THROMOBISIS RESEARCH Vol. 12. pp. 1177-l 194. 0 Pergamon Press Ltd.1978.PrinttdinGreatBrirain.
THE EFFECT OF REDUCING AGENTS ON FACTOR VIII AND OTHER COAGULATION FACTORS Birger Blomblck, Birgit Hessel, Geoffrey Savidge, Lena Wikstrijm and Margareta Blomb;ick From the Blood Coagulation Laboratories, Karolinska Institutet and Karolinska Sjukhuset, Stockholm,Sweden
(Received 8.3.1978: in revised form 24.4.1978. Accepted hy Editor H. Stormorken)
ABSTRACT The effect of various reducing agents on F VIII related activities (F VIII:C, F VIII:RAG and F VIII:RCF), and other coagulation factors was studied. Dithiothreitol (DTT) was found to be the most potent agent tested. Addition of DTT to blood or plasma induced a dose related activity reduction of all tested coagulation factors with the exception of F V. Pronounced reduction in activity of F XII, XI, IX, X and VII was seen, while F II and F VIII were found to be more resistant. F VIII was found to disaggregate in the presence of DTT at concentrations inducing only slight reductions in procoagulant activity (F VIII:C). At these concentrations, F VIII:RCF activity was decreased significantly while F VIII:RAG was only slightly reduced. After reduction the F VIII related activities were found to behave anomalously in ethanol-water mixtures. These activities were not recovered in Cohn's Fraction I, but were detectable in representative amounts in its supernatant. Addition of normal plasma and haemophilia A plasma to reduced normal plasma resulted in a significant shift of F VIII:C from the supernatant of Fraction I to Fraction I itself. Likewise, a significant shift of F VIII:C to the void volume peak was observedon gel filtration of these mixtures. These changes were not observed on the addition of plasma from patients with von Willebrand's disease.
INTRODUCTION Reducing agents such as dithiothreitol have been reported by Austen (1,2) to induce changes in the physical state of F VIII as studied by gel filtration. The author,however,with
regard
changes toa chemicalreductionof
to F VIII activity, could not ascribe
these
theF VIIIprotein,since a similar
phenomenon
FACTOR
1178
was In
observed the
with
present
factors
was
salt
solutions the
study,
effect
with
studied,
(F VIII:C),
X-111 AND
F VIII
of
high
of
reducing
special
related
ionic
Vol.l2,So.4
strength. agents
emphasis
antigen
AGESTS
REDUCISC
on several
on F VIII
(F VIII:RAC)
coagulation
procoagulant
and
activity
Ristocetin
cofactor
(F VIII:RCF).
MATERIALS Blood or
was
0.1
collected
M sodium
and platelet blood
for
Larger
in
polystyrene
oxalate
(9
at
30 and
15 min.
at
room
blood
(450
ml)
of
4825
collected
was
In
in this
Wisconsin, Germany
were
room
to
1 vol.
centrifugation
the
at
in and
and
blood
In
respectively. 65 ml
centrifuged
experiment,
blood
centrifuged
at
at
until
was maintained
of
containing
and
one
poor
volumes
g,
bags
subsequently
plasma
small
140
adenine)
citrate
Platelet
of
3250
collected
and
Y trisodium
anticoagulant).
temperature.
experiment
0.18
by
glucose
tubes,
groups
2)
4)
Lipoic
were
a pH of
Centre,
in
some
and
experiments
for
3.0
15 min.
for
(3),
at
was
Biochemicals,
Ascorbic
acid
form)
37’~
was
rh.,./.'Y.\ /. 10
i
\ . . ‘li
‘1.
/ 20
30
40
10
EFFLUENT
VOL!JME,ml
20
30
40
FIG. 6 Gel filtration of Mixtures of Non-reduced and Reduced Plasma. Chromatography on Sepharose 2B at +4'C. a) DTT-treated normal plasma. b) 90% DTT-treated normal plasma + 10% untreated normal plasma. c) 90% DTT-treated normal plasma + 10% haemophilia A plasma. d) 90% DTT-treated normal plasma + 10% von Willebrand plasma. Fraction volumes: a) and b): 1.6 ml, c) and d): 2.1 ml. The fresh plasmas were dialysed against 0.05 M Tris-0.1 M NaCl buffer pH 7.2 at 4 C prior to chromatography. The shaded areas represent the void volume peaks seen on gel filtration of normal untreated plasma.
The results of these experiments suggest that in the reduction process a factor is lost which is responsible for the precipitation of F VIII:C in Fraction I, and for the appearance of F VIII:C in the void volume on gelfiltration. This factor seems to be deficient in patientswithvonWillebrand'sdisease DISCUSSION The present study confirms Austen's observation (1,2) that, in the presence of DTT, disaggregation of the F VIII procoagulant component (F VIII:C) occurs. However, in fresh blood or plasma, before addition of DTT, the F VIII:C is already present in a more or less disaggregated form. These observations are consistent with the findings of Newman et al (22). On the other hand, Newman et al concluded that aggregate formation was more pronounced when plasma samples were studied at lower temperatures (25OC or 4'C) than at 37'C. In the present investigation, blood samples from the same subject were simultaneously collected, centrifuged and chromatographed at both 37'C and at room
tempera-
ture (20-25'C). F VITI:C profiles from these experiments were essentially the same, suggesting that the temperature difference under the prevailing experi-
~01*12,?x-0.6
FACTOR
VIII
AND
REDTXISG
XGE?;TS
1191
mental conditions exerted little influence on the aggregation phenomenon. On the other hand, this conclusion must be taken with some reservation, since, not only is F VIII:C more rapidly inactivated at 37'C, but it can be conjectured that the inactivation process may be preferential for either the aggregated or disaggregated forms of F VIII:C. The importance of the physicochemical environment should, however, be emphasized as illustrated by the ease with which aggregates are formed on freezing of non-reduced plasma. The disaggregation process does not seem to be dependent upon the cleavage of covalent bonds, since a similar effect can be attained by exposing the FVIII:C to salt solutions of high ionic strength (1,33,?4). The disaggregation caused by DTT is most probably related to a reduction of one or several of the possible protein components in the F VIII complex. Since the residual F VIII activity in plasma of patients with von Willebrand's disease is present in non-aggregated form, it can be assumed that a factor, susceptible to reduction, and directly or indirectly responsible for aggregate formation is deficient in these patients. Further evidence as to this relationship is obtained from the experiments in which mixtures of non-reduced and reduced plasmas were studied. Addition of both normal and haemophilia A plasma to reduced normal plasma resulted in the appearance of F VIII:C in Fraction I on ethanol fractionation, and in the void volume on gel filtration. The addition of plasma from patients with von Willebrand's disease did not produced these effects. The ristocetin cofactor (F VIII:RCF) is absent in plasma of patients with severe von Willebrand's disease. This activity is lost to a large extent during reductionofp1asma.T.t is
thereforepossiblethatthis factorplaysamajorrole
in the
formation of FVIIIaggregates. Inaddition to F VIII:RCF, several other clotting factors, including the Vitamin K dependent factors, are affected by DTT treatment. It is therefore feasable that one or several of these clotting factors may be responsible for the aggregation phenomenon. In any case, wt.ichever factor or factors are involved, it must be assumed that these are altered in some way in von Willebrand's disease. The fact that BaSO
adsorbed 4 plasma behaves in a similar manner as normal plasma on addition to reduced plasma suggests that at least the Vitamin K dependent factors are not important in the aggregation process. The interpretation of F VIII:RAG data from the gel-filtration studies is, in general, consistent with previous reports (23). The antigen profile mainly followed that of the procoagulant activity. Partial separation of these entities could be achieved under certain experimental conditions. These results
1192
FACTOR
suggest
that
before
and
F VIII:C
l-111 AM3
REDCCI?;G
and F VIII:RAG
subsequent
to
represent
aggregation,
are
vo1.12,?io.6
XGEXTS
two
separate
associated
as
entities,
which
a non-covalentlybound
complex. On ethanol were
fractionation
recovered
reduced
plasma,
Cohn’s
form. plasma
also
either
quantitatively It
was
tions
in
resulting
the
present valid.
relatively
varying
the
site
increase
of
in
sequential
In
of
aetiology,
do
processes,
since Red-ox
not
formation.
its
acid
maY be
of
in
platelets
the
is
with
formation. required and
Particular
the
These
in
such
thioredoxin since
this
less
based
of
that one
importance the
component
component
(e.g.
may serve
the
processes. system
as
Biological may be of
thioredoxin
their
has
byred-ox
and alkylated regulation
a lesion
of
intra-
thereby
carriers red-ox
importance. been
in
VIII:RAG/RCF)
F VIII:C) role
may re-
of
the
of
during
and
exchange
(e.g.F
no
rates
reduced in
site
that
aggregates
disulfide in
formed
specimens
different
occurs
at
fact
regardless
to
also
in
rapidly
the
the
is
present
blood
aggregates, related
be
are
on
of at
shown
interpretation
or
formation
be
F VIII prepara-
experiments
to
different
may occur
possible
another
relevance (25,26).
or
represented
The
appear
is
The
however,
Platelets in
that
with
aggregates
thus
purifica-
aggregation
more
weight
blood,
conclusion
directly
of
for
(22).
indicate
which
be
a non-aggregated
cryoprecipitation,
circulation
stream. to
in
non-reduced
subsequent
or
molecular
aggregates
occurred
may,
It
not
rearrangements
potentials
1iPoiC
seem
in
responsible
performed
incubation.
blood
interaction
aggregate
or
process,
On further
I. high
the
the
in
maintain,
I from
fractionation
the
This
on
a factor
In non-
precipitated
and remain
during
FVIII:RAC I.
complex.
Fraction
form
the
formation
processes
disulfide
the
in
reformation
aggregate
red-ox
amounts
of
fraction
do
opinion
the
molecular change
however,
or
parts
another
procedure
our
sampling
various
cing
the
with
Fraction
predominately
an aggregated
ethanol
that
from
venipuncture.
a physiological
plasma.
al
ethanol
aggregate
present
one
Fraction
by
study,
completely
at
in
by Newman et e.g.
of
removal
by
together of
F VIII:C and F VIII:RAG
that
destroyed
F VIII:C
supernatant are
a non-aggregated
recovered
stated
after
may indicate
been
obtained,
artefacts
in
the
form the to
F VIII:C
This have
in
plasma,
relationship
remains
process.
can
presumably
residual
plasma, in
and F VIII:RAG
reduced
a similar The
tion
I, of
reduced
exclusively
F VIII:C
Fraction
purification general,
of
almost
found
influen-
of
the
systems The to
may
like
latter
be present
Vol.12,So.h
FACTOR
\-ITT _1SD REDUCING
AGENTS
1193
ACKNOWLEDGEMENTS The authors wish to thank MrsHelga Messel, BirgittaStrimmeand
SonjaSiiderman
for their help in executing this work, supported by grants from the Swedish Medical Research Council (13X-02475 and 19X-520).
REFERENCES
1.
AUSTEN, D.E. Factor VIII of small molecular weight and its aggregation. Brit. J. Haematol. 27, 89, 1974.
2.
AUSTEN, D.E., CAREY, M. and HOWARD, M.A. Dissociation of Factor VIII related antigen into subunits. Nature 253, 55, 1975.
3.
YORK, L. and BLOMBACK, B. Interaction of fragments of fibrinogen with insolubilized fibrin monomers (activated fibrinogen). Thrombosis Research 8, 607, 1976.
4.
ZIMMERMAN, T.S., RATNOFF, O.D. and LITTELL, A.S. Detection of carriers of classic haemophilia using an immunologic assay for antihemophilic factor (factor VIII). J. Clin. Invu 50, 244, 1971.
5.
LAURELL, C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal.Biochem. 15, 45, 1966.
6.
NEWMAN, J., JOHNSON, A.L., KARPATKIN, M.H. and PUSZKIN, S. Methods for the production of clinically effective intermediate- and high-purity factor VIII concentrates. Brit. J. Haematol. 21, 1, 1971.
7.
SAVIDGE, G.F. and CARLEBJijRK, G. In preparation 1978.
8.
STIBBE, J. and KIRBY, E.P. The influence of haemaccel, fibrinogen and albumin on Ristocetin-induced platelet aggregation. Relevance to the quantitative measurement of the ristocetin cofactor. Thrombosis Research 8, 151, 1976.
9. NYMAN, D.
Personal communication.
10. NO&N, I. Clinical application of a new assay of factor VIII and factor VIII inhibitors. Thrombosis Research 1, 19, 1972. 11. NILSSON,I.M., BLOMBACK, M. and von FRANCKEN, I. On the inherited autosomal hemorrhagic diathesis with antihemophilic globulin (AHG) deficiency and prolonged bleeding time. Acta Med. Stand. 159, 35, 1957. 12. NOREN, I. Specific assay of prothrombin. Amethod using a freeze-dried reagent of intrinsic coagulation factors. Stand. J. Clin. Lab. Invest. 25, 47, 1970. 13.
KAPPELER, R. Das Verhalten von Faktor V in Serum unter normalen und patologischen Bedingungen. Zschr. Klin. Med. 153, 103, 1955.
14.
KOLLER, F., LOELIGER, A. and DUCKERT, F. Experiments on a new Clotting factor, Factor VII. Acta Haematol. 6, 1, 1951.
1194
FACTOR
VIII
A\.LrD REDTICIXG
AGENTS
L-01.12,X0.6
15. NILSSON, I.M., BLOMBACK, M., THI&N, A. and von FRANCKEN, I. Carriers of hemofilia A. Acta Med. Stand. 165, 357, 1959. 16. BACHMANN, F., DUCKERT, F. and KOLLER, F. The Stuart-Prover factor assay and its clinical significance. Thromb. Diathesis Haemorrh. 2, 24, 1958. 17. VELTKAMP, J.J., DRION, E.F. and LOELIGER, E.A. Detection of the carrier state in hereditary coagulation disorders. II. Thromb.Diathesis Haemorrh. 19, 403, 1968. 18. COHN. E.J., STRONG, L.E., HUGHES, W.L. Jr., MULFORD. D.J.. ASHWORTH, J-N., MELIN, M. and TAYLOR, H.L. Preparation and properties of serum and plasma proteins. J. Amer. Chem. Sot. 68, 459, 1946. 19. BLOM8ACK, M. Purification of antihemophilic globulin. I. Some studies on the itability of the antihemophilic globulin activity in fraction I-O and a method for its partial separation from fibrinogen. Arkiv Kemi 12. 387,1958, 20.
TANGEN, 0. and BERMAN, H.J. Gel filtration of Blood Platelets: A methodological report in platelet function and thrombosis. A review of Methods. (Eds. P.M. Mannucci and S. Gorini.) Plenum Press, New York, London 1972, p 235.
21.
LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with the Folin phenol reagent. 3. Biol. Chem. 193, 265, 1951.
22.
NEWMAN, J., HARRIS, R.B. and JOHNSON, A.J. Molecular weights of antihaemophilic factor and von Willebrand factor proteins in human plasma. Nature, 263, 612, 1976.
23.
RICK, M.E. and HOYER, L.W. Immunologic studies of antihemophilic factor (AHF, F VIII). V. Immunologic properties of AHF subunits produced by salt dissociation. Blood 42, 737, 1973.
24.
POON, M.C. and RATNOFF, O.D. Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds. Blood 48, 87, 1976.
25.
HOLMGREN, A. The enzymatic functions and folding of thioredoxin in relation to its tertiary structure. Proceedings of the Tenth FEBS Meeting, p. 35, FEBS, 1975.
26.
BLOMBXCK, B., BLOMBACK, M., FINKBEINER, W., HOLMGPEN, A,, KOWALSKA-LOTH, B. and OLOVSON, G. Enzymatic reduction of disulfide bonds in fibrin-ogen by the thioredoxin system. I. Identification of reduced bonds and studies on reoxidation process. Thrombosis Research 4, 55, 1974.
THE EFFECT OF REDUCING AGENTS ON FACTOR VIII AND OTHER COAGULATION FACTORS Birger Blomblck, Birgit Hessel, Geoffrey Savidge, Lena Wikstrijm and Margareta Blomb;ick From the Blood Coagulation Laboratories, Karolinska Institutet and Karolinska Sjukhuset, Stockholm,Sweden
(Received 8.3.1978: in revised form 24.4.1978. Accepted hy Editor H. Stormorken)
ABSTRACT The effect of various reducing agents on F VIII related activities (F VIII:C, F VIII:RAG and F VIII:RCF), and other coagulation factors was studied. Dithiothreitol (DTT) was found to be the most potent agent tested. Addition of DTT to blood or plasma induced a dose related activity reduction of all tested coagulation factors with the exception of F V. Pronounced reduction in activity of F XII, XI, IX, X and VII was seen, while F II and F VIII were found to be more resistant. F VIII was found to disaggregate in the presence of DTT at concentrations inducing only slight reductions in procoagulant activity (F VIII:C). At these concentrations, F VIII:RCF activity was decreased significantly while F VIII:RAG was only slightly reduced. After reduction the F VIII related activities were found to behave anomalously in ethanol-water mixtures. These activities were not recovered in Cohn's Fraction I, but were detectable in representative amounts in its supernatant. Addition of normal plasma and haemophilia A plasma to reduced normal plasma resulted in a significant shift of F VIII:C from the supernatant of Fraction I to Fraction I itself. Likewise, a significant shift of F VIII:C to the void volume peak was observedon gel filtration of these mixtures. These changes were not observed on the addition of plasma from patients with von Willebrand's disease.
INTRODUCTION Reducing agents such as dithiothreitol have been reported by Austen (1,2) to induce changes in the physical state of F VIII as studied by gel filtration. The author,however,with
regard
changes toa chemicalreductionof
to F VIII activity, could not ascribe
these
theF VIIIprotein,since a similar
phenomenon
FACTOR
1178
was In
observed the
with
present
factors
was
salt
solutions the
study,
effect
with
studied,
(F VIII:C),
X-111 AND
F VIII
of
high
of
reducing
special
related
ionic
Vol.l2,So.4
strength. agents
emphasis
antigen
AGESTS
REDUCISC
on several
on F VIII
(F VIII:RAC)
coagulation
procoagulant
and
activity
Ristocetin
cofactor
(F VIII:RCF).
MATERIALS Blood or
was
0.1
collected
M sodium
and platelet blood
for
Larger
in
polystyrene
oxalate
(9
at
30 and
15 min.
at
room
blood
(450
ml)
of
4825
collected
was
In
in this
Wisconsin, Germany
were
room
to
1 vol.
centrifugation
the
at
in and
and
blood
In
respectively. 65 ml
centrifuged
experiment,
blood
centrifuged
at
at
until
was maintained
of
containing
and
one
poor
volumes
g,
bags
subsequently
plasma
small
140
adenine)
citrate
Platelet
of
3250
collected
and
Y trisodium
anticoagulant).
temperature.
experiment
0.18
by
glucose
tubes,
groups
2)
4)
Lipoic
were
a pH of
Centre,
in
some
and
experiments
for
3.0
15 min.
for
(3),
at
was
Biochemicals,
Ascorbic
acid
form)
37’~
was
rh.,./.'Y.\ /. 10
i
\ . . ‘li
‘1.
/ 20
30
40
10
EFFLUENT
VOL!JME,ml
20
30
40
FIG. 6 Gel filtration of Mixtures of Non-reduced and Reduced Plasma. Chromatography on Sepharose 2B at +4'C. a) DTT-treated normal plasma. b) 90% DTT-treated normal plasma + 10% untreated normal plasma. c) 90% DTT-treated normal plasma + 10% haemophilia A plasma. d) 90% DTT-treated normal plasma + 10% von Willebrand plasma. Fraction volumes: a) and b): 1.6 ml, c) and d): 2.1 ml. The fresh plasmas were dialysed against 0.05 M Tris-0.1 M NaCl buffer pH 7.2 at 4 C prior to chromatography. The shaded areas represent the void volume peaks seen on gel filtration of normal untreated plasma.
The results of these experiments suggest that in the reduction process a factor is lost which is responsible for the precipitation of F VIII:C in Fraction I, and for the appearance of F VIII:C in the void volume on gelfiltration. This factor seems to be deficient in patientswithvonWillebrand'sdisease DISCUSSION The present study confirms Austen's observation (1,2) that, in the presence of DTT, disaggregation of the F VIII procoagulant component (F VIII:C) occurs. However, in fresh blood or plasma, before addition of DTT, the F VIII:C is already present in a more or less disaggregated form. These observations are consistent with the findings of Newman et al (22). On the other hand, Newman et al concluded that aggregate formation was more pronounced when plasma samples were studied at lower temperatures (25OC or 4'C) than at 37'C. In the present investigation, blood samples from the same subject were simultaneously collected, centrifuged and chromatographed at both 37'C and at room
tempera-
ture (20-25'C). F VITI:C profiles from these experiments were essentially the same, suggesting that the temperature difference under the prevailing experi-
~01*12,?x-0.6
FACTOR
VIII
AND
REDTXISG
XGE?;TS
1191
mental conditions exerted little influence on the aggregation phenomenon. On the other hand, this conclusion must be taken with some reservation, since, not only is F VIII:C more rapidly inactivated at 37'C, but it can be conjectured that the inactivation process may be preferential for either the aggregated or disaggregated forms of F VIII:C. The importance of the physicochemical environment should, however, be emphasized as illustrated by the ease with which aggregates are formed on freezing of non-reduced plasma. The disaggregation process does not seem to be dependent upon the cleavage of covalent bonds, since a similar effect can be attained by exposing the FVIII:C to salt solutions of high ionic strength (1,33,?4). The disaggregation caused by DTT is most probably related to a reduction of one or several of the possible protein components in the F VIII complex. Since the residual F VIII activity in plasma of patients with von Willebrand's disease is present in non-aggregated form, it can be assumed that a factor, susceptible to reduction, and directly or indirectly responsible for aggregate formation is deficient in these patients. Further evidence as to this relationship is obtained from the experiments in which mixtures of non-reduced and reduced plasmas were studied. Addition of both normal and haemophilia A plasma to reduced normal plasma resulted in the appearance of F VIII:C in Fraction I on ethanol fractionation, and in the void volume on gel filtration. The addition of plasma from patients with von Willebrand's disease did not produced these effects. The ristocetin cofactor (F VIII:RCF) is absent in plasma of patients with severe von Willebrand's disease. This activity is lost to a large extent during reductionofp1asma.T.t is
thereforepossiblethatthis factorplaysamajorrole
in the
formation of FVIIIaggregates. Inaddition to F VIII:RCF, several other clotting factors, including the Vitamin K dependent factors, are affected by DTT treatment. It is therefore feasable that one or several of these clotting factors may be responsible for the aggregation phenomenon. In any case, wt.ichever factor or factors are involved, it must be assumed that these are altered in some way in von Willebrand's disease. The fact that BaSO
adsorbed 4 plasma behaves in a similar manner as normal plasma on addition to reduced plasma suggests that at least the Vitamin K dependent factors are not important in the aggregation process. The interpretation of F VIII:RAG data from the gel-filtration studies is, in general, consistent with previous reports (23). The antigen profile mainly followed that of the procoagulant activity. Partial separation of these entities could be achieved under certain experimental conditions. These results
1192
FACTOR
suggest
that
before
and
F VIII:C
l-111 AM3
REDCCI?;G
and F VIII:RAG
subsequent
to
represent
aggregation,
are
vo1.12,?io.6
XGEXTS
two
separate
associated
as
entities,
which
a non-covalentlybound
complex. On ethanol were
fractionation
recovered
reduced
plasma,
Cohn’s
form. plasma
also
either
quantitatively It
was
tions
in
resulting
the
present valid.
relatively
varying
the
site
increase
of
in
sequential
In
of
aetiology,
do
processes,
since Red-ox
not
formation.
its
acid
maY be
of
in
platelets
the
is
with
formation. required and
Particular
the
These
in
such
thioredoxin since
this
less
based
of
that one
importance the
component
component
(e.g.
may serve
the
processes. system
as
Biological may be of
thioredoxin
their
has
byred-ox
and alkylated regulation
a lesion
of
intra-
thereby
carriers red-ox
importance. been
in
VIII:RAG/RCF)
F VIII:C) role
may re-
of
the
of
during
and
exchange
(e.g.F
no
rates
reduced in
site
that
aggregates
disulfide in
formed
specimens
different
occurs
at
fact
regardless
to
also
in
rapidly
the
the
is
present
blood
aggregates, related
be
are
on
of at
shown
interpretation
or
formation
be
F VIII prepara-
experiments
to
different
may occur
possible
another
relevance (25,26).
or
represented
The
appear
is
The
however,
Platelets in
that
with
aggregates
thus
purifica-
aggregation
more
weight
blood,
conclusion
directly
of
for
(22).
indicate
which
be
a non-aggregated
cryoprecipitation,
circulation
stream. to
in
non-reduced
subsequent
or
molecular
aggregates
occurred
may,
It
not
rearrangements
potentials
1iPoiC
seem
in
responsible
performed
incubation.
blood
interaction
aggregate
or
process,
On further
I. high
the
the
in
maintain,
I from
fractionation
the
This
on
a factor
In non-
precipitated
and remain
during
FVIII:RAC I.
complex.
Fraction
form
the
formation
processes
disulfide
the
in
reformation
aggregate
red-ox
amounts
of
fraction
do
opinion
the
molecular change
however,
or
parts
another
procedure
our
sampling
various
cing
the
with
Fraction
predominately
an aggregated
ethanol
that
from
venipuncture.
a physiological
plasma.
al
ethanol
aggregate
present
one
Fraction
by
study,
completely
at
in
by Newman et e.g.
of
removal
by
together of
F VIII:C and F VIII:RAG
that
destroyed
F VIII:C
supernatant are
a non-aggregated
recovered
stated
after
may indicate
been
obtained,
artefacts
in
the
form the to
F VIII:C
This have
in
plasma,
relationship
remains
process.
can
presumably
residual
plasma, in
and F VIII:RAG
reduced
a similar The
tion
I, of
reduced
exclusively
F VIII:C
Fraction
purification general,
of
almost
found
influen-
of
the
systems The to
may
like
latter
be present
Vol.12,So.h
FACTOR
\-ITT _1SD REDUCING
AGENTS
1193
ACKNOWLEDGEMENTS The authors wish to thank MrsHelga Messel, BirgittaStrimmeand
SonjaSiiderman
for their help in executing this work, supported by grants from the Swedish Medical Research Council (13X-02475 and 19X-520).
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2.
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L-01.12,X0.6
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