Proc. Nat. Acad. SCi. USA Vol. 70, No. 8, pp. 2326-2329, August 1973 Factor VIII Recombination After Dissociation by CaC12 (canine plasma fractions/antihemophilic factor/gel chromatography/molecular weight) HERBERT A. COOPER, THOMAS R. GRIGGS, AND ROBERT H. WAGNER Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514 Communicated by K. M. Brinkhous, May 16, 1973 Factor VIII is a large protein molecule of ABSTRACT molecular weight 2,000,000 or larger that elutes in the void volume on agarose gel chromatography. It has been shown previously that high concentrations of alkali halides and, more specifically, 0.25 M Ca2+ dissociate the molecule into a large carrier protein and a small fragment that retains the factor VIII activity. Factor VIII was prepared from normal canine plasma collected in sodium oxalate and heparin and adsorbed with BaSO4. Results with Ca2+ dissociation were the same as those obtained with fractions prepared from canine plasma collected in sodium citrate. The addition of 0.1 M e-aminocaproic acid in the dissociation step had no effect. Fractionation of canine hemophilic plasma produced preparations without activity, and no activity was found when these inert preparations were dissociated with Ca2+. These results indicate that the Ca2+ dissociation is a true dissociation and not caused by enzymatic degradation by plasmin, thrombin, or activated factors VII, IX, or X. The apparent molecular weight of the small active fragment of factor VIII determined by gel chromatography was about 100,000. Finally, when the large carrier protein and the small active fragment of factor VIII were separated by gel chromatography, mixed, and dialyzed free of Ca2 , they recombined to form a large active molecule that appeared in the void volume on agarose gel chromatography. Antihemophilic factor (AHF, factor VIII) is a glycoprotein which when highly purified has a molecular weight of about 2 X 106, as determined by ultracentrifugal techniques (1-4) and gel chromatographic studies (2-5). Its large size is also confirmed by its inability to enter standard polyacrylamide gels and its slow migration in agar during immunodiffusion. AHF is present in plasma at a concentration of <10 ,g/ml and has been purified 3,000-10,000 times by various procedures. Beginning with the work of Thelin and Wagner (1) in the early 1960s, evidence accumulated suggesting that under suitable conditions this macromolecule could be dissociated, with release of an active small-molecular-weight fragment(s). Weiss and coworkers (6) have shown that at high ionic strength, subunits of AHF can be separated by agarose gel chromatography. Most recently, Owen and Wagner (7) achieved dissociation of canine AHF with alkali halides, detergents, and most specifically 0.25 M Ca2+. Their data suggested that a small active AHF fragment(s) binds to a large carrier protein by both electrostatic and hydrophobic interactions. Rechromatography of the isolated small fragment after removal of calcium showed no reaggregation. The reversibility of the AHF dissociation was tested by incubating canine AHF in 0.25 M Ca2+, removing the Ca2+ by dialysis, and chromatographing the material on agarose 15m. The AHF activity was again eluted as a single peak in the void volume, suggesting that partially purified preparations are capable of recombination. This paper presents further studies on the active smallmolecular-weight fragment(s) of canine AHF. The previous studies of Owen and Wagner (7) were performed with citrated plasma. In this paper, citrated plasma was used as well as plasma collected in oxalate and heparin followed by BaSO4 adsorption. The calcium-dissociation step was also done in the presence of 0.1 M eaminocaproic acid, in order to minimize possible effects of plasmin degradation. The molecular weight of the small active fragment was estimated by the use of a calibrated Sephadex G-150 column. Finally, conditions were found for successful recombination of the small active fragment of AHF from normal canine plasma with the large inactive canine carrier protein. Remova of calcium fractions was essential before recombination could occur MATERIALS AND METHODS Chemicals were reagent grade unless otherwise specified. Water was deionized and then glass distilled. All blood was collected in silicone-treated glassware or plastic. Silicone (Siliclad, Clay Adams) treatment of all glassware including columns was performed according to the manufacturer's instructions. Blood Collection. Unanesthetized, healthy, fasting, normal, or AHF-deficient (8) dogs (30-40 kg), conditioned to venipuncture, were used. AHF-deficient animals had not been transfused for at least 6 weeks before the blood was drawn. Blood was collected by jugular venipuncture with a 16-gauge needle and a two-syringe technique, into either 1/8 volume of 0.11 M trisodium citrate or 1/9 volume of 0.1 M sodium oxalate containing 9 units of heparin (Sodium Heparin, Lilly) per ml of oxalate. The latter blood was centrifuged at 3000 X g for 30 min at 40, the plasma was removed by aspiration, 10 g of BaSO4 per 100 ml of plasma was added, and the mixture was adsorbed for 30 min at 4V. After centrifugation at 3000 X g for 30 min, the plasma was removed by decantation and further clarified by centrifugation at 10,000 X g for 30 min at 4°. The plasma was decanted, adjusted to pH 7.35 with 0.1 N acetic acid, and used within 15 min. The citrated blood was processed as described (9). Preparation of Dialysis Casing. Dialysis casing (Fisher Scientific Co.) was treated for 30 min at 600 with a solution of 0.01 M disodium EDTA and 0.2 M Na2CO3. The casing Abbreviation: AHF, antihemophidic factor. 2326 Froc. Nat. Acad. Sci. USA 7o Factor VIII Recombination (197s) was then washed in water, 70% ethanol, and again thoroughly with water. Buffers. Tris-buffered saline was 0.05 M Tris HCl-0.15 M NaCl (pH 7.35). A Tris-calcium buffer was used routinely for calcium dissociation and contained 0.05 M Tris HCl0.01 M NaCl-0.25 M CaCl2 (pH 7.35); for some experiments 0.10 M e-aminocaproic acid was also added. All buffers were freshly prepared for each gel chromatographic experiment, filtered through Millipore membrane filters, degassed under reduced pressure, and equilibrated to the appropriate temperature. Gel Chromatographic Procedures. Agarose 15m (Bio-Gel A-15m, 4% agarose beads, 200-40 mesh, Bio-Rad Laboratories) was used in all instances except for the molecular weight experiments in which Sephadex G-150 (Pharmacia, 40-120 um) was used. Upward flow was maintained with a peristaltic pump. Column void volumes were determined with Blue Dextran 2000 (Pharmacia Fine Chemicals); the Sephadex G-150 column was calibrated with nonenzymatic markers (Schwartz-Mann). The effluent fractions from each column were monitored at 278 nm with an LKB Uvicord ultraviolet analyzer and recorder and then measured at 280 and 220 nm with a Beckman DU-2 Spectrophotometer. Clotting Assays. AHF activity was assayed by a modification (10) of the one-stage method of Langdell et al. (11). One unit of canine AHF is defined as that amount present in 1 ml of normal canine plasma. When the test sample contained 0.25 M Ca2+, a further modification of the assay method was used (7). Preparation and Dissociation of AHF Fractions. AHF was prepared from canine plasma by the method of Owen and Wagner (9). 400 or 500 ml of fresh plasma were concentrated to 30-40% of the original volume by dialysis against 40% PREPARATION AND DISSOCIATION OF AHF PLASMA + PLASMA + SODIUM OXALATE & HEPARIN S DIUM CITRATE BaSO4 10 g/1 00 ml for 30 min 3000 x g for 30 mi, 40 pH 1. 02N Acetic Acid Concentrate versus 40% polyethylene glycol Collection of heavy phase, D PRECIPITATE, Si Dissolve, tris-buffered saline, 230 Chromatography (Agarose 15M) POOLED VOID VOLUME FRACTIONS, S2 Adjust to 0. 25M Ca2' Chromatography (Agarose 15M) INACTIVE VOID VOLUME FRACTIONS, S3 2.0 E . 1.5- < ID 3 -2 m Li. = 0.5 0 30 120 150 60 90 EFFLUENT VOLUME (ml) 180 210 FIG. 2. Dissociation of an AHF preparation at 40 in 0.25 M CaCl2. Preparation S2 (7 ml) was used. Bed dimensions were 2.5 X 35 cm, flow rate was 30 mi/hr (6.1 ml cm-' hr'1), and 3.0-ml fractions were collected. AHF concentration (QOO.); Anso (A- - -A); A22o (--- -) polyethylene glycol having an average molecular weight of 20,000 (Fisher Scientific Co.) or by use of a hollow fiber concentrator (Amicon, model DC-2). The concentrated plasma was incubated for 1 hr at 00. The heavy phase that formed was collected by centrifugation and dissolved in Tris-buffered saline at 230 to about 1/40 of the initial plasma volume. The solution, which contained over 80% of the original AHF activity, was chromatographed on a 2.5 X 100-cm agarose column. Fractions of 3.0 ml were collected and assayed immediately for AHF activity. Void volume fractions, S2 (Fig. 1), containing the AHF activity were brought to 0.25 M Ca2+, applied to a 2.5 X 40-cm column that had been equilibrated with Tris-calcium buffer, and eluted with the same buffer at 4°. The same dissociation step was also done with the Tris-calcium buffer containing 0.10 M e-aminocaproic acid. Molecular Weight Estimation. Fractions, S4, from the Ca2+dissociation step were pooled, concentrated, and chromatographed on a calibrated Sephadex G-150 column (1.0 X 30 cm, Pharmacia). The effluent fractions from this column were assayed for AHF activity, and the apparent molecular weight was calculated on the basis of the peak effluent fraction. These experiments were done with and without Ca2+ at 4 and 230. PRECIPITATE ADSORBED PLASMA SUPERNATANT 2327 SMALL ACTIVE PIECE FRACTIONS, S4 FIG. 1. Flow diagram for preparation and dissociation of canine AHF. Recombination Experiments. In preliminary studies, 1.0-ml aliquots from each 3.0.ml fraction eluted from the calcium dissociation column were pooled. The pooled fractions were dialyzed against Tris-buffered saline at 40 for 3.5 hr, concentrated by pervaporation, and chromatographed as in Fig. 3. In subsequent experiments only the peak fractions of S3 containing the inactive material from the void volume were mixed with the peak fractions of S4 containing the Ca2+-dissociated small active fragment. Samples (2-4 ml) of the mixture were dialyzed for various periods of time against Tris-buffered saline at 40 and chromatogaphed as above. In both instances the fractions after chromatography were assayed for AHF activity, and UV absorbance was monitored. The steps in the procedure followed one another without interruption. The time elapsed from the original venipuncture until the molecular weight or recombination experiments was 36-48 hr. Medical Sciences: Cooper et al. 2328 Proc. Nat. Acad. Sci. USA 70 (1973) 15m, no AHF activity was found in any of the column fractions. Apparent molecular weight of small active fragment 0.4 ~2.0- | 8 |k| Ib ARCta cm Fractions containing the small active fragment were concentrated and chromatographed on Sephadex G-150 at 4 and 230 both in the presence and in the absence of 0.25 M Ca2+. The small active fragment obtained from canine plasma had an apparent molecular weight of about 100,000 by this method*. Neither variable had any effect on the apparent molecular weight. Recombination experiments 02 1.0 0.5- 0 . 30 60 0.1 90 120 150 EFFLUENT VOLUME (ml) FIG. 3. Recombination experiment at 230. 3.4 ml of a Calfree mixture of equal volumes of S3 and S4 (7 units of AHF) were applied to the column. Bed dimensions were 1.5 X 25 cm, flow rate was 15 mi/hr (8.5 ml cm-' hr-'), and 1-ml fractions A); were collected. AHF concentration (O-O); A2so (A A,,. (e-)- RESULTS Effect of heparin and BaSO4 adsorption BaSO4 adsorption, which effectively removes factors II, VII, IX, and X from the plasma, and heparin were used in the preparation of the starting plasma. This was an effort to minimize any possible effects that these factors, in an activated state, might have on the formation of the small active fragment. The treated plasma was then processed according to Fig. 1. The AHF activity appeared in the void volume fractions, S2, of an agarose 15m column indicating a molecule of large molecular weight. When these fractions were pooled, brought to 0.25 M in Ca2+, and chromatographed on an agarose 15m column' equilibrated with Tris-calcium buffer, essentially complete dissociation of the large-molecular-weight complex was achieved (Fig. 2). The bulk of the protein eluted in the void volume, S3, but contained only a trace of AHF activity. The small active fragment(s), S3, was well separated from the void volume and contained little protein. Effect of e-aminocaproic acid The Ca2+ dissociation step was also done in the presence of 0.1 M e-aminocaproic acid, which inhibits plasminogen activation and the action of plasmin (12). Thus, the possibility of the proteolytic effect of plasmin on the appearance of the small active piece was minimized. The results were the same as shown in Fig. 2, namely, the appearance of only a trace of activity in the void volume, with the bulk of the activity eluting as a small active fragment. Preparations from hemophilia canine plasma As an additional control of the possibility that the small active fragment represents activity other than AHF, we prepared AHF-deficient canine plasma as described in Fig. 1. Void volume fractions, S2, contained the usual protein peak, but no AHF activity. When these fractions were pooled, brought to 0.25 M Ca2+, and chromatographed on agarose In the preliminary experiments, with aliquots from all the eluted fractions, partial recombination was achieved as evidenced by the reappearance of AHF activity in the void volume fractions after agarose 15m chromatography. In the subsequent experiments in which only the peak fractions were used, 90% of the activity was again found in the void volume fractions (Fig. 3). Results obtained with shorter dialysis periods indicated that the degree of recombination was dependent upon the efficiency of Ca2+ removal. Peak fractions of S3 and S4 samples were also made free of calcium before they were mixed. The calcium-free mixture was then chromatographed immediately and the results obtained were the same as those shown in Fig. 3. DISCUSSION AHF activity from canine plasma is associated with a relatively small molecule that, in plasma and partially purified preparations, is associated with a particle of very high molecular weight (7). Under suitable conditions, the complex can be dissociated, producing a small active molecule with no tendency to reaggregate. Investigators have used polyacrylamide gel electrophoresis in the presence. of sodium dodecyl sulfate to study preparations of highly purified human and bovine7;AHF. In each instance the large size of the AHF molecule has prevented its entrance into 3.5-10% gels even in the presence of 8 M urea (4). When sulfhydryl reducing agents such as 2-mercaptoethanol or dithioerythritol were used, major subunit bands of 22,000-240,000 were obtained with and without the appearance of minor bands. Schmer and coworkers (4) observed a single band with bovine preparations and suggested that AHF is made up of subunits of very similar or perhaps identical size held together by disulfide bonds. Marchesi et al. (13), using human preparations, found a major subunit band of 240,000 with four minor bands of 80,000-160,000 molecular weight. They suggested that the large size of the AHF molecule represents aggregation of monomers when other proteins that usually complex with the monomer in plasma are removed. McKee (14) reported for human preparations a major band of 260,000 molecuar weight and seven minor bands. Hershgold (15) found his purified human AHF material to have a subunit size of 22,000 on sodium dodecyl sulfate gels and 30,000 by fingerprinting of tryptic peptides. Under the denaturing conditions used by all of these investigators, one has lost the ability to relate these major or minor subunits to AHF activity. Three observations suggest that AHF is not solely a re* 105,000 ± 5,000 at 40 in 0.25 M Ca2+; 93,000 A= 5,000 at 230 in 0.25 M Ca2+; 103,000 i 3,000 at 230 in Trisbuffered saline. Proc. Nat. Acad. Sci. USA 70 (1973) peating monomeric subunit and support the concept of -a carrier molecule and a small active fragment. First, rechromatography of the small active fragment in the absence of calcium does not indicate any increase in size and hence tendency to repolymerize (7). Second, the molecular weight estimations reported here do not change with temperature or with removal of the dissociating agent*. Third, reassociation of the dissociated piece with the carrier protein molecule is possible after removal of the dissociating agent (Fig. 3). It would appear that the major subunit reported by other investigators may represent the major subunit of the large carrier molecule with the active small fragment having been lost, obscured by the major band, or represented by one of the minor bands. There is no difference between the results obtained with citrated plasma and those with oxalated plasma collected in the presence of heparin and adsorbed with BaSO4. Addition of 0.1 M e-aminocaproic acid does not affect the calcium dissociation step, thus suggesting that fibrinolytic activity does not play a role in the appearance of the small-molecularweight material possessing AHF activity. More conclusive evidence that the small-molecular-weight activity is actually AHF is the fact that AHF-deficient plasma, when processed in the same manner as normal plasma, results in no activity even after dissociation in 0.25 M Ca2 . In addition to being a negative control, these findings also show that if there is in canine hemophilia plasma a complex of a large-molecularweight carrier and a small piece with potential activity, the activity is not unmasked by calcium dissociation. These findings lend further support to the hypothesis that the release of the small active fragment(s) under conditions of high calcium concentration is a consequence of alteration of the electrostatic and hydrophobic bonding between the small fragment and a large carrier molecule, rather than of proteolytic action of thrombin or plasmin, or activation of the pro- thrombin complex. Using a Sephadex G-150 column, we determined the apparent molecular weight of the small active fragment(s) to be about 100,000. Earlier estimations of the molecular weight of the small active fragment as 25,000 (7) probably represented the limitations of the particular gel used in performing the estimation. Another possible explanation could be retention of the small fragment in the column. The lack of aggregation of the small fragment upon the removal of Ca2+ as previously described was confirmed in our studies by obtaining the same molecular weight both in the presence and absence of 0.25 IV Ca2+. Temperature also had no effect on the molecular weight since similar measurements were obtained at 4 and 230. The ability to reconstruct the large-molecular-weight complex from its dissociated fragments was possible only after adequate calcium removal. Essentially complete recombination to form the large AHF molecule was obtained when the peak fractions of S3 and S4 were mixed. No other fractions were necessary for recombination. In most of the recombination experiments, the fractions were mixed and then dialyzed overnight to remove Ca2+. However, if the calcium was re- Factor VIII Recombination 2329 moved first and then the fractions were mixed and chromatographed immediately, the results were the same, indicating that the recombination is quite rapid. Successful dissociation of preparations of normal canine factor VIII and recombination of the fragments suggest further experiments. The use of normal plasma preparations from other species as well as preparations from canine hemophilic plasma and human hemophilic and von Willebrand's disease plasma is our immediate goal. This work was supported by Research Grants HL-06350 and HL-01648 from the National Heart and Lung Institute. H.A.C. was supported by Training Grant HL-05652 from the National Heart and Lung Institute and T.R.G. was supported by Training Grant GM-00092 from the National Institute of General Medical Sciences. 1. Thelin, G. M. & Wagner, R. H. (1961) "Sedimentation of plasma antihemophilic factor," Arch. Biochem. Biophys. 95, 70-76. 2. Ratnoff, 0. D., Kass, L. & Lang, P. D. (1969) "Studies on the purification of antihemophilic factor (Factor VIII). II. Separation of partially purified antihemophilic factor by gel filtration of plasma," J. Clin. Invest. 48, 957-962. 3. Hershgold, E. J., Davison, A. M. & Janszen, M. E. (1971) "Isolation and some chemical characterization of human factor VIII (antihemophilic factor)," J. Lab. Clin. Med. 77, 185-205. 4. Schmer, G., Kirby, E. P., Teller, D. C. & Davie, E. W. (1972) "The isolation and characterization of bovine factor VIII (antihemophilic factor)," J. Biol. Chem. 247, 25122521. 5. Van Mourik, J. A. & Mochtar, I. A. (1970) "Purification of human antihemophilic factor (factor VIII) by gel chromatography," Biochim. Biophys. Acta 221, 677-679. 6. Weiss, H. J., Phillips, L. L., & Rosner, W. (1972) "Separation of subunits of antihemophilic factor (AHF) by agarose gel chromatography," Thromb. Diath. Haemorrh. 27, 212-219. 7. Owen, W. G. & Wagner, R. H. (1972) "Antihemophilic factor: Separation of an active fragment following dissociation by salts or detergents," Thromb. Diath. Haemorrh. 27, 502-515. 8. Graham, J. B., Buckwalter, J. A., Hartley, L. J. & Brinkhous, K. M. (1949) "Canine hemophilia," J. Exp. Med. 90, 97-111. 9. Owen, W. G. & Wagner, R. H. (1972) "Antihemophilic factor. A new method of purification," Thromb. Res. 1, 7187. 10. Margolis, J. (1958) "The kaolin clotting time. A rapid onestage method for diagnosis of coagulation defects," J. Clin. Pathol. 11, 406-409. 11. Langdell, R. D., Wagner, R. H. & Brinkhous, K. M. (1953) "Effect of antihemophilic factor on one-stage clotting tests," J. Lab. Clin. Med. 41, 637-647. 12. Alkjaersig, N., Fletcher, A. P. & Sherry, S. (1959) "eAminocaproic acid: An inhibitor of plasminogen activation," J. Biol. Chem. 234, 832-837. 13. Marchesi, S. L., Shulman, N. R. & Gralnick, H. R. (1972) "Studies on the purification and characterization of human factor VIII," J. Clin. Invest. 51, 2151-2161. 14. McKee, P. A. (1970) "Purification and electrophoretic analysis of human antihemophilic factor (factor VIII)," Fed. Proc. 30, 540. 15. Hershgold, E. J. (1971) "The subunit structure of human factor VIII (antihemophilic factor)," Fed. Proc. 29, 647.