Factor VIII Recombination After Dissociation by CaC12

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)
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
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
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
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.
Froc. Nat. Acad. Sci. USA 7o
Factor VIII Recombination
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%
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
Dissolve, tris-buffered
saline, 230
Chromatography (Agarose 15M)
Adjust to 0. 25M Ca2'
Chromatography (Agarose 15M)
< ID
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.
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.
Proc. Nat. Acad. Sci. USA 70
15m, no AHF activity was found in any of the column fractions.
Apparent molecular weight of small active fragment
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
Recombination experiments
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
were collected. AHF concentration (O-O); A2so (A
A,,. (e-)-
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.
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
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
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
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,
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,
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,
8. Graham, J. B., Buckwalter, J. A., Hartley, L. J. & Brinkhous, K. M. (1949) "Canine hemophilia," J. Exp. Med. 90,
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.