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Separation of human plasma fibrin stabilizing factor (factor XIII) from fibrinogen by fibrinogen polymer formation
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 678-684
January 29, 1986
SEPARATION OF HUMAN PLASMA FIBRIN STABIEIZING FACTOR (FACTOR X I I I ) FROM FIBRINOGEN BY FIBRINOGEN POLYMER FORMATION Carter M. Becker Department of Pathology and Laboratory Medicine University of Cincinnati Cincinnati, Ohio 45267 Received December 6, 1985
Human plasma f i b r i n s t a b i l i z i n g factor ( f a c t o r X I I I ) may be separated from fibrinogen through reversible fibrinogen polymer formation at pH 6.6, F/2 0.3, O°C, and subsequent Bio-Gel A 1.5m f i l t r a t i o n . Factor XIII a c t i v i t y is eluted a f t e r the monomer fibrinogen peak. Polymer fractions from eight preparations, processed in duplicate, contain a mean 0.002 units factor XIII per mg fibrinogen, or about 0.7% the factor X I I I content of standard plasma. Factor X111-free fibrinogen polymers are easily dissociated (>98%) to the monomer form by incubation at 37°C, 18 hours. The fibrinogen preparations u t i l i z e d were devoid of plasma f i b r o n e c t i n ; thus these studies also show that reversible human fibrinogen polymer formation occurs in i t s absence. ® 1986 Academic Press, Inc.
Highly p u r i f i e d contain
preparations
plasma factor
XIII
of fibrinogen
(fibrin
(clottability
stabilizing
factor).
>97%) generally Factor X l l l a ,
the
activated form induced by thrombin and Ca++, promotes y - y chain crosslinking and covalent of
polymerization
fibrinogen
approached in
of
~ chains
purified
in
fibrin
preparations
may also act as a substrate for factor X l l l a (2). would be useful
(I).
At concentrations
(>30 mg/ml),
fibrinogen
Factor X l l l - f r e e
fibrinogen
in the study of the dependence on factor X l l l a of fibrinogen
polymer formation under physiologic conditions
(2,3),
and as a substrate for
q u a n t i t a t i v e assay of factor XIII in plasma. Factor XIII 3.3M urea (4); necessarily
activity
may be eliminated
however, this
from fibrinogen
method primarily
removes the a c t i v i t y
the
factor
XIII
to a denaturing
agent,
although not s i g n i f i c a n t l y
of the protein,
could possibly e f f e c t
action with f i b r o n e c t i n
(6),
protein.
In addition,
its
by d i a l y s i s
exposure of
altering
self-association
678
and not
fibrinogen
the c l o t t a b i l i t y (5),
or with the p l a t e l e t membrane (7).
0006-29tX/86 $1.50 Copyright @ 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
vs
its
inter-
Removal of
Vol. 134, No. 2, 1986
factor X l l l
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from fibrinogen has also been achieved by a f f i n i t y
chromatography
on organomercurial agarose (8). At low temperature,
e.g.
shown to undergo reversible
O°C, pH 6.6 and F/2 0.3, polymer formation
of polymer are a function of the t o t a l duced in a period of weeks. n e g l i g i b l e level of polymer.
(5).
fibrinogen
Near-equilibrium
At 37°C, dissociation
within
hours leads to a
Although fibrinogen and factor XIII have similar respectively
(9,10),
formation of
fibrinogen polymer permits separation of the two by gel f i l t r a t i o n . of
In addition, of
factor
XIII
from fibrinogen
by t h i s
because the human fibrinogen
plasma f i b r o n e c t i n ,
the
levels
fibrinogen concentration and are pro-
molecular weights of 340,000 and 320,000,
ration
has been
study
method is
preparations
shows that
reversible
The sepa-
herein reported.
utilized
were devoid
fibrinogen
polymer
formation occurs in i t s absence, Materials and Methods Reagents. Sources of BaSO4 powder, 95% ethanol, and enzyme grade (NH4)2S04; and treatment of the general supply of d i s t i l l e d water and d i a l y s i s tublng were as previously reported ( I I ) . P u r i f i c a t i o n of human fibrinogen, Human fibrinogen Fraction-ld was obtained by the method previously described for bovine fibrinogen ( I I ) , with the following modifications. Human blood was obtained from volunteer donors; generally 120 ml per donor. Informed consent was obtained as per protocol #83-I-12-2 approved by the University of Cincinnati Committee on Human Research, To remove plasma f i b r o n e c t i n , the BaSO4-adsorbed plasma was passed at 4 ml/min through a 2.8 cm diameter by 4.0 cm long column of gelatin agarose (Sigma Chem. No. G-5384). The e f f l u e n t was precipitated at 8% ethanol, O°C for 1 hour. The remainder of the procedure was similar to that for bovine preparations ( I I ) . Fibrinogens p u r i f i e d in this manner have a plasma f i b r o nectin content, as determined by assay u t i l i z i n g a polyclonal antibody and an alkaline phosphatase labeled tracer (Biomedical Technologies Inc.),awhich is 0,001% the molar concentration of fibrinogen. This represents a IOT-fold decrease of the f i b r o n e c t i n / f i b r i n o g e n r a t i o in plasma. For this study, the f i n a l (NH~)gSOm p r e c i p i t a t e s of the preparations were dissolved in and dialyzed v s ~h~sp~ate-KCI-KCN buffer (28.5 mM sodium phosphate, 250 mM KCl, and 1 mM KCN, pH 6,6, F/2 0.30), The eight preparations had c l o t t a b i l i t i e s ( i . e . the portion of total absorbance which is c l o t table by thrombin) ranging 96.6 to 97.9% (mean 97.1%). Gel f i l t r a t i o n . Analytical gel f i l t r a t i o n to assess polymer content of a preparation and preparative gel f i l t r a t i o n to produce factor X l l l - f r e e fibrinogen were conducted in a water-jacketed column on a 2.5 X 88 cm bed of Bio-Gel A 1.5m ( I I ) , developed at 4°C with phosphate-KCl-KCN buffer, Fractions of about 3.4 ml were collected in tared polypropylene tubes, permitting volume determination by weight. Absorbances were determined at 280 nm. For analytical purposes, the column load was about I0 AU. The polymer appears as a narrow leading peak, and even at a magnitude of >0.35 AU/ml, the trough preceding the monomer peak is <0.02 AU/ml, The f r a c t i o n of total c l o t t a b l e absorbance present in the polymer peak is designated the polymer mass f r a c tion, Pmf" Conditions for preparative gel f i l t r a t i o n are given below. 679
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Assay for factor X I I I a c t i v i t y . The factor X I I I a c t i v i t y of preparations was determined by modification of the monodansylcadaverine fluorometric method of Lorand et a l , ( 1 2 ) . Modifications included: reducing the concentration of casein from 0.4% to 0,2%, which did not s i g n i f i c a n t l y a l t e r binding of dansylcadaverine but greatly f a c i l i t a t e d resuspension of the p r e c i p i t a t e ; decreasing the thrombin concentration for activation to 16 NIH units/ml; using an acetone-ethanol ( I : I ) wash (3 X I0 ml); and s o l u b i l i z i n g the f i n a l prec i p i t a t e in 0,5% sodium dodecylsulfate, which proved adequate and had much less background fluorescence than the recommended solution which also contained 8 M urea, Purified bovine and human thrombins were obtained by the method of Rosenberg and Waugh (13) as modified by Wolfe and Waugh (14), and had equivalent a c t i v i t y in the assay, One unit of factor XIII is defined as the amount present in one ml of pooled c i t r a t e d plasma which also contained 3,45 mg fibrinogen, or 0.29 units factor Xlll/mg fibrinogen. In addition to the q u a n t i t a t i v e monodansylcadaverine incorporation assay, clots formed with various samples at a fibrinogen concentration of 0.7 to 1.4 mg/ml were tested for s o l u b i l i t y in I% monochloroacetic acid within 5 minutes at room temperature and in 5 M urea within 24 hours at 37°C ( ~ ) . Clots were formed with 1NIH unit/ml thrombin in the presence of 2,5 mM Ca . Results and Discussion Separation of factor × I I I
from fibrinogen has been carried out with eight
preparations of human Fraction l-d, tions
and factor XIII
Pertinent data regarding these prepara-
separation are given in Table I.
of a preparation was reduced i n i t i a l l y for
18 hours,
For 7 preparations
1 had an i n i t i a l
Pmf of 0.02.
The polymer content
to a low level by incubation at 37°C
the i n i t i a l
Pmf was <0,005.
Preparation
To promote polymer development, preparations
were then incubated at O°C for variable and in some cases an extensive period of
time
prior
to preparative
filtration
(column
3).
The presence of 1 mM
KCN and the low temperature of incubation prevented bacterial Recognizable
degradation
products
were
not
present
in
contamination.
any gel
filtration
effluents. Given the high concentration of fibrinogen extended period of incubation of fibrinogen
for
preparations
in the preparations, 1 through 4,
however, are completely reversible.
the p r e c i p i t a t e
dissolves
to dissociate.
For such preparations,
tration
the s o l u b i l i t y
polymer was in these cases exceeded, producing a p r e c i p i t a t e .
These reactions,
prior
and the
to sampling for (column
5)
gel
within
15 minutes and polymer would be expected p r e c i p i t a t e was dissolved immediately
filtration,
may be
lower
With incubation at 37°C,
Thus,
than
(column 2) and incubation time (column 3). 680
the Pmf with preparative f i l -
anticipated
for
the
concentration
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I PREPARATIVE SEPARATIONOF FACTORX I I I AND FIBRINOGENa Prep
Conc, mg/ml
Incub, (days)
Load (mg)
Pmf
I n i t i a l Material units/mg ~
Polymer Peak units/mg
la b
38.3
55 57
32,5 35,8
0,28 0,32
0.266
=0 =0
(0,001) (0.001)
2a b
39,4
55 57
36.7 36.7
0.26 0.40
0.263
=0 =0
(0.002) (0.001)
3a b
37,5
55 57
33,9 35,5
0.27 0.24
0.173
4a b
40.7
55 57
36.6 38.5
0,22 0,20
0.165
5a b
35,6
13 14
40,8 33,6
0.39 0,41
0.125
0.007 0.007
6a b
32.3
13 14
37,7 29.9
0,36 0,36
0.154
=0 0.005
7a b
31.9
12 26
30,0 30.0
0.33 0.40
0.063
=0 =0
8a b
32.3
14 26
31,1 30,0
0.35 0,45
0.078
=0 =0
0,003 (0.002) =0 ( =0 ) =0 =0
(0,003) ( =0 )
aColumn 1 indicates the fibrinogen preparation and the individual gel f i l t r a tions a and b for each preparation. Columns 2-5 give the fibrinogen concent r a t i o n , the days incubation at O°C prior to gel f i l t r a t i o n , the column load, and the Pmf' respectively, Pm# is the fraction of total clottable absorbance present as polymer, Columns"B and 7 give the factor X I I I content (units/mg fibrinogen) of the i n i t i a l unfractionated material and polymer peak fractions, respectively. One unit factor X I I I a c t i v i t y is the amount present in one ml of pooled citrated plasma, Results indicated as =0 had no detectable fluorescence, thus representing amounts below the s e n s i t i v i t y of the assay (<0,005 units). The values in parentheses followed concentration of sample by (NH4)2SO4 precipitation, and dissolution to about 5% the original volume, thus increasing the s e n s i t i v i t y for detecting factor X I I I a c t i v i t y .
A typical figure I,
preparative
Fractionation
monomer absorbance peaks,
gel of
filtration
the
pattern
initial
material
Note t h a t the t a l l
a Pmf of 0.35 and having a maximum of monomer by a trough of <0.04 AU/ml,
I,II
preparation
7a,
For the
other
polymer peak were pooled to 1,0
AU/ml,
along w i t h
P r i o r to a dilution
concentration
were
of
the
dialyzed
solutions
factor original at
in
is
clearly
shown
polymer
in and
separated from
in the f i g u r e ,
with dilutions
5 to
II
individual
of the c o n t r o l
fractions
of c o n c e n t r a t i o n
XIII
content,
preparation
room 681
results
is
S i m i l a r r e s u l t s were obtained w i t h
preparations,
give
assay f o r
AU/ml
content,
plasma made in phosphate-KCl-KCN b u f f e r ,
8a)
narrow polymer peak r e p r e s e n t i n g
As i n d i c a t e d
f r a c t i o n s were assayed f o r f a c t o r X I I I
(prep
aliquots to
temperature
within
the
ranging 0.5 to of
the
pools
the same approximate vs
Tris-NaCl
buffer
Vol. 134, No. 2, 1 9 8 6
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
0,05
I
f'\
/ i.c E
::
I
DD4 I
1
0 CO
OD3 ~ hi "._) Z
2
//
Q~
0
x
/,:/, aol
,I,'
/
"
t/
150
~ieoo • eei--i-&-i-&-&-i-A-i
"/
",,
/
I
,
~o I
200 250 EFFLUENT VOLUME (ml)
i i ol
\
I
300
Figure I: Bio-Gel A 1,5m effluent pattern for preparative separation 8a, The 2.5 X 88 cm column is developed at 4°C with phosphate-KCl-KCN buffer at pH 6,6, F/2 0,3 (see text), The ordinate gives the absorbance at 280 nm or units/ml factor XIII a c t i v i t y ; and the abscissa, the effluent volume in ml, The void volume is indicated by the arrow, The resulting Pmf' the fraction of total clottable absorbance representing polymer, is 0.35, (0.02 M T r i s , was u t i l i z e d
0.13 M NaCl, to d i l u t e
1,0 mM KCN, pH 7,4,
the control
plasma.
r / 2 0.15),
The same b u f f e r
D i l u t i o n s of samples and control
plasma in e i t h e r Tris-NaCl or phosphate-KCl-KCN gave equivalent r e s u l t s , Factor for
XIII
activities
for
the polymer peak f r a c t i o n s
the
initial
(unfractionated)
p r e p a r a t i v e mate-
p o s s i b l y r e f l e c t i n g v a r i a t i o n among the s t a r t i n g plasma and/or in f r a c -
tionation ever,
and
are given in columns 6 and 7, r e s p e c t i v e l y ,
Note the v a r i a t i o n in f a c t o r X I I I content among the i n i t i a l rials,
materials
during the f i b r i n o g e n
the f a c t o r
XIII
contents
p r e p a r a t i v e steps ( I I ) , of
By comparison,
polymer peak f r a c t i o n s
how-
are s i g n i f i c a n t l y
reduced, ranging =0 to 0,007 units/mg f i b r i n o g e n (mean of 0.002), In
spite
fractions
of
the
variations
(column 7),
clot
in
quantitative
solubility
test
values
results
among polymer peak
showed l i t t l e
variation,
Without exception, c l o t s formed with polymer peak m a t e r i a l s dissolved in monochloroacetic rials
as well
acid and urea,
Those formed with i n i t i a l
as pooled f r a c t i o n s
( u n f r a c t i o n a t e d ) mate-
from the monomer absorbance peaks did not
d i s s o l v e in e i t h e r . The a c q u i s i t i o n
of monomeric
units factor Xlll/mg fibrinogen
fibrinogen
with =0.7% of plasma l e v e l s f o r
is accomplished with l i t t l e 682
or no s i g n i f i c a n t
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
effect on fibrinogen c l o t t a b i l i t y . and monomer fractions
for
For example, the c l o t t a b i l i t i e s
preparation
5 were 95.9 and 96.6%, respectively,
Corresponding values for preparation favorably with (prep 6). XIII
values for
of polymer
6 were 96,5 and 95.7%.
the i n i t i a l
materials:
These compare
97.4% (prep 5) and 96.6%
To demonstrate regeneration of monomer fibrinogen following factor
separation,
6 and 7 ( a l l
polymer fractions
from gel
filtration
of preparations
5,
at Pmf >0.33) were incubated at 37°C for 18 hours, and then sub-
jected to gel f i l t r a t i o n .
In all
cases >98% of the fibrinogen
the monomer peaks, indicating dissociation of the polymer.
appeared in
Complete dissoci-
ation can be achieved at lower temperatures, e.g. 29°C (5). According to Greenberg and Shuman (16), bind to fibrinogen ever,
binding
at 0.3 M NaCl. factor
XIII.
is
by the a2 subunit. progressively
With
the
and plasma factor XIII
With increasing ionic strength,
inhibited,
At that point,
platelet
with maximal i n h i b i t i o n
however, there is some residual
preparations
reported
herein,
polymer
occurred at O°C and ionic strength 0.30 (0.25 due to KCl).
attained binding of formation
Apparently the
findings which Greenberg and Shuman obtained at 22°C also apply at O°C. figure 1 i l l u s t r a t e s , monomer fibrinogen, elution
of
fibrinogen
factor XIII appears as a d i s t i n c t consistent
fibrinogen
polymer
and factor X I I I ,
with
its
assures
As
peak, eluting beyond
lower molecular weight. essentially
how-
complete
The early
separation
of
with a mean residual of 0,002 units factor XIII
per mg fibrinogen )resent in polymer fractions.
Acknowledgement My deep appreclation is expressed to Ms. Joan M. Fisher for her excellent technical assistance. References I. 2. 3. 4. 5.
McDonagh, R.P., McDonagh, J., and Blomback, B. (1972) Proc. Natl. Acad. Sci. U,S. 69, 3648-3652. Kanaide, H., and Shainoff, J.R. (1975) J. Lab. Clin. Med. 85, 574-597. Becker, C.M. (1985) Fed. Proc. 44, 1656. Schwartz, M.L., Pizzo, S.V., H i l l , R.L., and McKee, P.A. (1971) J. Clin. Invest. 50, 1506-1513. Becker, C,M., and Waugh, D,F. (1980) Arch. Biochem. Biophys. 204, 101-108, 683
VOI. 134, No. 2, 1 9 8 6 6. 7. 8, 9. I0. II. 12. 13. 14. 15. 16.
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Stathakis, N.E., Mosesson, M.W., Chen, A.B., and Galanakis, D.K. (1978) Blood 51, 1211-1222. Marguerie, G.A., and Plow, E.F. (1983) Annals N.Y. Academy of Science 408, 556-566. McDonagh, J., Waggoner, W.G., Hamilton, E.G., Hindenach, B., and McDonagh, R.P. (1976) Biochim. Biophys. Acta 446, 345-357. Doolittle, R.F. (1984) Ann. Rev. Biochem. 53, 195-229. Schwartz, M.L., Pizzo, S.V., H i l l , R.L,, and McKee, P.A. (1973) J, Biol. Chem. 248, 1395-1407. Becker, C.M., and Waugh, D.F. (1980) Arch. Biochem, Biophys. 204, 88-100. Lorand, L., Urayama, T., de Kiewiet, J.W.C., and Nossel, H.L. (1969) J. Clin, Invest. 48, 1054-1064, Rosenberg, R.D., and Waugh, D.F. (1970) J. Biol. Chem. 245, 5049-5056. Wolfe, J.K., and Waugh, D.F. (1981) Arch. Biochem. Biophys. 211, 125-142. Triplett, D . A . (1982) In: Laboratory Evaluation of Coagulation, D.A. T r i p l e t t , Ed. p. 95, American Society of Clinical Pathologists Press, Chicago. Greenberg, C.S., and Shuman, M.A. (1982) J. Biol. Chem. 257, 6096-6101.
684
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 678-684
January 29, 1986
SEPARATION OF HUMAN PLASMA FIBRIN STABIEIZING FACTOR (FACTOR X I I I ) FROM FIBRINOGEN BY FIBRINOGEN POLYMER FORMATION Carter M. Becker Department of Pathology and Laboratory Medicine University of Cincinnati Cincinnati, Ohio 45267 Received December 6, 1985
Human plasma f i b r i n s t a b i l i z i n g factor ( f a c t o r X I I I ) may be separated from fibrinogen through reversible fibrinogen polymer formation at pH 6.6, F/2 0.3, O°C, and subsequent Bio-Gel A 1.5m f i l t r a t i o n . Factor XIII a c t i v i t y is eluted a f t e r the monomer fibrinogen peak. Polymer fractions from eight preparations, processed in duplicate, contain a mean 0.002 units factor XIII per mg fibrinogen, or about 0.7% the factor X I I I content of standard plasma. Factor X111-free fibrinogen polymers are easily dissociated (>98%) to the monomer form by incubation at 37°C, 18 hours. The fibrinogen preparations u t i l i z e d were devoid of plasma f i b r o n e c t i n ; thus these studies also show that reversible human fibrinogen polymer formation occurs in i t s absence. ® 1986 Academic Press, Inc.
Highly p u r i f i e d contain
preparations
plasma factor
XIII
of fibrinogen
(fibrin
(clottability
stabilizing
factor).
>97%) generally Factor X l l l a ,
the
activated form induced by thrombin and Ca++, promotes y - y chain crosslinking and covalent of
polymerization
fibrinogen
approached in
of
~ chains
purified
in
fibrin
preparations
may also act as a substrate for factor X l l l a (2). would be useful
(I).
At concentrations
(>30 mg/ml),
fibrinogen
Factor X l l l - f r e e
fibrinogen
in the study of the dependence on factor X l l l a of fibrinogen
polymer formation under physiologic conditions
(2,3),
and as a substrate for
q u a n t i t a t i v e assay of factor XIII in plasma. Factor XIII 3.3M urea (4); necessarily
activity
may be eliminated
however, this
from fibrinogen
method primarily
removes the a c t i v i t y
the
factor
XIII
to a denaturing
agent,
although not s i g n i f i c a n t l y
of the protein,
could possibly e f f e c t
action with f i b r o n e c t i n
(6),
protein.
In addition,
its
by d i a l y s i s
exposure of
altering
self-association
678
and not
fibrinogen
the c l o t t a b i l i t y (5),
or with the p l a t e l e t membrane (7).
0006-29tX/86 $1.50 Copyright @ 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
vs
its
inter-
Removal of
Vol. 134, No. 2, 1986
factor X l l l
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from fibrinogen has also been achieved by a f f i n i t y
chromatography
on organomercurial agarose (8). At low temperature,
e.g.
shown to undergo reversible
O°C, pH 6.6 and F/2 0.3, polymer formation
of polymer are a function of the t o t a l duced in a period of weeks. n e g l i g i b l e level of polymer.
(5).
fibrinogen
Near-equilibrium
At 37°C, dissociation
within
hours leads to a
Although fibrinogen and factor XIII have similar respectively
(9,10),
formation of
fibrinogen polymer permits separation of the two by gel f i l t r a t i o n . of
In addition, of
factor
XIII
from fibrinogen
by t h i s
because the human fibrinogen
plasma f i b r o n e c t i n ,
the
levels
fibrinogen concentration and are pro-
molecular weights of 340,000 and 320,000,
ration
has been
study
method is
preparations
shows that
reversible
The sepa-
herein reported.
utilized
were devoid
fibrinogen
polymer
formation occurs in i t s absence, Materials and Methods Reagents. Sources of BaSO4 powder, 95% ethanol, and enzyme grade (NH4)2S04; and treatment of the general supply of d i s t i l l e d water and d i a l y s i s tublng were as previously reported ( I I ) . P u r i f i c a t i o n of human fibrinogen, Human fibrinogen Fraction-ld was obtained by the method previously described for bovine fibrinogen ( I I ) , with the following modifications. Human blood was obtained from volunteer donors; generally 120 ml per donor. Informed consent was obtained as per protocol #83-I-12-2 approved by the University of Cincinnati Committee on Human Research, To remove plasma f i b r o n e c t i n , the BaSO4-adsorbed plasma was passed at 4 ml/min through a 2.8 cm diameter by 4.0 cm long column of gelatin agarose (Sigma Chem. No. G-5384). The e f f l u e n t was precipitated at 8% ethanol, O°C for 1 hour. The remainder of the procedure was similar to that for bovine preparations ( I I ) . Fibrinogens p u r i f i e d in this manner have a plasma f i b r o nectin content, as determined by assay u t i l i z i n g a polyclonal antibody and an alkaline phosphatase labeled tracer (Biomedical Technologies Inc.),awhich is 0,001% the molar concentration of fibrinogen. This represents a IOT-fold decrease of the f i b r o n e c t i n / f i b r i n o g e n r a t i o in plasma. For this study, the f i n a l (NH~)gSOm p r e c i p i t a t e s of the preparations were dissolved in and dialyzed v s ~h~sp~ate-KCI-KCN buffer (28.5 mM sodium phosphate, 250 mM KCl, and 1 mM KCN, pH 6,6, F/2 0.30), The eight preparations had c l o t t a b i l i t i e s ( i . e . the portion of total absorbance which is c l o t table by thrombin) ranging 96.6 to 97.9% (mean 97.1%). Gel f i l t r a t i o n . Analytical gel f i l t r a t i o n to assess polymer content of a preparation and preparative gel f i l t r a t i o n to produce factor X l l l - f r e e fibrinogen were conducted in a water-jacketed column on a 2.5 X 88 cm bed of Bio-Gel A 1.5m ( I I ) , developed at 4°C with phosphate-KCl-KCN buffer, Fractions of about 3.4 ml were collected in tared polypropylene tubes, permitting volume determination by weight. Absorbances were determined at 280 nm. For analytical purposes, the column load was about I0 AU. The polymer appears as a narrow leading peak, and even at a magnitude of >0.35 AU/ml, the trough preceding the monomer peak is <0.02 AU/ml, The f r a c t i o n of total c l o t t a b l e absorbance present in the polymer peak is designated the polymer mass f r a c tion, Pmf" Conditions for preparative gel f i l t r a t i o n are given below. 679
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Assay for factor X I I I a c t i v i t y . The factor X I I I a c t i v i t y of preparations was determined by modification of the monodansylcadaverine fluorometric method of Lorand et a l , ( 1 2 ) . Modifications included: reducing the concentration of casein from 0.4% to 0,2%, which did not s i g n i f i c a n t l y a l t e r binding of dansylcadaverine but greatly f a c i l i t a t e d resuspension of the p r e c i p i t a t e ; decreasing the thrombin concentration for activation to 16 NIH units/ml; using an acetone-ethanol ( I : I ) wash (3 X I0 ml); and s o l u b i l i z i n g the f i n a l prec i p i t a t e in 0,5% sodium dodecylsulfate, which proved adequate and had much less background fluorescence than the recommended solution which also contained 8 M urea, Purified bovine and human thrombins were obtained by the method of Rosenberg and Waugh (13) as modified by Wolfe and Waugh (14), and had equivalent a c t i v i t y in the assay, One unit of factor XIII is defined as the amount present in one ml of pooled c i t r a t e d plasma which also contained 3,45 mg fibrinogen, or 0.29 units factor Xlll/mg fibrinogen. In addition to the q u a n t i t a t i v e monodansylcadaverine incorporation assay, clots formed with various samples at a fibrinogen concentration of 0.7 to 1.4 mg/ml were tested for s o l u b i l i t y in I% monochloroacetic acid within 5 minutes at room temperature and in 5 M urea within 24 hours at 37°C ( ~ ) . Clots were formed with 1NIH unit/ml thrombin in the presence of 2,5 mM Ca . Results and Discussion Separation of factor × I I I
from fibrinogen has been carried out with eight
preparations of human Fraction l-d, tions
and factor XIII
Pertinent data regarding these prepara-
separation are given in Table I.
of a preparation was reduced i n i t i a l l y for
18 hours,
For 7 preparations
1 had an i n i t i a l
Pmf of 0.02.
The polymer content
to a low level by incubation at 37°C
the i n i t i a l
Pmf was <0,005.
Preparation
To promote polymer development, preparations
were then incubated at O°C for variable and in some cases an extensive period of
time
prior
to preparative
filtration
(column
3).
The presence of 1 mM
KCN and the low temperature of incubation prevented bacterial Recognizable
degradation
products
were
not
present
in
contamination.
any gel
filtration
effluents. Given the high concentration of fibrinogen extended period of incubation of fibrinogen
for
preparations
in the preparations, 1 through 4,
however, are completely reversible.
the p r e c i p i t a t e
dissolves
to dissociate.
For such preparations,
tration
the s o l u b i l i t y
polymer was in these cases exceeded, producing a p r e c i p i t a t e .
These reactions,
prior
and the
to sampling for (column
5)
gel
within
15 minutes and polymer would be expected p r e c i p i t a t e was dissolved immediately
filtration,
may be
lower
With incubation at 37°C,
Thus,
than
(column 2) and incubation time (column 3). 680
the Pmf with preparative f i l -
anticipated
for
the
concentration
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I PREPARATIVE SEPARATIONOF FACTORX I I I AND FIBRINOGENa Prep
Conc, mg/ml
Incub, (days)
Load (mg)
Pmf
I n i t i a l Material units/mg ~
Polymer Peak units/mg
la b
38.3
55 57
32,5 35,8
0,28 0,32
0.266
=0 =0
(0,001) (0.001)
2a b
39,4
55 57
36.7 36.7
0.26 0.40
0.263
=0 =0
(0.002) (0.001)
3a b
37,5
55 57
33,9 35,5
0.27 0.24
0.173
4a b
40.7
55 57
36.6 38.5
0,22 0,20
0.165
5a b
35,6
13 14
40,8 33,6
0.39 0,41
0.125
0.007 0.007
6a b
32.3
13 14
37,7 29.9
0,36 0,36
0.154
=0 0.005
7a b
31.9
12 26
30,0 30.0
0.33 0.40
0.063
=0 =0
8a b
32.3
14 26
31,1 30,0
0.35 0,45
0.078
=0 =0
0,003 (0.002) =0 ( =0 ) =0 =0
(0,003) ( =0 )
aColumn 1 indicates the fibrinogen preparation and the individual gel f i l t r a tions a and b for each preparation. Columns 2-5 give the fibrinogen concent r a t i o n , the days incubation at O°C prior to gel f i l t r a t i o n , the column load, and the Pmf' respectively, Pm# is the fraction of total clottable absorbance present as polymer, Columns"B and 7 give the factor X I I I content (units/mg fibrinogen) of the i n i t i a l unfractionated material and polymer peak fractions, respectively. One unit factor X I I I a c t i v i t y is the amount present in one ml of pooled citrated plasma, Results indicated as =0 had no detectable fluorescence, thus representing amounts below the s e n s i t i v i t y of the assay (<0,005 units). The values in parentheses followed concentration of sample by (NH4)2SO4 precipitation, and dissolution to about 5% the original volume, thus increasing the s e n s i t i v i t y for detecting factor X I I I a c t i v i t y .
A typical figure I,
preparative
Fractionation
monomer absorbance peaks,
gel of
filtration
the
pattern
initial
material
Note t h a t the t a l l
a Pmf of 0.35 and having a maximum of monomer by a trough of <0.04 AU/ml,
I,II
preparation
7a,
For the
other
polymer peak were pooled to 1,0
AU/ml,
along w i t h
P r i o r to a dilution
concentration
were
of
the
dialyzed
solutions
factor original at
in
is
clearly
shown
polymer
in and
separated from
in the f i g u r e ,
with dilutions
5 to
II
individual
of the c o n t r o l
fractions
of c o n c e n t r a t i o n
XIII
content,
preparation
room 681
results
is
S i m i l a r r e s u l t s were obtained w i t h
preparations,
give
assay f o r
AU/ml
content,
plasma made in phosphate-KCl-KCN b u f f e r ,
8a)
narrow polymer peak r e p r e s e n t i n g
As i n d i c a t e d
f r a c t i o n s were assayed f o r f a c t o r X I I I
(prep
aliquots to
temperature
within
the
ranging 0.5 to of
the
pools
the same approximate vs
Tris-NaCl
buffer
Vol. 134, No. 2, 1 9 8 6
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
0,05
I
f'\
/ i.c E
::
I
DD4 I
1
0 CO
OD3 ~ hi "._) Z
2
//
Q~
0
x
/,:/, aol
,I,'
/
"
t/
150
~ieoo • eei--i-&-i-&-&-i-A-i
"/
",,
/
I
,
~o I
200 250 EFFLUENT VOLUME (ml)
i i ol
\
I
300
Figure I: Bio-Gel A 1,5m effluent pattern for preparative separation 8a, The 2.5 X 88 cm column is developed at 4°C with phosphate-KCl-KCN buffer at pH 6,6, F/2 0,3 (see text), The ordinate gives the absorbance at 280 nm or units/ml factor XIII a c t i v i t y ; and the abscissa, the effluent volume in ml, The void volume is indicated by the arrow, The resulting Pmf' the fraction of total clottable absorbance representing polymer, is 0.35, (0.02 M T r i s , was u t i l i z e d
0.13 M NaCl, to d i l u t e
1,0 mM KCN, pH 7,4,
the control
plasma.
r / 2 0.15),
The same b u f f e r
D i l u t i o n s of samples and control
plasma in e i t h e r Tris-NaCl or phosphate-KCl-KCN gave equivalent r e s u l t s , Factor for
XIII
activities
for
the polymer peak f r a c t i o n s
the
initial
(unfractionated)
p r e p a r a t i v e mate-
p o s s i b l y r e f l e c t i n g v a r i a t i o n among the s t a r t i n g plasma and/or in f r a c -
tionation ever,
and
are given in columns 6 and 7, r e s p e c t i v e l y ,
Note the v a r i a t i o n in f a c t o r X I I I content among the i n i t i a l rials,
materials
during the f i b r i n o g e n
the f a c t o r
XIII
contents
p r e p a r a t i v e steps ( I I ) , of
By comparison,
polymer peak f r a c t i o n s
how-
are s i g n i f i c a n t l y
reduced, ranging =0 to 0,007 units/mg f i b r i n o g e n (mean of 0.002), In
spite
fractions
of
the
variations
(column 7),
clot
in
quantitative
solubility
test
values
results
among polymer peak
showed l i t t l e
variation,
Without exception, c l o t s formed with polymer peak m a t e r i a l s dissolved in monochloroacetic rials
as well
acid and urea,
Those formed with i n i t i a l
as pooled f r a c t i o n s
( u n f r a c t i o n a t e d ) mate-
from the monomer absorbance peaks did not
d i s s o l v e in e i t h e r . The a c q u i s i t i o n
of monomeric
units factor Xlll/mg fibrinogen
fibrinogen
with =0.7% of plasma l e v e l s f o r
is accomplished with l i t t l e 682
or no s i g n i f i c a n t
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
effect on fibrinogen c l o t t a b i l i t y . and monomer fractions
for
For example, the c l o t t a b i l i t i e s
preparation
5 were 95.9 and 96.6%, respectively,
Corresponding values for preparation favorably with (prep 6). XIII
values for
of polymer
6 were 96,5 and 95.7%.
the i n i t i a l
materials:
These compare
97.4% (prep 5) and 96.6%
To demonstrate regeneration of monomer fibrinogen following factor
separation,
6 and 7 ( a l l
polymer fractions
from gel
filtration
of preparations
5,
at Pmf >0.33) were incubated at 37°C for 18 hours, and then sub-
jected to gel f i l t r a t i o n .
In all
cases >98% of the fibrinogen
the monomer peaks, indicating dissociation of the polymer.
appeared in
Complete dissoci-
ation can be achieved at lower temperatures, e.g. 29°C (5). According to Greenberg and Shuman (16), bind to fibrinogen ever,
binding
at 0.3 M NaCl. factor
XIII.
is
by the a2 subunit. progressively
With
the
and plasma factor XIII
With increasing ionic strength,
inhibited,
At that point,
platelet
with maximal i n h i b i t i o n
however, there is some residual
preparations
reported
herein,
polymer
occurred at O°C and ionic strength 0.30 (0.25 due to KCl).
attained binding of formation
Apparently the
findings which Greenberg and Shuman obtained at 22°C also apply at O°C. figure 1 i l l u s t r a t e s , monomer fibrinogen, elution
of
fibrinogen
factor XIII appears as a d i s t i n c t consistent
fibrinogen
polymer
and factor X I I I ,
with
its
assures
As
peak, eluting beyond
lower molecular weight. essentially
how-
complete
The early
separation
of
with a mean residual of 0,002 units factor XIII
per mg fibrinogen )resent in polymer fractions.
Acknowledgement My deep appreclation is expressed to Ms. Joan M. Fisher for her excellent technical assistance. References I. 2. 3. 4. 5.
McDonagh, R.P., McDonagh, J., and Blomback, B. (1972) Proc. Natl. Acad. Sci. U,S. 69, 3648-3652. Kanaide, H., and Shainoff, J.R. (1975) J. Lab. Clin. Med. 85, 574-597. Becker, C.M. (1985) Fed. Proc. 44, 1656. Schwartz, M.L., Pizzo, S.V., H i l l , R.L., and McKee, P.A. (1971) J. Clin. Invest. 50, 1506-1513. Becker, C,M., and Waugh, D,F. (1980) Arch. Biochem. Biophys. 204, 101-108, 683
VOI. 134, No. 2, 1 9 8 6 6. 7. 8, 9. I0. II. 12. 13. 14. 15. 16.
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Stathakis, N.E., Mosesson, M.W., Chen, A.B., and Galanakis, D.K. (1978) Blood 51, 1211-1222. Marguerie, G.A., and Plow, E.F. (1983) Annals N.Y. Academy of Science 408, 556-566. McDonagh, J., Waggoner, W.G., Hamilton, E.G., Hindenach, B., and McDonagh, R.P. (1976) Biochim. Biophys. Acta 446, 345-357. Doolittle, R.F. (1984) Ann. Rev. Biochem. 53, 195-229. Schwartz, M.L., Pizzo, S.V., H i l l , R.L,, and McKee, P.A. (1973) J, Biol. Chem. 248, 1395-1407. Becker, C.M., and Waugh, D.F. (1980) Arch. Biochem, Biophys. 204, 88-100. Lorand, L., Urayama, T., de Kiewiet, J.W.C., and Nossel, H.L. (1969) J. Clin, Invest. 48, 1054-1064, Rosenberg, R.D., and Waugh, D.F. (1970) J. Biol. Chem. 245, 5049-5056. Wolfe, J.K., and Waugh, D.F. (1981) Arch. Biochem. Biophys. 211, 125-142. Triplett, D . A . (1982) In: Laboratory Evaluation of Coagulation, D.A. T r i p l e t t , Ed. p. 95, American Society of Clinical Pathologists Press, Chicago. Greenberg, C.S., and Shuman, M.A. (1982) J. Biol. Chem. 257, 6096-6101.
684