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Circulating Heparan Sulfate Proteoglycan Anticoagulant from a Patient with a Plasma Cell Disorder

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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. Research Article 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. Find the latest version: https://jci.me/109712/pdf 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 B S T R A C T A womiiani, aged 68, with miiultiple polymerization (3-5). The patienit described in this imiyelomiia (im munoglobulin [IgJA kappa type) developed study appears to be unii(que in that she developed a an anticoagulanit with properties suggestive of heparin. coagulopathy not directly related to the miyeloma The anticoagulanit proloniged the thromiibini tiunle but not protein, but clharacterized by the presence of a circulat- the reptilase timiie and(l was resistanit to boilinig, proteo- ing proteoglycan with properties similar to heparini in lytic enizymie dligestioni, anid tricliloracetic acid precipi- that it functionied as cofiictor to antithrombin III. tation. The thrombin timiie was corrected by the additioni The acid mnucopolysaceharides are presenit as stirLctual (in vitro) of protaminie sulfhate or the additioni of purified miiembers of conniective aiud other supportinig tissues platelet Factor 4 (PF4) to the plasmiia. The aniticoagulanit of mammiiiials, anid their preseniee in a variety of cell linles was isolated by PF4-Sepharose affinity chromnatography has been described (6, 7). The presenice of heparini in and analyzed in terms of' its miiolecular weight, uroniic blood has beeni reportedl (8) altlhouigh this observation acid, anid aminio acid coimlpositionl. The proteoglycan was bx'iindirect proceduires anid lhas nlot beeni conifirmiied. isolated had a miiol wt of 116,000 anid appears to consist The presenlt stu(ly clearly demiionistrattes the presence of of two 38,000 daltoln polysacchari(le unlits initerconi- ani antitlhromiibini III cofactor active miiaterial in humilan nected by peptide miiaterial totalinig 39,000 daltonis. plasmiia and shovs that it was nlot associated withl the Electrophoretic analysis of the proinase digested pep- plasmacytes or the immunoglobtlini fractioni of the tidoglycan using the lithiumii acetate-agarose techniique plasmiia. The isolationi anid partial characterizationi of tlhe suggested the miiaterial was of the heparani sulfate type. material is also presen-itedl. The peptidoglycan ha(l about onie-ten-thl the specific activity of commercially available heparini onl a weight METHODS basis. The isolated was proteoglycani inidistinguishable Aminohexvl Sepharose was obtained fromil Phariiaciaii Finie from commercial heparin wlheni analyzed in termiis of its Chemilicals, Inic., Piscataway, N. J. 10% Agarose and(l P-150 ability to act as a cof'actor in the anitithrolmpbini III were obtainied fromil Bio-Rad Laboratories, Richilloild, Calif. inhibitioni of thrombini. The comimillercial heparini used for routinle assays was froml1 Upjohn Co., Kalamilazoo, Mich. (heef lunlg), wlhereas reference stanidards of the acid mucopolysaccharides were provided by INTRODUCTION Dr. Martiii NMatthewvs,' Universitv of Chicago. Agarose for electrophoretic analysis was SeaKemil (ME), ob)tained firolim Mlultiple myeloma may interfere with hemiiostasis in MIarine Colloids, Inc.' Rockland, Maine. Azoruhiin S dye, several ways (1). These incltilde the dlirect comiiplexinig D-glucuromo-3,6-lactone, and( carbazolq wvere fi'oiln Aldrich of the paraprotein with a coagtulationi f;actor (2), or in- Chemiiical Co., Milwaukee, Wise. The water soluble r-e- direct interaction by interferenice with fibrin miioniomiier agenit I-ethl\1-3-(3-d imllethvlai\tinopropvl-)carl)odiiidi(le was fromil Sigmila Chemiiical Co., St. Louiis, Mo. Crtulde thromhin foi routinie aniticoaggulanit analysis was oltained fromii Parke, D)avis A preliminary report of this studv was presenited ata imieetinlg & Co., Detroit, Mich. Proniase was obtaine(d fromim Sigmiia of the Federation for Amiierican Societies for Experimiienital Chemical Co., (protease IV). Platelet Factor 4 was purifie(d bh Biologv, April 1978, Atlaniitic City, N. J. 1978. Fed. Proc. 37: the methlo(d of Ruiz et al. (9). Purifie(d bovine a-thrombinm use(d 3. (Abstr. 618) for examiiniationi of initeractioni of the inhihitor of antithromhlln Dr. Khoorv wvas supporte(d bh Bloo(o Banikinig anid lemiiostasis III was prepared 1y the miiethlodl of Lunidblad et al. (10). Bovine traininlg granit HL-07069 fromii the National Heart, Ltiung, and(l antithrombin III was prepared uisinig the miietlho(d of Datmns atn(d Blood Institute. Dr. Mann is ani Established Investigator of the Am-ierican Heart Associationi. Address reprinit re(luests to Dr. Manni. I Acidl mucopolysaccharide referenice stanl(lar-ds, April Receive(ifor puiblicatiom 9 Mlarch 1979 ani(I ini revtise(lform 1977, MI. B. Mlatthews, J. A. Ciff)oielli, D)epartmnent of Pediatrics, 9 Novemnber 1979. University of Chicago, Chicago, Ill. 666 J. Clitn. Inoest. © The Americani Society foirtCClinical Intvestig(ation), Inc. 0021-9738I80I0'3I066609) $1.(00 Voltumiie 65 March 1980 6661-674 Rosenberg (11). Analyses of thrombin concentrations were of acid mucopolysaccharide were performed according to the performed by the modified National Institutes of Health (NIH) procedure of Homer (18) in lithium acetate buffer and 0.6 or procedure (12) described by Mann et al. (13). 1.2% agarose gels. Staining was accomplished using toluidine Coagulation tests were performed by published methods blue 0. (14). The appearance of the anticoagulant material throughout Antithrombin III cofactor activitv of the isolated anticoagulant the different steps of the isolation and purification procedures was evaluated with purified coomponents. Solutions of the were monitored by the thrombin time using crude bovine isolated inhibitor at concentrations equivalent to 0.66 U thrombin reconstituted to yield a normal thrombin time of heparin/ml (deten-nined from thrombin-time assays onl nlorimnal 16-18 s in the absence of added inhibitor. As a substrate, human plasma) were incubated at 220C in 0.02 M Tris-HCl, 50 ,ul of a 0.15 M NaCl solution in which the unknown material 0.15 NI NaCl, pH 7.4 with bovine antithrombin III (2.44 ,utM) was dissolved was added to 150 ,ul of normal pool of fresh and human a-thrombin (0.856 KLM). At regular intervals after plasma collected in oxalate; as a control, 50 ,ul of 0. 15 M NaCl the addition of thrombin aliquots of the reaction mixture were was added to 150 ,ul of normal plasma. The anticoagulant withdrawn and assayed for residual thrombin-catalyzed clotting activity of the material was measured against a standard curve activitv according to the NIH procedure (12) as modified by prepared with increasing dilutions of beef lung heparin with Mann et al. (13). Control experiments were also performedl 0.15 M NaCl as a diluent. Because of the variation of the using an eqtuivalent amount of commercial heparin in place thrombin solution from day to day, such a standard curve was of the isolated anticoagulant. In some instances human PF4 freshly plotted every time an assay ofthe anticoagulant activity at a final concentration of 0.14 mg/!lll was included to compare was to be done. the neutralization of the heparin-like cofactor activitv of the Proteolytic digestion was accomplished by incubating the patient-derived inhibitor to the neutralizationi of commercial thawed patient plasma, freshly collected in acid citrate dextrose heparin. and stored at -700C, or the isolated proteoglycan from platelet The patient's leukocvtes, obtained by leukophoresis, were Factor 4 (PF4)2-Sepharose column, with pronase (3 mg/100 ml) pelleted by centrifugation at 800 g for 8 min. Cells were then for 48 h at 450C. At the end of this period, solid trichloroacetic prepared for either short-tenrm culture or homogenization. For acid was added to 5% (wt/vol) and incubation continued for 2 h short-term cultures, the cells were resuspended in RPMI at 40C. The supernate from this as well as two washes of the 1640 medium and cultured in Marbrook vessels (19) as precipitate with deionized water were exhaustively dialyzed described by Katzmann (20). Cell pellets were homogenized against the water and lyophilized. in a pestle tissue grinder maintained at 0°C. Nuclei and PF4 was coupled to aminohexyl Sepharose 4B in the follow- cellular debris were removed by centrifugation at 1,000 g for ing manner: to 1 ml of packed resin was added 2.5 ml human 10 min. The supernate of the short-term leukocyte culture did PF4 (2.6 mg/ml) in 0.5 M NaCl, pH 5.5, 22°C. 20 mg of solid not possess anticoagulant activity, nor did the extracts of water-soluble carbodiimide was then added with stirring and homogenized cells.
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