatedpatientanticoagulant under

three sets of conditions. The data are summarized in Table III. Two sets of experiments were performed

withtheintact, proteasenondigested anticoagulant. In

dilute aqueous buffers, the molecular weight of the

anticoagulantand any noncovalentlyassociated

mate-rials are measured. Under this set of conditions, an average molecular weight of 111,000 was obtained,

presuming apartialspecific volume of0.61cm3/g. The sedimentation equilibrium dataobtained under these

sets of conditions showed some evidence of nonideal behavior nearthecell bottom. However, this problem

wasnotsufficientlysevereso as toprecludeanaccurate

interpretation ofthe data. Sedimentation equilibrium

experiments were also performed with the isolated proteoglycaninthepresenceof6 Mguanidinium chlo-ride. Under this set ofconditions, noncovalent inter-actions between the covalent proteoglycan unit and

any other noncovalent associated entities would be

destroyed and the molecular weight obtained should reflect the mass of the covalent anticoagulant unit.

TABLE III

Molecular WVeight of Antticoagulatit

Preparation Solvent MolecularM weight Amiiino acid

C1113/g %

PF4chromatography 0.15M NaCl 0.61 111,000 38-41

P-150chromatography 0.02 MTris pH7.4

PF4chromatography 6 M Guanidinium 0.61 116,000 38-41

P-150chromatography Chloride

PF4chromatography 0.15MINaCl 0.53 42,000 8-9

P-150chromiatography 0.02 NI Tris Pronase digestion pH 7.4 TCAprecipitationi

Under these sets ofconditions, an average mol wt of 116,000 wasobtained,again presum-ing that the partial

specific volume of the proteoglycan is 0.61 cm113/g.

Pronase digestion of the proteoglycan anticoagulanit

removed -86% of the amino acid imiaterial iniitially associated with the anticoagulant and the resulting

polymer gave a mlol wt of42,000 when examinied in the analytical ultracentrifuge,presumiinigapartial specific

volume of0.53Cm113/g. The relativehomogeneity of the

polysaccharide unit is illustrated inthe sedimenitationi

equilibriumdatapresentedinFig.4.These data clearly

show that the polysaccharideisnothighly polydisperse. In addition the NIzvalue determiined for this analysis was 48,000, inidicating a relatively narrow ranige of

molecularweights for the polysaccharidemass distribu-tion. Approximately 9% of this imiaterial is aminio aci(d

by compositioin data, and thus thepolysacchari(le miiol wt is 38,000.

Acomposite of analysis of the molecularweightanid comipositioin data obtained for the proteoglycan isolated

1500 _r2>

1000

750

- 3500-350

250/

150 /

50.4 r2(cm2)

FIGURE4 Sedimentationi equilibrium clistributioni for

pep-tidoglycan,at18,000rpm (22.3°C).Concentration (c)isplotted

vs.radial distancesqtuared.The cellbottomissignifiedlby_r2,.

from the patient's plasmna wouldsuggestthe structure

containinlgtwopolysaccharidechains of38,000 mol wvt

linikedto a peptide chaiin (or chains) wlhose total mass

would be -39,000. Such a structure wouild be -66%

carbohydrate and34%peptide.Thesevaltues are within 4-7%of theestimated composition values based uponl thepartialcoimipositioindataavailable. Thecoinsisteincy betweeni thevaluesobtained from theproteoglycan in the nativestate in diluteaqueouis buffer ( 111,000) and that obtaiinedl in 6NI guaniidiniiumii chloride (116,000),

ani( the consistency between the values obtained for

relativefractioin ofpepticletocarbohydrate basedupoIn

both chemiiical analysis and physical analysis strongly

suggest that the appropriate assumilptionis have been made with respect to partial specific volume for the intact proteoglycan basedUponlthe partiallyassumptive

analysis of

comlipositioni

of the patient-derived aniti-coagulanit.

Functioinal assays of the relative anticoagulaint potency of the patient-derived peptidoglycani were coinducted by comiipariing its effects to that of commiiiiercial heparini

oIn a plasmiia-thromiibini clot timiie. The patienit-derivedl anticoagulanithad aspecificactivity of -8 U/miig, which is substantially less thani the specific activity of the

commlllercial beef luIng heparin, which has a specific activity of -120 U/miig.

The niatture ofthe initeraction of the patient-derived ainticoagtulanit peptidoglycan with antithrombin III,

thrombin, and PF4 vere comparedto that of beeflunig

heparin. The resultsoftheseexperimeintsarepresented

in Fig. 5.The thrombin inihibition datafor each of the

experiments presenite(din Fig. 5 areexpresse(daspseuldo

first-or(ler rectetionis. The aiimounlt of patient-dierived anticoagulanit or beef lutnig heparini adIdledI to

aniti-thrombin III (closedl an(l openi circles, respectivelv),

are

eqdiivalenit

basedl uponi the plasmiia thromiibini clot

timne assay(lescribed in Niethods. Upon a(diniistration

of PF4 to the mixture of heparin, antithrombin III, aindthromnbini orthatconitaininlgpatient-derived

.t

o s

100

50

20- I \\

1 2 3 4 5 6

Time (min)

FIGuRE 5 Thrombin inhibition by antithrombin III in the

presence of commercial beef lung heparin orpatient-derived

anticoagulant. The reaction isanalyzedas apseudofirst-order reaction in terms of the logarithm of the percent thrombin remaining as a function oftime(minutes).Thetime-dependent

decrease of thrombin activity with antithrombin IIIalone is

shown (A). The mixture ofantithrbinbin III, thrombin, and patient-derived anticoagulant (0.6Bheparin equivalent U/ml)

is shown (0) and a mixture of antithrombin III, thrombin,

andheparin (0.66 units/ml) isgiven (0).Results obtainedby

adding PF4 (0.14 mg/ml)to these twosamplesaredesignated

by AandL, respectively.

oglycan anticoagulant,antithrombin III, and thrombin,

equivalent rates of inactivation for thrombin were

observed. These rates obtained after neutralization of

heparin and heparin-like anticoagulant are

experi-mentally equivalenttotherate of neutralization ofthe

equivalent amount of thrombin by the equivalent

amountofantithrombinIIIalone (Fig.5, opentriangles).

It should be noted here that the rate observed and presented for Fig. 5 inhibition of thrombin in the presence of heparin is notthe ultimate rate that could be observedupon saturationof thereaction withheparin. The concentration ofheparin used inthis experiment was chosen such that the rate of decrease of thrombin

activity could be measured. If saturating amounts of

patientproteoglycan orcommercial heparin are added tothe amd'untof antithrombin III present in the reac-tion mixture,theexperimental data would show

inhibi-tion of all thrombin present at the earliest time of analysis. Under conditions of thesaturationof the

anti-thrombin IIIpresenteitherby patient-derived

proteo-glycan or by heparin, complete return to the

anti-thrombin III alone inhibition rate is Observed upon

administration

ofPF4. Therateconstantsderived from these experiments are presented in Table IV.

TABLE IV

Rate of Thromnbin Inihibition

Componentadded k* Halt:life

liter*wini-'

ntiol-V x10(-5 "?li

Antithrombin III + PF4 1.22 2.33

Antithrombin III + heparint 6.51 0.44

Antithrombin III +anticoagulant§ 6.91 0.41 Antithrombin III +heparin + PF4 1.03 2.76

Antithrombin III +anticoagulant

+PF4 1.10 2.57

* The second-order rate constant (at 22°C)

determined from

pseudo first-order plots wvith AT-III in 2.85-fold mol excess

over Ila.

IHeparin (intestinal mucosa)obtained from Eli Lilly&Co.,

Indianapolis, Ind.

§ Patient-derived proteoglycananticoagulanit.

DISCUSSION

The anticoagulant activity present in this patient's plasma could be quantitatively bound to and subse-quentlyeluted from PF4 immobilized on agarose. The chemicalandphysical data derived from studies of the

anticoagulantsuggestthat it is likely composed of two

polysaccharidechains of nearlyidenticalsize

intercon-nected by peptide material. Whereas current data

indicates that the polysaccharide chains present in the

anticoagulant proteoglycanare of nearly identicalsize, no knowledge is presently available as to whether a

singleormultiple polypeptidesareresponsiblefor the protein constituent of the proteoglyean. In any event,

theprotein constituentsdonot appear to havean

influ-ence ontheanticoagulantactivityof theproteoglycan,

inthatpronasedigestion leaves thisactivity intactwith the peptidoglycan. Further, the anticoagulant activity

associated with the peptidoglycan is consistent with

itsbehavioras anantithrombin IIIcofactorinamannier completely analogous toheparin. Electrophoretic

analy-sisofthepeptidoglycananticoagulant indicates that the

isolated material behaves more like heparan sulfate than heparin.

The molecular weight of commercially available

heparin isreportedtobe intherange of6,000-16,000 (21-23). The molecular weight of the reference

stanclardl

heparan sulfate used for electrophoretic and chromato-graphiccomparisons canbeestimatedtobeintherange

of30,000-40,000 based upon intrinsic viscosity data' supplied with the material. Both of these materials

wereisolatedwithharshchemicalextractionprocedures

and proteolytic digestion of the animaltissue. In con-trast, macromolecular heparin has been observed on moregentleextractionof cultures of mast cells (24, 25), and from rat skin (26, 27). In these cases, the major

heparin fraction obtained was resistent to protease digestion but could be reduced in size by alkaline hydrolysis, oxidative-reductive

depolyimerization

with ascorbic acid, or by cleavage by heparinases. The products obtained by the chemical treatments had mol wt near 40,000, whereastheenzymatic digestion products had a mol wt of about 14,500.

Heparan sulfates are found in a variety of tissues including the aorta (28, 29), and apparently are

sus-ceptible to change with bothage(30)and disease(31), including cellular transformation (32-34). In contrast to heparin, which appears to be a complex

macromo-lecularpolysaccharide structure with additional protein attached, heparan sulfate appears tobe aproteoglycan with a peptide backbone to which a series of polysac-charide chains is connected. Janssonand Lindahl (35) reported theisolation of a heparan sulfate proteoglycan fromn aorta that had an apparent mol wt of 70,000 and couldbecleavedltoproducepolysaccharide chains with

a mol wt of10,000. Chiarugi and(l Urbano (36) reported the isolation of a heparan sulfate proteoglycani for normiial and Rotis sarcoma virus transformed baby

hampster kidney cells which was -40% protein and

had a mol wt of 130,000, somewhat similar tothe value obtained for ourmaterial.

Circulating anticoagulantshave been known to occur in abroad variety of conditions. Some of these

antico-agulants, such as the lupus anticoagulant, cause an

immediate inhibition of the coagulation mechanism whereas others are directed against specific coagula-tion factors, particularly factor VIII (37). In multiple

myeloma, however, specific abnormalitiesinthe clotting

mechanisms have not frequently been characterized. Prolongation of prothrombin andpartialthromboplastin

times have generally been attributed to nonspecific interference ofthe monoclonal proteins,

although

in

certain instances specificcoagulation factorantibodies

have been elaboratedinmyelomna(1).Inseveralstudies (4, 5,38), an antithrombin role has been assigned to a

proteinintheplasmawhichmay,ormaynot, have been the

monoclonial

immunoglobulini

molecule. In two

studies (39, 40) heparin-likeactivity forananticoagulant

wassuggested but fu-rtheranalyseswere notconducted

to specifically identify thenature ofthe anticoagulant

activity. Inthe patient whose anticoagulantisthe sub-ject of this investigation, the anticoagulant material

appears to havenospecificrelationshiptothe

immuno-globulin elaborated by the myeloma cell line, and, in

fact, the patient's disease was in remission and IgAat anormal level when thespontaneously occurring

anti-coagulant activity developed. The observation of a

circulating

heparani

sulfateproteoglyeaninthispatient

isprovocativeinviewof reports suggesting differences

with respect to heparansulf:ate in mucopolysaccharide componentsaftercell transformation (32-34).Although

this particular patient had aplasmacell leukemia, the

anticoagulant activitywas notelaboratedin short-term cultures of her peripheral leukocytes, nor was it present in the extracts of homogenates of the cultured cells. Thus,although this anticoagulant molecule was generated intheblood of the individual suffering from plasmacell

leukemia, no direct connections between the disease state andthe anticoagulant molecule can be identified. Two potential routes by which the anticoagulant molecule may have appeared in the plasma appear worthy of some speculation. These are: (a) that the

anticoagulant molecule is a normal constituent of plasma, ordinarily present only at very low concentra-tions, which was released

4t

a greater rate as a

conse-quenice ofthe disease state; and (b) that tissue damage secondary to the primary disease state has led to the release of the proteoglycan. Heparan sulfate and re-lated substances have been reported to be produced by endothelial cells in culture (6); in addition, Muller

etal. (41) have reporteda heparin-like inhibitor in the

blood of the newborn, and it is conceivable that the anticoagulant described in the present work is related

tothese materials.

ACKNOWLEDGMENTS

WewouldliketothankDr.JerryA.Katzmannforperforming the short-term cell culture experiments described in this paper. We would also like to thank Dr. Matthews and Dr.

Ciffonelli for providing the mucopolysaccharide reference

standards used for comparison. Finally, we would like to

acknowledge Dr. Robert Kyle and Dr. Philip Greipp for

makingthe plasma of this patient available for our studies. Thisresearch by grant HL-07049 from the National Heart, Lung,andBlood Institute.

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