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From www.bloodjournal.org by guest on August 11, 2017. For personal use only. CONCISE REPORT Factor VIlI-Related Protein High The size of agarose was it human identified forms VIll-related was identified with plasmas venipuncture plasma as methods. the a population ANY of very the rabbit The distribution the pattern was complete. multimers and that the have factor VIII sought an structure and issue has been the protein (VIIIR), of the factor VIII complex that is heterologous antibodies to factor VIII and is responsible for ristocetin cofactor (von Willebrand factor) gel filtration indicated that this concept and ultraV1IIR is has been by the results of recent gel filtration4 ultrafiltration5 studies carried out directly While it is possible that anticoagulation at low temperatures artifactual might aggregation of and on or be responsible data. reason, VIIIR studies conditions, plasma (SDS) were agarose plasma as in carried vitro, apparent of has multimers x of 0.85 Mr out same observations distribution the of The 106. University and Hematology with Under fresh Supported 16872, in part and Macy. Jr. ilL Address Medicine, Conn. University by Grune 31, 1 979, requests of an and at 37’C VlllR:Ag from induced citrate the circulates artifact and of multimeric heparin in glyoxyl agarose, in by time normal purification In the previously course HL Scholar of is Medicine. Farmington. Cleveland, 16361, Award and by Grant C’onn. Ohio. HL of these Inc. studies, for a new purified method was bilizing gel, readily glyoxyl agarose,”’2 controllable reactivity which by used initially was for separating as a gel matrix through covalent inert support medium neutral pH and then proteins were fixed virtue of as an proteins at a to which the attachment. VIIIR, having been separated according to size by SDS electrophoresis and then immobilized, was washed free of SDS, incubated with labeled antiVIIIR:Ag and identified by autoradiography. MATERIALS Standard agarose lots type, 474 and from Sepharose Glycidol (2,3 545) 16626, Co. Co. (St. Louis, obtained from (West Orange, rabbit described.#{176} Before from a patient U/mI) ture. The HL 1gM N.J.). use nemia, from proteins and for 1gM of molecular the plasmas crosslinked the of weight two with (VIIIR:Ag at room < tempera- markers with patients at of plasma included Sweden), dimethyl borate-buffered incubated disease Stockholm, grade. previously volume 5 mm sodium Organon, reagent it was g for (IMCO, thickness) and as an equal Willebrand’s at 8730 used were Co. Chem- from purified experiments, von (7-mil obtained was and Sigma Me.) chemicals 16 hr with fibrinogen from composition was in these East- Chemical film mucosa, centrifuged human polyester other N.J.). from from (Rockland, at 4#{176}C for severe Aldrich Colloids All obtained wasobtained bond (HSA was prepared cyanoborohydride from anti-VIIIR:Ag with and purified Gel Marine intestinal 251-Iabeled 0.01 X-100 Denmark Piscataway, were sodium obtained Triton Mo.), porcine SDS N.Y.); were ical Co., agarose Fine Chemicals, and (Rochester, Wise.); Litex glyoxyl-moditied 4B (Pharmacia was purified of the Josiah I3X-2475 and METHODS from epoxypropanol-1) Kodak Inc. AND obtained was human macroglobuli- suberimidate.’4 saline (BS) The has been published.’3 accepted (onnecticui demonstrated developed for measuring the size of this trace protein. The method employs a newly developed protein-immo- heparin, Council. to Leon #{231} Stratton, 0006-497I/80/5506-0029$0I.00/0 1056 Clinic, Grants 06032. 1980 Medicine, by a Faculty Research January reprint of to L. W. Hover, Medical Submitted kept is not the same as that VIIIR.6’#{176} (Milwaukee, in an pattern Department Cleveland by USPHS 19767, Foundation the Swedish School Division, same that (SDS) A series the dimethylsuberimidate these been identified a protein with multimenic Division, of Connecticut the Research sulfate modified as in plasma indicate 37#{176}C for 2 hr and From was freezing dodecyl it to the (VlllR:Ag). multimers after size sodium antigen as it was separated by sodium dode- electrophoresis. VIIIR a series by by coupling VIll-related man human plasma kept at 37#{176}Cwhile from cellular elements and analyzed cyl sulfate determined electrophoresis for suggested by the two studies, it is also possible that the differences reflect problems associated with size measurements inferred from agarose gel filtration and ultrafiltration For this been These as R. Shainoff of VlllR:Ag the Plasma Multimers anti-factor x 10’. was STUDIES of platelet-related questioned analytic plasma. an purified and large has after Human chelation. activities. While most agarose centrifugation studies have very large (> 106 daltons),’ storage in plasma immobilized using important unresolved of factor-VIlI-related VIIIR:Ag) other and John of 0.85-12 or calcium RECENT the component identified by and By Leon W. Hoyer electrophoresis understanding function. An size in plasma Weight was in serum. in Normal Molecular protein protein M and until freezing. M The by autoradiography anticoagulated of factor electrophoresis. Circulates February W. Hover, Health M.D.. (enter, 6, 1980. Department Farmington. Glyoxyl of agarose” 4% agarose was prepared glycidol at room temperature agarose was washed and suspended pH 7. After by incubating 100 ml of I M NaOH, with then in 500 60 mm for with ml 0.06 incubation 0.002 16 hr with distilled M sodium at room water 200 gentle and 30 ml agitation. The on a B#{252}chner funnel metaperiodate temperature, Blood. Vol. ml of washed M NaBH4, adjusted the to modified 55, No. 6 (June), 1980 From www.bloodjournal.org by guest on August 11, 2017. For personal use only. FACTOR VIII agarose was washed transferred equal MULTIMERS mately of the 1/3 Blood ture in B#{252}chner settled were obtained ( I /50 blood) in aliquot a protocol tubes centrifuged 6.88 sodium was added after to glass dissolved SDS, Serum, at 37#{176}C for 1.25% and 0.05 Electrophoresis cm. electrode chambers acetic acid, They were agarose, 0.25% pH backing using with dye 0.27% containing 0.1% a mold methanol, After marker and Blue water in acetic liter the (1:4:4 were a metal punch. surface blue marker of 0.1 to M the sodium Ultra-Wicks (Bio electrophoresis protein R-250 acid, assembly connected by cellulose Calif.). Coomassie with (Eastman by and were Kodak) volume)7 methanol, and lanes for water 30 mm. by volume). The samples hr at room examined carbonate, pH 1% Triton X-100. room for The cpm) total hr gently with I liter deionized out for with -70#{176}C for 16-24 No Screen film were fixed ml of 0.2 dish normal was 3 changes Kodak The XR-I at 4#{176}C for 3-7 BS and film results for 2 human lgG a magnetic (IMCO); the were gels were dried. using then each), washed over washed with Autoradiography two were Lanex obtained was screens using at Kodak days. RESULTS SDS-agarose electrophoresis revealed a series of VIIIR:Ag 0.85-12 x 106 daltons (Fig. of fresh human plasma bands of M, spanning 1). The most rapidly migrating VIIIR:Ag had slightly faster 1gM; no smaller forms were detected ment. The multimeric pattern was mobility than in any experi- identical ture, or at incubation the glyoxyl was 37#{176}C.The buffer agarose in which examined. inclusion of 8 M urea in the also had no effect. Studies in which or the fixation step (sodium cyano- at pH patterns: borohydride satisfactory 10) were omitted gave much less only a faint outline of the largest be detected. of the polymers to that of highly in each purified experiment fibrinogen VIIIR:Ag 1gM (0.95 x 106),16 and 1gM polymers x 106). The migration of the smallest band was slightly faster than that of the monomeric obtained from (1.9-4.8 form of patients two different 1gM with Waldenstrom’s globulinemia. The Mr, estimated from made in 12 separate experiments, was 106 (mean ± SEM). As many as VIIIR:Ag bands could be detected in The 0.8-1.2 In addition, poorly samples macro- measurements ± 0.03 eight separate the autoradio0.85 in M, of successive differences x 106. bands resolved x were VIIIR:Ag migrated with an Mr of ca. 8 x I 06 to I 2 x 1 06. The complete multimeric pattern was identified in plasma diluted as much as 1:16 (ca. 2 ng VIIIR). DISCUSSION Although most chromatographic and in seven vitro aggregation temperatures.5 VIIIR:Ag that is a have favored by decalcification4 We report demonstrate and low here studies of plasma a pattern of multimers, all greater than 0.85 x 106 daltons, in material kept at 37#{176}C until mixed with an anionic detergent (SDS), an alkylating agent (iodoacetamide), and urea-agents that would tend to prevent VIIIR:Ag aggregation. Taken together with recent agarose gel filtration studies,’7 our data, and similar results obtained independently is very by Ruggieri unlikely gates might SDS, until the electrophoresis ments in which citrate polymer size. The method in 7 expenianticoagulated ultracentniVIIIR:Ag reports been interpreted as evidence that VIII:Ag circulates in plasma as a molecule that is smaller than 106 daltons. They suggest that the larger forms are the result of in separate experiments in which fresh plasma was anticoagulated with citrate or heparin and kept at 37#{176}C was initiated, or hepanin of 1 hr to 3 fuge studies have suggested that plasma large heterogeneous protein, two recent temperature ml, and the contents (1 liter periods in 2 experiments chamber in which the surface temperature at 10#{176}C (running tap water), room tempera- and for 2 hr at (100,000-500,000 10-30 of pressed, hr. Equivalent with at room anti-VIIIR:AG I mg/mI 6 hr. agitated -70#{176}C for and M sodium cyanoborohydride incubated the incubation. with water M sodium BS gently ‘25l-labeled during period 0.02 subsequently in the plastic rocked a 48-hr carried were in BS containing volume migration in 400 The gels were then rinsed and washed with gels 16-24 VIIIR:Ag by immersion 10, containing temperature stirrer. for temperature held grams. in (1:4:4 was (340,000), 18 x samples were I SDS) 10 x bromphenol gels migration, destained the slab agarose, The at serum polymers could The migration was compared manner. glyoxyl 7.0. in the agarose so that added proteins in horizontal Twenty-microliter Richmond, of with out phosphate, agarose (each 7.0, Laboratories, stained mm The blood in the same out on a temperature-controlled 60-80 measurement from blue I hr. and marker was carried for pH obtained carried M bromphenol cut 4-4.5 5 V/cm mixture 0.01 SDS, I .0 cm slots migrated Rad unmodified spacer. at 37#{176}C. This analyzed was M sodium U-frame to 0. 1 x phosphate, were cast on a polyester a plastic added mg/mI, an to 4 mg/mI of 0.5% and held 0.2 cm gels were with volume was and added prepared phoresis U/ml blood 15 mm), in BS-was held incubation. with ( 10 frozen to analysis, In other studies of conditions that might affect the electrophoretic separations, similar patterns were obtained for fresh plasma samples run in an electro- University 37#{176}C.The 12.5 2-hr the heparin g for tubes electrophoresis containing 0.1% or to mixture 7.0. A 1/25 in BS at 2-3 SDS-agarose citrate) iodoacetamide, pH dissolved anticoagulated was prior freshly by venipunc- by were of plasma incubation mg/mI phosphate, directly approved 37#{176}C(3000 of a dilution of the SDS included mo mixture-approxi- personnel prewarmed at of plasma-or volumes laboratory of 0.5 M sodium polystyrene immediately water all of the agarose Committee, volume The plasma water, ofdistilled at 4#{176}C. from with distilled with in a volume until stored Experimentation citrate funnel volume. heated aliquots accordance Human at a agarose samples, 1057 PLASMA and suspended 2% in agarose-was 20-SO-mI gels on to a beaker, to and IN and that the Zimmerman,’8 large suggest multimers are that it aggre- formed in vitro. Although aggregated forms be dissociated in vitro by the incubation with there is no basis for an artifactual increase in used for protein identification in these From www.bloodjournal.org by guest on August 11, 2017. For personal use only. HOVER 1058 studies, autoradiography antibody glyoxyl with agarose. analytic after separated has several technique. incubation proteins major Protein of labeled immobilized advantages resolution gM is excellent in trophoretic 0.95x10 agarose angles gel that removes SDS to immunoprecipitate approach, protein mixtures of acids factory, but dissolution, body. The lems through from proteins be relied on to prevent antigen especially in the presence of excess glyoxyl agarose method avoids these and permits precise analysis of proteins by their migration in SDS-agarose. note that this method does not rely tation to identify The absence the specific of detectable antiprob- separated It is important on immunoprecipi- proteins. protein trailing to in Fig. 1 demonstrates that there is little interaction of proteins with glyoxyl agarose at neutral pH values, and standard electrophoretic separations can be carried out in the presence or absence of SDS.” Above pH 10, the aldehyde ible groups Schiff proteins and reversible interaction can be converted NPP s l06 Fig. i . SDS-agarose electrophoresis of VlllR:Ag. The migration of marker proteins. 1gM and 1gM polymers. is indicated in the Coomassie Blue-stained gel on the left and their molecular weights are noted. The proteins of other samples. each a i :3 dilution of plasma in BS. were separated by electrophoresis. immobilized in the glyoxyl agarose. washed free of SDS. incubated with “I-labeled rabbit anti-VlllR:Ag. and examined by autoradiography. The samples include citrate anticoagulated plasma from a patient with von Willebrand’s disease (VWD). frozen normal human plasma anticoagulated with citrate (CF) or heparin (HF). fresh citrate (C). or heparin (H) anticoagulated plasma. kept at 37’C until electrophoresis. and pooled (n = 24) normal citrate anticoagulated plasma (NPP). determinants remain readily available after the immobilization. The basic approach has a rather wide potential for application and has been of special antigenic agarose form reversgroups of adjacent is prevented. This of the modified with amino further migration bases H prior formation.’9 An alternative fixation after electrophoresis using and alcohols,20’2’ might be as satis- cannot C .9 x 106 a two-layered the HF 3.8 x l06 2.8 at right F SHAINOFF in as an this method, since there is direct immunologic identification of the relevant bands instead of a second elecstep VWD AND value in the analysis of fibrin complexes.’2 to a covalent ACKNOWLEDGMENT bond by mild reductive amination with sodium cyanoborohydnide.22 Thus, proteins are immobilized in agarose at the point they have reached at the end of the electrophoretic separation. They can then be washed to remove SDS and incubated with identifying reagents. All of the steps are relatively gentle, and We thank Carl Joanne Randall and Carta Blomb#{228}ck kindly and Lena provided of Drs. the manuscript. facilities for Coagulation Research, studies were initiated. The courtesy Wikstr#{246}m for technical for preparing 1gM G#{246}ranHoIm for Dr. Karolinska samples Hoyer F. Hinz, Birger at the Laboratory Institutet, were obtained and Carl assistance Professor where these through the Jr. REFERENCES I . Ratnoff OD, antihemophilic purified Kass L, Lang factor (factor antihemophilic Invest 48:957, 1969 2. Marchesi SL, purification and factor M: of Molecular Thromb 5. human 6. van VllI-von quaternary Invest 8. factor: Fass tateand on related Studies factor VIII. A population the J Clin VIII Davie EW: Isolation VIII (antihemophilic factor). 37:1375, J 12. IM, Holmberg of factor Johnson L, Miller-Anderson VIII in native von AJ: Molecular Willebrand factor 263:612, 1976 Bolhuis PA: factor. Thromb SL, of factor Furlan oligomers. JR: 1978 (abstr) Shainoff JR. Elgee weights of proteins Clin 14. in and Dispersity Res SK: VIII/von of 13:15, human factor bonds Willebrand factor. and the J Clin GJ, of multimers. Bowie J Lab EJW: Clin Porcine Med Willebrand 91 :307, 1978 LW: Vannier Immunochem linking proteins. Complete Beck EA: Studies Biophys immunoelectrophoresis. BN: Acta on Zonal of fibrin factor differences size Biochem 578:164, VIIIbetween 1979 Fed immobilization complexes. studies of Radioimmunoassay Proc procedure Thromb Haemo- antihemophilic of AHF Bryan WP, Campbell a hapten-cellulose factor antigen. J Lab DH: antibody The preparation adsorbent. Int J 1964 GE, reagent, immunoglobulin. 1979 1972 of Stark in NatI amino cryoprecipi- 53:1095, Cascade IV. WE, Putnam VIlI-related human of molecular Immunologic 2:1, Proc M, Dardik VIII). properties of (abstr) 80:822, I 5. Davies 1978 Disulfide factor Mcd Blood analysis 1979 factor from prepared 11. Estimation 13. Hoyer plasma. D: Comparison proteins concentrate. BA, stas 42: 120, 16. Knutson Deykin VIII protein. factor and 1978 DN, factor (AHF, and Paskell structure 62:702, M, factor for chromatographic RB, JA, RB, Weinstein Willebrand J Clin 1979 Willebrand 7. Counts RG, factor distribution Nature Mourik HR: human G, Counts factor plasma. von I 1. Shainoff Harris antihaemophilic 9. of partially of plasma. Gralnick of MJ, Nilsson J, filtration of 1973 14:589, Newman NR, human size Res gel on the purification Separation 10. Perret 248:3926, 4. Seghatchian Studies II. characterization characterization Chem by Shulman Invest 51:2151, 1972 3. Legaz ME, Schmer Biol PD: VIII). GR: studying Acad FW, Science the subunit Sci USA Florent acid Use of dimethylsuberimidate, G, sequence 182:287, 66:65 Paul of the 1973 a cross- structure of oligomeric 1 , I 970 C, mu Shinoda heavy T, Shimizu chain of A: a 1gM From www.bloodjournal.org by guest on August 11, 2017. For personal use only. FACTOR 17. ofhuman 18. structure brand VIII MULTIMERS Bolhuis PA, plasma Ruggeri IN PLASMA Beeser-Visser factor ZM, VIII. NH, I):300a, 1979 (abstr) 19. Converse CA, Papermaster 20. Olden Yamada factor Willebrand’s Molecular weight sodium 1979 dodecyl sulfate-polyacrylamide gels. Anal Biochem 78:481. 1977 in multimeric VIII/von 21. Wille- Burridge mation: disease. Blood 54 K: Changes Identification sodium dodecyl of sulfate in cellular specific gels. Proc glycoproteins after glycoproteins NatI Acad and transfor- antigens Sci USA 73:4457, 1976 DS: immunoelectrophoresis. K, ii: 16:497, Variation ofplasma types ofvon (Suppl by two-dimensional Res TS: expression factor in different Sixma Thromb Zimmerman and antigenic 1059 KM: Direct Membrane Science detection protein analysis 189:469, of antigens 22. 1975 rate in 1971 Borch anion RF, Bernstein as a selective MD, reducing Durst agent. HD: J Am The cyanohydridobo- Chem Soc 93:2897, in From www.bloodjournal.org by guest on August 11, 2017. For personal use only. 1980 55: 1056-1059 Factor VIII-related protein circulates in normal human plasma as high molecular weight multimers LW Hoyer and JR Shainoff Updated information and services can be found at: http://www.bloodjournal.org/content/55/6/1056.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.