Biochimica et Biophysica Acta, 996 (1989) 95-102 95

Elsevier

BBA 33369

Conformafional changes in human prothrornbin as detected

by antibody populations
H e r b e r t K . F . L a u * a n d R o b e r t D. R o s e n b e r g
Charles A. Dana Research Institute and Harvard Medical School, Boston, and the Department of Biology and Whitaker College,
Massachusetts Institute of Technology, Cambridge, MA (U.S.A.)

(Received 7 July 1988)

Key words: Prothrombin fragment 1; Calcium; Conformational change; (Human)

The amino-terminal pepfides of human prothrombin corresponding to residues 1-51 and 52-156 have been isolated
from a thrombin digest of prothrombin fragment 1. The products of digestion were purified by means of barium citrate
and ammonium sulfate precipitations, followed by gel filtration and hydroxyapatite chromatographies. They were
identified by their molecular sizes as well as their amino acid compositions. Peptides 1-51 (FIA) and 52-156 (F~B)
were used as affinity iigands for the isolation of antibody populations from antisera that were elicited against human
prothrombin or prothrombin fragment 1. These antibody populations displayed restricted specificity for the respective
ligands as shown by competitive radioinununoassays. They were used to study the confonnafional changes in
prothrombin and fragment 1. The F~A-specific antibody populations d:tected a confonnafional change which is
stabilized by calcium ions and which has a transition midpoint at --0.2 mM calcium ion concentration. The
Ft B-specific antibody populations identified a different confonnafional change which is destabilized by calcium ions and
which has a transition midpoint at ~ 0.5 mM calcium.

introduction prothrombin by factor Xa [14]. It has been shown that

Human prothrombin has a molecular mass of ap-
prox. 70 kDa and like other coagulation zymogens, is
composed of modular structures related to different
functions. These are fragment 1 which contains ten
3,-earboxyglutamic acids whose post-translational for-
mation requires vitamin K and whose function is be-
lieved to be binding of metal ions [1-6], fragment 2
whose function is to bind factor V/Va [7], and pre-
thrombin 2, which gives rise to thrombin after cleavage
at a disulfide bond [8,9]. The amino acid sequence of
prothrombin has been delineated [1] and the crystal
structure of fragment 1 has been reported [10-13].
Metal ions are important, in the presence of factor
V/Va and phospholipid surface, for the activation of
Abbreviations: F1, human prothrombin activation fragment 1; 1:1+2,
prothrombin activation fragmt:at 1 and 2; FIA, prothrombin N-termi-
nal peptide 1-51; FIB, prothrombin N-terminal peptide 52-156;
SDS-PAGE, sodium dodecyl sulfate-polyacrylam/de gel electrophore-
sis; PBS, phosphate buffered saline (0.05 M sodium phosphate/0.5 M
NaCI (pH 7.5); SDS, sodium dodecyl sulfate.
Correspondence (and present address): H.K.F. Lau, Department of
Biochemistry, University of Hong Kong, Sassoon Road, Hong Kong.
the interactions of the membrane surface with vitamin
K-dependent proteins are affected by metal ions [15-17].
On the other hand, prothrombin itself also undergoes
metal-dependent conformational changes when studied
by physical methods [18-20] or immunochemical means
[21]. The use of antibodies as structural probes for
various antigenic determinants have been successfully
applied to study the activation of fragment 2 and frag-
ment 1 + 2 of human prothrombin [22] and the human
thrombin-antithrombin complex [23]. Similar approach
has also been adopted to delineate alterations of the
three-dimensional structure of bovine and human pro..
thrombins [24-28]. We report here the isolation of the
N-terminal residues 1-51 and 52-156 of human pro-
thrombin, the use of these peptides in affinity chro-
matography to obtain four different antibody subpopu-
lations, and the use of these antibodies in studies of the
interaction of calcium ions with prothrombin and pro-
thrombin activation fragment 1.
Materials and Methods
All chemicals employed were of reagent grade.
DEAE-cellulose and hydroxyapatite were purchased
from Bio-Rad. Sephadex G-25, G-100, Sepharose 4B

0167-4838/89/$03.50 © 1989 Elsevier Science Pubfishers B.V. (Biomedical Division)

96
and QAE-50 Sephadex were obtained from Pharmacia.
Echis carinatus venom were purchased from Sigma.
Hirudin was obtained from Pentapharm (Basel). Rabbit
IgG and complete Freund's adjuvant were bought from
Miles. Rabbit brain thromboplastin was provided by
Ortho Diagnostics and purified as described [29].
Purification of prothrombin and prothrombin activation
fragments
Human prothrombin was prepared according to
Shapiro et al. [30] with minor modifications [22]. The
product appeared homogeneous and protein of the
highest specific activity was used. Human thrombin was
purified to homogeneity with a specific activity of 2800
N.I.H. units/mg by a procedure published before [31].
Prothrombin fragment F 1+2 was prepared according
to Aronson et al. [32] and the trace amounts of con-
taminating prothrombin and prethrombin 2 were re-
moved by means of heparin-bound Sepharose and
QAE-50 Sephadex [22].
To obtain F 1, a by-product of large-scale preparation
of human thrombin was used. This material was kindly
provided by Dr, J.W. Fenton of the New York State
Department of Health, Albany, N.Y., and contained a
mixture of prothrombin activation fragments and inter-
mediates. A typical experiment started by precipitating
1.74 1 of this material with 14.5 mM in barium citrate.
The precipitate was washed with ice-cold water and
dissolved in 400 ml of 0.2 M sodium citrate. The
solution was then made 40~ saturated ammonium
sulfate and the resulting precipitate discarded. The su-
pernatant was then made 80~ in saturated ammonium
sulfate, and the protein precipitated was dissolved in 30
ml 0.1 M NaCI/0.02 M Tris (pH 7.4) 6 ml of this
material containing ~ 6 mg protein was chromato-
graphed on a column of Sephadex G-100 (1.6 × 100 cm)
which was developed at 8 m l / h in the same buffer.
Fractions of 2.5 ml were collected. The slower-moving
component contained a protein of approx. 21 kDa and
was rechromatographed under the same experimental
conditions to remove traces of contaminating proteins.
To obtain prothrombin N-terminal residues 1-51
(FIA) and prothrombin N-terminal residues 52-156
(FIB), the method of Walz et al. [33,34] was adopted
with modifications. 45 ml of Fl (1 mg/ml) was digested
for 24 h at 37°C with 0.9 ml thrombin (4.6 mg/ml).
Afterwards 20 mM dithiothreitol was added, the pH
adjusted to 8.0, and the reaction continued for 20 h at
37 ° C. The products were carboxymethylated with 22
mM iodoacetamide for 3 h at 37 °C and at pH 8.9, and
then precipitated with 20 mM barium citrate. This
separated the reaction mixture into precipitate and su-
pernatant fractions. No further purification was taken
for the precipitate but the supernatant (47 ml) was
extensively di~ysed against 0.1 M Tris/0.1 M NaC1
(pH 7.4) and chromatographed on a hydroxyapatite
column (0.9 x 3 cm). After loading the sample, the
column was washed with the dialysis buffer followed by
0.1 M potassium phosphate (pH 6.8) at a flow rate of 15
m l / h and 2 ml fractions were collected.
Immunization and processing of antisera
Four New Zealand white rabbits were immunized
with 100/tg of F1 in complete Freund's adjuvant. Two
goats were injected with 500/~g of prothrombin without
the use of adjuvant. Hyperimmune antisera from these
animals were obtained by repeated injections and anti-
sera IgG fractions were obtained as described previ-
ously [22,23].
In order to obtain specific antibody populations
against F1A or F1B, two rabbit antisera IgG raised
against F I (R5 and R9) were affinity-fractionated. 4 mg
of FlA and 6 mg of F1B were separaltely bound to 10 ml
of Sepharose 4B using cyanogen bromide [35]. The gels
were washed with 1 M acetic acid/0.5 M NaCI (pH 2.4)
and then equilibrated with 0.05 M sodium phosphate/
0.5 M NaCI (pH 7.5) (PBS) before use. Then 150 mg R5
and 100 mg R9 anti-F1 were filtered through the F1A-
and FIB-bound Sepharose, respectively. The columns
were washed with PBS until the 280 nm absorbance of
the eluates were below 0.02, and the bound species were
eluted with the acetic acid/NaCl buffer. The eluted
materials were neutralized and dialysed against 0.13 M
NaCI/0.02 M Tris (pH 7.4) (buffer A) and were desig-
nated R5 anti-FiA and R9 anti-FiB, respectively.
In order to isolate goat anti-F1A or anti-FiB, the F
fractions of a goat anti-prothrombin were used as the
starting material. Two aliquots of 30 mg goat IgG were
separately affinity-fractionated on the same F~A- or
F l B-bound Sepharose columns, followed by the washing
and elution procedure as above.
Buffers
Buffers used in the binding studies were prepared
with double-distilled water after passing through the
Ultra Pure Water System (Hydro Services and Supplies,
Braintree, MA) and contained -0.075 ppb Ca 2+ or
Mg 2+ according to the manufacturer. Buffers prepared
with this water had been used to study the Ca 2+-
dependent intrinsic fluorescence of F~ according to
Nelsestuen [17] and Prendergast and Mann [18]. The
fluorescence of F 1 in buffer A described a sigmoidal
curve which plateaued at a fluorescence change of - 41%
of the original fluorescence and had a transition mid-
point at 0.25 mM C a C I 2. These observations were in
agreement with those published before [17] and indi-
cated that the quality of the water was of adequate
purity.
Competitive assays
These were carried out in radioimmunoassays using a
second antibody system to separate free from bound

antigen [22,23]. F~ was iodinated to ~ 7.1.10 6 cpm/#g
according to Greenwood et al. [36]. 50 #l 125I-labeled F 1
containing - 4 0 0 0 cpm (~ 1.8.10 -1° M) was prein-
cubated with 5 0 / d of different concentrations of com-
peting antigens for 60 min at room temperature. Then
i00 ~I Gf ~_5 anti-F,A (1.10 -8 M) or R9 anti-F1B
(3.8- 10 -8 M) was added, and the incubation eenfinued
for 20 to 24 h at 4 ° C. The second antibody system
wh:'.ch consisted of 200 #1 of sheep anti-rabbit IgG and
40/~g of nonimmune rabbit IgG, was added the next
day. The precipitates were washed and counted after
overnight incubation. The buffer in this assay was 0.155
M NaCl/0.005 M EDTA/0.0255 M sodium phos-
phate/20 mg per ml bovine serum albumin (pH 7.4).
The competing antigens used were prothrombin, F 1,
F 1A, F !B and F 1+ 2, in concentrations between 10-10 M
to 10 -5 M. The molecular masses of prothrombin,
thrombin, F l, F1 + 2 were assumed to be 70, 36, 21 and 32
kDa, respectively. Estimation of crossreacfivity and
computation of the slopes of the respective logit-log
dose response curves were obtained by fitting the raw
data to a 'four parameter' model as described before
[37,38].
Binding studies
The same radioimmunoassay was utilized, except that
no competing antigens were added. For antibody popu-
lations derived from goat, the second antibody system
consisted of 200/~1 of rabbit anti-goat IgG and 40 #g of
nonimmune goat IgG. All radiolabeled ligands, specific
antibodies and the second antibody systems were either
diluted into or dialysed against buffer A containing the
appropriate concentrations of Ca 2+ or EDTA before
use. The assays were carried out in triplicates and the
results averaged. Three types of binding assays were
performed:
(1) Antibody concentration-dependent binding experi-
ments. Various concentrations of anti-F~A or anti-F~B
were reacted with a fixed concentration of radiolabeled
F~ or prothrombin, in the presence of either 10 mM
EDTA or 10 mM CaCI 2. The fixed amount of 12SI-F1
used in this assay contained ~ 4000 cpm ( ~ 8.5.10-10
M) and was mixed with various concentrations of R5
anti-FlA (1.25.10 - 9 M to 1.25.10 -7 M), R9 anti-F1B
(1.1.10 -l° M to 1.1.10 -8 M), goat anti-FiA (5.3.
10 -1° M to 5.3-10 -8 M), or goat anti-FiB (7.5.10 -1°
M to 7.5.10 -8 M). The fixed amount of ~25I-labeled
prothrombin contained -- 4000 cpm ( - 1.7.10-10 M),
and was mixed with the same amounts of goat anti-F1A
or goat anti-F~B antibodies as given above.
(2) Ca 2 +-dependent binding experiments. Fixed con-
centrations of anti-F~A or anti-F~B antibodies were
reacted with fixed concentrations of either radiolabeled
F 1 or prothrombin, in the presence of 10 mM EDTA or
0.1 to 10 mM of CaC12. 1251-F1 (1.8.10 -1° M) was
97
diluted into buffers containing the various amounts of
Ca 2+ and was allowed to react with R5 anti-F~ (1.7.
10 -l° M), R5 anti-F1A (2.2.10 ..9 M), R9 anti-F1 (1.7.
10 -9 M), R9 anti-FiB (1.3.10 -.9 M), goat anti-pro-
thrombin (1.7.10 -8 M), goat anti-FiA (1.8.10 ..7 M),
or goat anti-F~B (2.8- 10 -7 M). ~25I-prothrombin ( - 1.8
• 10 -1° M) was reacted similarly with goat anti-FlA
(5.3• 10k 8) or goat anti-FiB (7.5.10 -9 M).
(3) Radioiodinated ligand concentration-aependent
binding experiments. Fixed concentrations of anti-F~A
or anti-F~B were reacted with various amounts of ~25I-F~
or ~25I-prothrombin in buffers containing either 10 mM
EDTA or 10 mM CaCI z. ~25I-F~ of 2.10 3 cpm to 5 • 10 5
cpm (1.5.10 -~° M to 3.8-10 -8 M) were diluted into
buffer A containing 10 mM EDTA or 10 mM CaCI:
and incubated with R5 anti-F~A (2.10 -9 M), R9 anti-
FIB (1.1 "10 -9 M), goat anti-F1A (1.9.10 -8 M), or
goat anti-F~B (2.4.10-7 M). For binding of antibodies
to radioactive prothrombin, 4.7.10 -~1 M to 9.4.10 -9
M of 125I-prothrombin were used to react with 8.10 -8
M of goat anti-F~A or 7.5.10 -9 M of goat anti-F~B.
The data were plotted according to Scatchard [39] in the
form of r/c versus r, where r is the concentration of
the bound ligand per mole of the specific antibody and
c is the concentration of the free ligand. We have tested
the stabilities of the radioactively labeled F1 and pro-
thrombin by incubating them for 48 h with the usual
reagents used in the binding assay (including both sec-
ond antibody systems) and examining their apparent
molecular weights in SDS-polyacrylarrfide gel electro-
phoresis. In either case, a single radioactive peak corre-
sponding to the original protein or peptide was ob-
served, indicating no degradation had occurred during
the binding experiments.
Gel electrophoresis
The disc gel electrophoresis procedure of Davies et
al. [40] as modified by Rosenberg and Waugh [29] was
utilized to establish the homogeneity of polypeptides
with respect to charge. SDS-polyacrylamide gel electro-
phoresis (SDS-PAGE) was performed according to
Laemmli [41] using a 15% crosslinked gel. Proteins were
stained with Coomassie blue or by the periodic acid
Schiff's base method [42,43]. Molecular weight markers
included bovine serum albumin (68 kDa), ovalbumin
(40 kDa), myoglobin (16 kDa). cytochrome c (12 kDa)
and cyanogen bromide-cleaved peptides of myoglobin
and cytochrome c.
Protein determination
Protein concentrations were obtained by measure-
ments at 280 nm, assuming 13.2, 16.2, 11.9 and 12.5 for
1 mg/ml of human prothrombin, thrombin, F1 and
F1 ÷2, respectively [22], or determined by the method of
Lowry et al. [44].
98

Amino acid analysis a!kylated and then precipitated with barium citrate. The
The amino acid compositions of F 1, F1A and FiB precipitate displayed a single band upon SDS-PAGE
were determined by treatment wi ~'~ 6 M HC1 for 24, 48 under nonreducing conditions with an apparent molecu-
and 72 h at l l 0 ° C , and the,, examination of the lar mass of ~ 6 kDa. But like F1, there was a minor
products in a Beckman 121M amino acid analyzer. stained band migrating slightly faster than the major
one when it was electrophoresed under reducing condi-
Results tions. The amino acid composition of this peptide is
shown in Table I. Glutamic acids accounted for -- 1 / 4
Preparation of FI, F~A and F~B of the total amino acid residues, most of which were
F 1 was obtained from a n-,~xture of prothrombin presumably y-carboxylated. The observation that this
fragments by means of barium citrate and ammonium molecule was precipitated by barium ions, its molecular
sulfate precipitations, followed by gel filtration on size and its amino acid composition were consistent
Sephadex G-100. The matrix separated the sample into with the assignment of this peptide being FlA.
two peaks. Fractions containing the smaller molecules The supernatant of the thrombin-digested F1 con-
were pooled, concentrated and rechromatographed on tained two polypeptides when analyzed by SDS-PAGE
the same column to yield an apparently homogeneous and these were separated by chromatography on a
F 1 preparation which migrated with an apparent molec- hydroxyapatite column. The material not adsorbed con-
ular mass of ~ 35 kDa. Upon SDS-PAGE under nonre- tained a single peptide which showed an apparent
duced conditions, a single Coomassie blue-staining band molecular mass of ~ 15 kDa on SDS-PAGE under both
was visible which showed an apparent molecular mass reduced and nonreduced conditions. Upon gel filtration
of ~ 21 kDa. This observation agreed with Bensen and on Sephadex G-100, it also migrated with the same
Hanahan [45] who found that prothrombin fragments molecular mass. Its amino acid composition (Table I),
containing "t-carboxyglutamic acids migrated with a apparent molecular mass and solubility in barium salt
higher apparent molecular mass on gel filtration. In the were consistent with this peptide being FiB.
presence of reducing agent, a second faintly siained
band appeared immediately below the major one. This
second band had been observed before [33], and may be Preparation and specificities of anti-F1A and anti-F1B
due to partial proteolysis of F l within one or more of its Using FIA and FIB obtained above, affinity chro-
five disulfide bonds [10,11]. The amino acid composi- matographic columns were set up to isolate specific
tion of the Fl prepared is shown in Table I which is in antibody populations against these fragments. - 0.15 to
excellent agreement with the published data [10,11,33, 0.2% of rabbit R5 and R9 anti-F l were adsorbed, but
341. -270 of the goat anti-prothrombin antibodies were
F I A and FtB were obtained from thrombin digestion adsorbed by either FIA- or FiB-bound Sepharose col-
of Ft. The proteolytic fragments were reduced and umns under these experimental conditions.
To test for the specificities of these antibodies, we
used prothrombin, F1, F1 +2, FIA and FIB to displace
I ABLE 1 125I-Fi in the competitive radioimmunoassay for R5
A~aino acid compositions of prothrombin fragments Fi, F!A and FIB anti-FlA. As expected, F 1 was the most effective in
displacing the radioactive ligand. Prothrombin was quite
An~no acid a F! F1A 1:1B effective, as only twice the molar excess was needed to
Asp 15.3 3.6 13.0 displace 50~ 12SI-F1. It took 40-times molar excess of
Thr 19.7 6.7 13.3 F IA and 400-times of FIB to compete in the same assay,
Ser 10.8 3.6 7.1 while F l +2 did not react with R5 anti-F1A at all. The
Glu 24.3 12.5 12.9 average slopes of their logit-log dose response curves,
Pro 9.4 0 10.5
Gly 10.8 1.0 9.7 except for Fl + 2, were -0.7.
Ala 13.0 7.0 5.9 The same prethrombin fragments were used to test
Val 8,9 3.3 4,8 for their crossreactivities for R9 anti-FiB. In this case,
Met 0.9 0 0.7 prothrombin and F 1+ 2 had similar slopes of -0.55 and
lie 4,9 0 3.8
were, respectively, 50- and 100-times less effective than
Leu 8,1 3,8 5.3
Tyr 5.5 1.6 3.3 F1 in competing with 125I-F1. FiB, however, competed
Phe 4.7 3,7 1.3 with 125I-F1 for the antibody population only at a large
Lys 4.7 1.8 2.4 molar excess (2000-fold), and with a reduced slope of
His 2.1 0 2.3 -0.4. This crossreactivity was still superior to FIA
Arg 12.7 2.5 9.0
which did not react with anti-FiB at all. Therefo~'e these
a Number of amino acid residues per mole of prothrombin fragment. antibodies showed restricted specificities towards the
Tryptophan not determined. particular antigen that was used as the affinity ligand.
 99

Binding studies of R.5 anti-Fi A and R9 anti-F1B antibod- A

20 //-.-//~
ies o
1.8
y - C a r b o x y g l u t a m i c acids are believed to be responsi-
~ 1.6
ble for binding Ca 2 + in prothrombin, and F 1A contains Ca
1.4
all ten of these residues while Fz B does not contain any.
1.2
It would therefore be of interest to utilize the specific
antibodies as molecular probes to study Ca2+-induced 1.0 , , //--t-/P-r---

conformational changes in F~. We first investigated the 1.6] B

b i n d i n g of the antibodies to ~251 F~ in the presence of
either E D T A or Ca 2+. Fig. 1A shows that each con- '.24./
centration of R5 anti-F~A precipitated more F~ in the
presence of Ca 2+ t h a n in the absence of Ca 2+. On the
contrary, the opposite was true for R9 anti-F~B (Fig. • i'-¢/-r-¢;, =
1B). 0 1.0 2.0 3.0 5.0 iO.O

N e x t we investigated the bindings of ~25I-F] to un- CALCIUM CONCENTRATION (mM)

fractionated R5 a n t i - F t, R5 anti-F]A, unfractionated
R9 anti-F] and R9 anti-FiB, as a function of Ca 2+
concentrations. Fig. 2A shows Ca 2 +-dependent bi~ dings
of F~ to R5 anti-F~ and R5 anti-F t A, with the ~raasition
midpoints at 0.28 m M C a 2+, although / 0 anti-F~ ap-
peared to precipitate m o r e F~ than R5 anti-F] A. Fig. 2B
shows the binding of F~ to R9 anti-F~ also increased
Fig. 2. Calcium dependent binding of R5 and R9 antibodies to F~. (A)
R5 anti-F] (¢) or R5 anti-F]A (o) was reacted with ]251-F] in buffers
containing 0 to 10 mM CaCI 2 in a radioimmunoassay as detailed in
Materials and Methods. The data are expressed as the ratio of the
percentage bound F] to total F] in the presence of Ca 2+ divided by
the percentage of bound F] to total F~ in the absence of Ca 2+. (B) R9
anti-F1 (O) or R9 anti-FiB (0) was reacted with 12Sl-F] in buffers
containing 0 to 10 mM CaC! 2. The experimental procedure and
calculation were performed as given above.

with increasing amounts of Ca 2+, reaching a half-maxi-

mal point at 0.18 m M Ca 2+. But the binding of F ! to R9
60 A
anti-F] B, in contrast, decreased with increasing a m o u n t s
of Ca 2+, and its transition midpoint was higher at 0.45
m M C a 2+.

tll A

' o 2- Q

K
,o
7

0 ! !
2 0.2 0.02
Antibody concentration ( 108M )
100 0., i , t

6-
80 q --"""o B

6O x
:3
0 7
m
or qO
O 2-

!
0 0 I 2 3
1.0 0.1 0.01 r
Antibody concentration ( 108M )
Fig. 3. Interaction of R5 and R9 antibodies with FI in the presence of
Fig. 1. Effect of calcium on the antibody-dependent binding to 1::1. CaCI 2 or EDTA. These data are expressed using a Scatchard analysis
Various concentrations of R5 anti-FiA (A) and R9 anti-FiB (B) were where c is the concentration of free F! and r is the concentration of
allowed to react with 125I-Fi in buffers containing either CaCI2 (O) or F! divided by the concentration of the respective antibody population.
EDTA (0) in a radioimmunoassay as detailed in Materials and (A) R5 anti-FlA was reacted with leSI-F! in the presence of CaCl2 (e)
Methods. The percentage bound F1 per total F] was expressed as a or EDTA (o). (B) R9 anti-FiB was reacted with lesI-F1 in the
function of the antibody concentrations. presence of CaCI 2 (O) or I~DTA (o).
100
1.6
In order to quantitate these Ca2+-dependent effects,
fixed concentrations of antibodies were incubated with 1.4
various concentrations of 125I-F1 in either EDTA- or
CaCl2-containing buffers. As shown in Fig. 3, the O
1.2
rn
Scatchard plots were all nonlinear. R5 anti-FtA could
be described to display two classes of binding sites in m 1.0
the presence of C a 2+, one with an average association
constant (KA) of 1.4.10 9 M -1 and the other 1.4.108 0.8 , o

M-1. The high-affinity binding sites constituted - 38% 0.6

of the total available binding sites (Fig. 3A, Table IIA). 1.0 2.0 3.0 5.0
In the absence of Ca 2 +, R5 anti-F~ A also displayed two Calcium c o n c e n t r a t i o n (mMI
classes of binding sites. The high-affinity sites had the 1.6 m
same K A as when Ca 2 + were present, but they accounted B
• I/'"®
for only - 2~ of the total available sites, with low-af-
finity binding predominated in this case (Fig. 3A, Table
IIA). R9 anti-FIB, on the other hand, bound 1251-FI with
the same high- and low-affinity constants in the pres-
o
CO
,-....
1.2 /
m
ence or absence of Ca 2+, except that the number of 1"0' O~..x:k ~
high-affinity sites was bigger in the absence of Ca 2+
0.8 o
(Fig. 3B, Table IIA). #-o
0.6 ! m ~#..a
1.0 2.0 3.0 5.0
Binding studies of goat anti-F! A and anti-Fj B
When various concentrations of goat anti-F~A was Calcium concentration (raM)
diluted into buffers containing either 10 mM EDTA or Fig. 4. Calcium-dependent binding of goat anti-FiA and anti-FiB to
F ! and prothrombin. These experiments were carried out and calcula-
CaCI 2, it was found that the antibody population bound tion expressed as given in Fig. 2. (A) Goat anti-FiA (0) or anti-FIB
more 125I-F~ in the presence of Ca 2+ than in its absence. ( 0 ) was reacted with tasI-FI in buffers containing 0 to 5 mM CaCI 2.
The opposite was true for goat anti-FmB (data not (B) Goat anti-FIA (e) or anti-FiB ( 0 ) was reacted wi:h t251-pro-
shown). thrombin in buffers containing 0 to 5 mM CaCI 2-
The binding of goat anti-FiA to 125I-F1 was depen-
dent on Ca 2+ with a half-maximal point at 0.24 mM
Ca 2+. On the other hand, the binding of goat anti-F! B In order to determine whether prothrombin also ex-
tO 1251-F! decreased as Ca 2+ increased, while the half- hibited these Ca 2+-dependent effects, ~25l-prothrombin
maximal point was also higher than at 0.51 mM Ca 2+ was used as the radioactive ligand for binding goat
(Fig. 4). antibodies. Similar results to those using ~251-F! as the
radioactive ligand were obtained (Fig. 4B).
Scatchard analyses of the binding between the goat
TABLE Ii antibodies with F1 and prothrombin were then per-
Scott'hard analysis of the interaction of either 1251.FI or l"51-prothrom- formed. The results are presented in Table If. Again all
bin with different antibody populations Scatchard plots were nonlinear and two classes of bind-
ing sites could be used to describe each curve. F 1 bound
High K^ Approx. ,~ Low K A
(M - ! ) total sites ( M - n) goat anti-FiA with about 8-fold higher affinity in the
having presence of Ca 2+ (1.3.101° vs. 1.7.10 9 M - I ) than in
high K^ its absence. On the other hand F 1 bound goat anti-F~B
(A) Using 12Sl-Fi as the ligand for binding antibodies: with twice as many high-affinity sites in the absence of
R5 anti-FiA Ca 2+ 1.4,10 ~ 38 1.4. l0 s C a 2+ (31 vs. 15t~).
EDTA 1,3,10 9 2 1.4.10 s Prothrombin also bound goat anti-F~A with slightly
R9 anti-F, B Ca 2 + 3.5.10 9 33 1.6. l0 s
higher affinity in the presence of Ca 2+. In contrast,
EDTA 3.1.10 9 39 2.1.10 s
Goat anti-FtA Ca 2+ 1.3,101° 30 1,5.10 ~ prothrombin bound goat anti-F~B with 2.5-times higher
EDTA 1.7.10 9 30 1.6.10 8 affinity (4.7.10 9 vs. 1.8-10 9 M - l ) in the absence of
Goat anti-FzB Ca 2+ 2.0.10 9 15 1.5.107 Ca 2+.
EDTA 1.8.10 9 31 7.3-107

(B) Using 1251-prothrombin as the ligand for binding antibodies: Discussion

Goat anti-FiA Ca 2+ 3.8-10 9 30 6.0. l0 s
EDTA 2.5. l0 9 35 4.1. l0 s We have purified human prothrombin fragment 1 to
Goat anti-FiB Ca 2+ 1.8- l0 9 33 1.8. l0 s
apparent homogeneity and from it, the amin:~ terminal
EDTA 4.7-109 40 5.2.10 s
peptides FIA and F1B. They were identified by binding

properties to barium salt, SDS-PAGE, gel filtration and
amino acid compositions. F1A and FiB were utilized as
affiPAty ligands to isolate specific antibodies which
showed restricted specificities toward the respective
ligands and were useful probes to study the conforma-
tional changes induced by Ca 2+ in native 1::1 and pro-
thrombin.
Two types of conformational change could be de-
tected and both were Ca2+-dependent. In the first type,
Ca 2+ was able to stabilize a conformation in F1 when F 1
reacted with R5 anti-FlA (Fig. 1A), or when 1"1 reacted
with unfractionated R5 and R9 anti-F1 (Fig. 2), or when
Ft or prothrombin reacted with goat anti-prothrombin
or goat anti-FlA (Fig. 4). Presumably Ca 2+ bound to
some common conformations in prothrombin, F~ and
F1A and induced conformational changes in these mole-
cules. The binary complex¢s having the new conforma-
tion were more stable and could precipitate more of the
specific antibodies. The interactions were the results of
either higher affinity or larger number of high-affinity
binding sites in the presence of Ca 2+. The half maxima
of this transition were between 0.18-0.28 mM Ca 2+ and
corresponded closely the dissociation constants ob-
tained by direct binding of Ca 2+ to F1 [15], with circular
dichroism [19], fluorescence [16,18] or immunochemical
means [24,25]. Furthermore, the common epitopes on
these molecules were necessarily immunodominant fea-
tures and probably involve the y-carboxyglutamic acid
residues or regions surrounding them. This conforma-
tional change is probably similar to that observed in the
study of F 1 crystal structure. There was no density in
the amino acid sequence 1-35 in the absence of metal
ions [12,13], indicating a disorganized structure in the
y-carboxyglutamic acid-containing region. However, ad-
dition of Sr 2+ ',ransformed the region to become elec-
tron dense and therefore presumably an ordered struc-
ture [46].
The second type of conformational change was de-
tected by R9 anti-FiB and goat anti-F~B. It is also
Ca2+-dependent, but Ca 2÷ in this case destabilized the
new conformation. Ca 2+ probably formed binary com-
plexes with F~ and prothrombin first, lowered the stabil-
ity of the subsequent ternary complexes when anti-F~B
combined with them, and thus gave rise to less precipi-
tates. These interactions were resulted from either lower
affinity binding or smaller number of high-affinity
binding sites in the presence of Ca 2+. The conformer
recognized in prothrombin and F t was probably local-
ized on the FIB portion of these molecules, since the
antibodies which recognized this conformational change
did not recognize F~A in the competitive radioim-
munoassay. However, it cannot be excluded that
neoantigens could have arisen when F~A joins F~B in
native F~ or prothrombin, and that it was this new
format that was destabilized by Ca 2+. Tiffs conforma-
tional change took place with transition midpoints at
101
considerably higher Ca 2+ (0.45-0o61 raM). It is prob-
ably a weaker transition since it was completely masked
by the first type of conformational change when un-
fractionated R5 and R9 anti-F1 interacted with F 1 in the
presence of Ca a+ (Fig. 2). It is of interest to note that
FIB consists of a short helical region and a compact,
organized kringle structure [12,13], in which no
metal-dependent changes have been described before.
The significance of this conformational change is not
known, but it could be involved in the factor Xa-depen-
dent cleavage of prothrombin or in the binding of the
zymogen of the phospholipid surface.
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