Báo cáo khoa học: Identification of amino acids in antiplasmin involved in its noncovalent ‘lysine-binding-site’-dependent interaction with plasmin pptx

Identification of amino acids in antiplasmin involved in its noncovalent
‘lysine-binding-site’-dependent interaction with plasmin
Haiyao Wang, Anna Yu, Bjo¨ rn Wiman and Sarolta Pap
Department of Clinical Chemistry and Blood Coagulation, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden
The lysine-binding-site-mediated interaction between plas-
min and antiplasmin is of great importance for the fast rate
of this reaction. It also plays an important part in regulat-
ing the fibrinolytic enzyme system. To identify structures
important for its noncovalent interaction with plasmin, we
constructed seven single-site mutants of antiplasmin by
modifying charged amino acids in the C-terminal part of the
molecule. All the variants were expressed in the Drosophila
S2 cell system, purified, and shown to form stable complexes
with plasmin. A kinetic evaluation revealed that two mutants
of the C-terminal lysine (K452E or K452T) did not dif-
fer significantly from wild-type antiplasmin in their reac-
tions with plasmin, in either the presence or absence of
6-aminohexanoic acid, suggesting that this C-terminal lysine
is not important for this reaction. On the other hand,
modification of Lys436 to Glu decreased the reaction rate
about fivefold compared with wild-type. In addition, in the
presence of 6-aminohexanoic acid, only a small decrease in
the reaction rate was observed, suggesting that Lys436 is
important for the lysine-binding-site-mediated interaction
between plasmin and antiplasmin. Results from computer-
ized molecular modelling of the C-terminal 40 amino acids
support our experimental data.
Keywords: antiplasmin; fibrinolysis; lysine-binding site; muta-
genesis; plasmin.
The reaction between plasmin and its natural inhibitor in
blood plasma, antiplasmin, normally occurs in several

sequential steps [1–4]. The first step, which is rate limiting,
takes place between one of the so called ‘lysine-binding sites’
in the plasmin molecule and a complementary site in the
antiplasmin molecule. The second step is a noncovalent
interaction between the substrate-binding pocket in the
plasmin active site and the scissile peptide bond in the
reactive centre loop of antiplasmin. Subsequently, peptide
bond cleavage occurs, and, after formation of an ester bond
between the carboxy group of the arginine in the newly
cleaved peptide bond in antiplasmin and the hydroxy group
of the active site serine in plasmin [4,5], major conforma-
tional changes probably occur in both the enzyme and
inhibitor, in a similar manner to that described for other
serine proteinase–serpin reactions [6]. The interaction
between a lysine-binding site in plasmin and a complement-
ary site in antiplasmin is also of importance in regulating the
fibrinolytic system. The same sites in the plasmin molecules
are used in the interaction between plasmin and fibrin [7].
Fibrin-bound plasmin molecules therefore react much more
slowly with antiplasmin compared to free plasmin, thereby
keeping the fibrinolytic process localized [8].
Antiplasmininplasmaisslowlyconvertedintoa
nonplasminogen-binding form, by proteolytic cleavage
and removal of a C-terminal peptide [9,10]. This suggests
that the lysine-binding-site-dependent interaction between
antiplasmin and plasmin occurs in the C-terminal part of
antiplasmin. It is also known that antiplasmin has a lysine as
the C-terminal amino acid [11,12] and that proteins with an
exposed C-terminal lysine typically interact with the lysine-
binding sites in plasmin(ogen) [13,14]. Therefore it is usually

accepted that the C-terminal lysine in antiplasmin is
responsible for its interaction with the plasmin(ogen)
lysine-binding sites [15]. From kinetic experiments using
different natural or synthesized peptides that mimic the
C-terminal part of antiplasmin as inhibitors of the plasmin–
antiplasmin reaction, it was not possible to clearly determine
the specific amino acids involved in this interaction [16–18].
However, it has been suggested that Lys436 in antiplasmin
may be, at least partly, involved in the lysine-binding-site-
mediated interaction with plasmin [17].
We investigated this in more detail, using site-directed
mutagenesis of charged amino acids in the C-terminal
portion of antiplasmin. Our first goal was to produce
mutants in which the C-terminal lysine was replaced by
amino acids without a positive charge. We also produced
variants in which other charged amino acids in this portion
of the molecule were changed to either uncharged residues
or residues of opposite charge. All the antiplasmin variants
were expressed in insect cells (Drosophila S2 cells), purified,
and characterized with regard to their reactions with
plasmin.
Materials and methods
Chemicals and reagents
The vector pMT/BiP/V5 (Invitrogen, Stockholm, Sweden)
was used to express antiplasmin, using the efficient
Correspondence to B. Wiman, Department of Clinical Chemistry,
Karolinska Hospital, SE-17176 Stockholm, Sweden.
Fax: + 46 851776150, Tel.: + 46 851773124,
E-mail:
Enzyme: Human plasmin (EC 3.4.21.7).

(Received 12 February 2003, accepted 17 March 2003)
Eur. J. Biochem. 270, 2023–2029 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03578.x
Drosophila metallothionein (MT) promoter. pMT/BiP/V5
also contains the Drosophila Bip secretion signal, which
efficiently targets high levels of BiP to the endoplasmic
reticulum in the S2 cell line. The Drosophila Schneider S2
cell (DES system, Invitrogen) was cultured in Schneider
medium with
L
-glutamine, heat-inactivated fetal bovine
serum at a final concentration of 10% (v/v), and penicillin–
streptomycin at final concentrations of 50 UÆmL
)1
penicil-
lin G and 50 lgÆmL
)1
streptomycin sulfate. For selection,
the pCoHygro vector (Invitrogen) was used in medium
containing Hygromycin-B (Roche Diagnostics Scandinavia
AB, Stockholm, Sweden). Goat antiplasmin polyclonal
antibody (Biopool AB, Umea
˚
, Sweden) was conjugated
with horseradish peroxidase as described elsewhere [19].
DEAE-Sepharose CL6B and anhydrotrypsin–agarose were
purchased from Amersham Pharmacia Biotech (Uppsala,
Sweden) and Sigma-Aldrich Sweden AB (Stockholm,
Sweden), respectively. Human plasmin was prepared from
purified human plasminogen by activation with strepto-
kinase as described [20]. Native antiplasmin was purified

from human plasma as described [21]. The plasmin
substrate Flavigen Pli was purchased from Biopool AB,
and the inhibitor 6-aminohexanoic acid was obtained from
Sigma-Aldrich Sweden AB.
Construction and expression of wild-type antiplasmin
(wt-antiplasmin)
Plasmid cDNA of wt-antiplasmin (a gift from Roger Lijnen,
Center for Vascular Research, University of Leuven,
Belgium) was subcloned into the expression vector pMT/
Bip/V5 using the BglII and XhoI sites. The nucleotide
sequence of the insert was confirmed by DNA sequencing
and the recombinant protein expressed by transient trans-
fection. The wt-antiplasmin plasmid (19 lg) was transfected
into Drosophila Schneider S2 cells, and CuSO
4
was added
to the medium (final concentration 500 l
M
) to induce
expression. After 24 h, the supernatant was harvested and
the presence of antiplasmin was detected by ELISA. To
obtain a stable cell line, the wt-antiplasmin plasmid and the
selection plasmid pCoHygro were cotransfected into Droso-
phila Schneider S2 cells (3 · 10
6
mL
)1
, in a total volume
of 3 mL), using a concentration ratio between expression
plasmid and selection plasmid of 19 : 1 (19 lg expression

plasmid and 1 lg selection plasmid). The cells were then
grown in the presence of Hygromycin-B at a final concen-
tration of 250 lgÆmL
)1
. The medium was changed every
fourth day, and after 3 weeks a stable cell line that could
express wt-antiplasmin was established.
Mutagenesis of antiplasmin
The cDNA of wt-antiplasmin, subcloned into the expression
vector pMT/Bip/V5 at the BglII and XhoI sites, was used to
produce the selected mutants of antiplasmin. Site-directed
mutagenesis was performed using modified primers result-
ing in the desired modification (Quick Change Mutagenesis
Method; Stratagene, Stockholm, Sweden). The primers,
each complementary to opposite strands of the template
cDNA, were extended during temperature cycling using
PfuTurbo DNA polymerase. The PCR product was then
treated with DpnI endonuclease, which is specific for
methylatedandhemimethylatedDNA.Itisusedtodigest
the parental DNA template, but not newly synthesized
DNA copies. The nicked plasmid DNA containing the
desired mutations was transformed into XL1-Blue super
competent cells. Seven constructs were made to obtain
the following mutants of antiplasmin: K429E; K436E;
E442G; E443G; D444G; K452E; K452T (Table 1, Fig. 1).
The nucleotide sequences of all the constructs were
confirmed. All mutants were then transfected as described
for wt-antiplasmin.
Expression of antiplasmin variants
To express the antiplasmin variants, transfected Drosophila

Schneider S2 cells were cultured and extended in Schneider
medium containing
L
-glutamine, heat-inactivated fetal
bovine serum (final concentration 10%, v/v), 50 UÆmL
)1
penicillin G and 50 lgÆmL
)1
streptomycin sulfate. After the
volume had been increased to 500 mL with a cell concen-
tration of about 3 · 10
6
mL
)1
, the cells were transferred to
thesamevolumeofDrosophila serum-free medium con-
taining
L
-glutamine and penicillin/streptomycin at the same
concentrations as above. Pluronic F68 (final concentration
0.05%) and CuSO
4
at a final concentration of 500 l
M
were
also added. The cells were cultured at room temperature in
darkness for 3 days with gentle stirring. The cells were
removed by centrifugation at 2000 g for 30 min, and the
supernatant containing antiplasmin was stored at )70 °C.
Table 1. Identification of oligonucleotides used to introduce mutations in

antiplasmin cDNA and sequencing antiplasmin variants.
Amino acid
exchanged Nucleotides exchanged Primer length
K429E A1285G G1270–C1303
K436E A1306G, A1308G T1291–G1324
E442G A1324G, G1325C C1309–G1342
E443G A1327G, G1328C G1312–T1345
D444G A1330G, T1331C C1315–C1348
K452E A1354G C1339–G1372
K452T A1354C C1339–G1372
Fig. 1. Schematic presentation of the antiplasmin structure and sites
where mutations were introduced.
2024 H. Wang et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Determination of antiplasmin activity and antigen
concentration
Antiplasmin activity was determined by a titration method
against plasmin of known concentration, essentially as
described [20]. Antiplasmin antigen concentration was
determined by an ELISA method. For this purpose,
Maxisorp microtiter plates (96 wells; Nunc, Labdesign
AB, Stockholm, Sweden) were coated for 2 h at room
temperature with goat anti-antiplasmin IgG, diluted in
0.1
M
NaHCO
3
buffer, pH 9.6. The plates were then
incubated at room temperature for 30 min with 0.04
M
sodium phosphate buffer, pH 7.3, containing 0.1

M
NaCl
and 1 mgÆmL
)1
BSA and washed three times with the
phosphate/NaCl buffer without BSA. The samples (200 lL)
to be analysed were compared with an antiplasmin standard
(purified native antiplasmin from human plasma at the
following concentrations: 40, 20, 10, 5, 2.5 and 0 lgÆL
)1
).
The microtiter plates were incubated for 2 h at room
temperature and then washed four times with the phos-
phate/NaCl buffer. Then, the anti-antiplasmin IgG conju-
gated with horseradish peroxidase was added to the
samples. After incubation for 1 h and washing the plates
four times with phosphate/NaCl buffer, the horseradish
peroxidase substrate o-phenylenediamine (Sigma) in the
presence of H
2
O
2
was added. After another incubation for
10 min, 50 lL stop solution (3
M
H
2
SO
4
) was added to each

well, and A
492
recorded by a microtiter plate reader.
Antiplasmin antigen concentration in the samples was then
calculated from the standard curve obtained.
Purification of antiplasmin variants
Three steps were used for purification of the antiplasmin
variants from the S2 cell cultures. First, the supernatants
(about 500 mL) were incubated with DEAE-Sepharose
CL6B in a batch procedure (about 70 mL, equilibrated with
0.05
M
Tris/HCl buffer, pH 8.0) with slow stirring for 2 h at
4 °C. Each suspension was then filtered through a Bu
¨
chner
funnel and washed with about 1 L 0.05
M
Tris/HCl buffer,
pH 8.0 (until the A
280
was less than 0.1), and then with the
equilibration buffer, containing 0.2
M
NaCl. Anti-
plasmin was then eluted from the Bu
¨
chner funnel using about
100 mL equilibration buffer containing 0.4
M

NaCl. The
fractions containing antiplasmin (detected by ELISA) were
dialysed overnight at 4 °C against 0.05
M
Tris/HCl buffer,
pH 8.0. The dialysate was then applied to a DEAE-
Sepharose CL6B column (2.5 cm diameter · 5 cm), equili-
brated with 0.05
M
Tris/HCl buffer, pH 8.0. After a wash
with this buffer, the column was eluted with a linear gradient
from 0 to 0.4
M
NaCl in the Tris/HCl buffer [5]. The
antiplasmin concentration was determined by ELISA (see
above), and the fractions containing antiplasmin were pooled
and dialysed overnight against 0.04
M
sodium phosphate
buffer, pH 7.5, containing 0.1
M
NaCl. The dialysate was
applied to an anhydrotrypsin–agarose column (column
volume  2 mL), equilibrated with the phosphate/NaCl
buffer. The column was washed with the same buffer until the
A
280
was less than 0.1. Elution was performed with the
equilibration buffer, containing 0.3
M

arginine. Fractions
containing antiplasmin were dialysed against 0.04
M
sodium
phosphate buffer, pH 7.3, and then stored at )70 °C.
SDS/PAGE
SDS/PAGE was performed in a Mini-protean II electro-
phoresis apparatus (Bio-Rad, Stockholm, Sweden) as
described by Laemmli [22]. Proteins were separated in
10% (w/v) polyacrylamide gels and stained with Coomassie
Brilliant Blue R-250.
Determination of rate constants in the reaction
between plasmin and the antiplasmin variants
The two reactants were mixed at low concentrations, in
either 0.1
M
sodium phosphate buffer, pH 7.3, or the same
buffer containing 1.0 m
M
6-aminohexanoic acid. The final
plasmin concentration (active site titrated) used in these
experiments was 0.6 n
M
, whereas the antiplasmin concen-
tration varied between 1 and 5 n
M
. After specified times
of reaction (0–300 s), samples were withdrawn into tubes
containing high concentration of a plasmin substrate
(0.6 m

M
Flavigen Pli, final concentration), 20 m
M
6-aminohexanoic acid and polyclonal rabbit anti-(human
antiplasmin) IgG (1 mgÆmL
)1
). By this procedure further
inhibition of plasmin was rapidly and efficiently decreased,
allowing long incubation times with the plasmin substrate,
which is necessary to accurately measure the low plasmin
concentrations. After incubation for 1.5 h, plasmin cleavage
of the chromogenic substrate was stopped by addition
of acetic acid (final concentration 1%, v/v) and the
A
405
recorded. A
405
is thus a reliable measure of the residual
plasmin concentration at the time of sampling. Then, the
reaction rate constants were calculated from the results
using the classic formula for second-order reactions [1],
using data obtained before 50% of the plasmin was
inhibited. In the experiments performed in the presence of
6-aminohexanoic acid, in which the antiplasmin concentra-
tion was almost 10-fold higher than the plasmin concentra-
tion, pseudo-first-order conditions were assumed and the
reaction rate constants were calculated from the half-lives of
plasmin in these experiments (also before 50% of the
plasmin activity was inhibited).
Computer model of the C-terminal 40 amino acids

in antiplasmin
A computer model of the C-terminal 40 amino acids in
antiplasmin [11] was constructed by
CS CHEM
3
DULTRA
,
version 7.0 (Cambridge Soft, Cambridge, MA, USA),
followed by energy minimization with the MM2 protocol.
Modelling was performed on different lengths of the
C-terminal portion of the antiplasmin molecule, ranging
from 30 to 50 residues from Lys452. However, energy
minimization did not work well on structures with more
than 40 amino acids.
Results
Generation of antiplasmin mutants
Using the QuickChange mutagenesis method, seven anti-
plasmin mutants (K429E, K436E, E442G, E443G, D444G,
K452E and K452T) were constructed. The cDNA structure
was confirmed by nucleotide sequencing. The mutant
Ó FEBS 2003 Antiplasmin–plasmin interaction (Eur. J. Biochem. 270) 2025
K452T contained an unwanted mutation in position 180,
changing the expected Phe to a Leu. As the functional
behaviour of this mutant was found to be almost identical
with the other mutant of this specific residue, K452E, and
with wt-antiplasmin, we did not correct this mistake. The
conservative mutation from one hydrophobic to another
hydrophobic amino acid distant from the C-terminal
portion of antiplasmin seems therefore to be of little
importance.

Expression and purification of antiplasmin variants
wt-antiplasmin and seven antiplasmin mutants were
expressed in Drosophila S2 cells. With the expression
plasmid used (see Material and methods), the antiplasmin
variants were exported via the endoplasmic reticulum to the
conditioned medium, where they are found in soluble
forms. The concentrations of the different antiplasmin
variants in the conditioned medium were typically quite
high, from 5 to 70 lgÆmL
)1
(Table 2). After purification by
DEAE-Sepharose CL6B and anhydrotrypsin–agarose chro-
matography, the typical yield from the harvested condi-
tioned medium was  20%. All antiplasmin variants could
be purified using this procedure. Activity determination by
titration against plasmin of known concentration and
measuring free plasmin with a chromogenic plasmin sub-
strate suggested that all antiplasmin variants were fully or
close to fully active (data not shown).
Formation of SDS-stable complexes between
antiplasmin variants and plasmin
The ability of the different variants to form stable complexes
with plasmin was studied by SDS/PAGE. As shown in
Fig. 2, the most important antiplasmin variants (wt,
K436E, K452E and K452T) were  80% pure after the
described purification procedure and could almost quanti-
tatively form stable complexes with plasmin.
Influence of the antiplasmin variants
on the plasmin–antiplasmin reaction
To study the reaction between the antiplasmin variants and

plasmin in the absence of 6-aminohexanoic acid, plasmin
(final concentration 0.6 n
M
) was mixed with antiplasmin
(final concentration 1.3 n
M
). Samples were taken from 0
to 60 s and added to a mixture of plasmin substrate,
6-aminohexanoic acid and anti-antiplasmin IgG as des-
cribed in Material and methods. After incubation for
90 min at room temperature and addition of acetic acid,
A
405
was recorded. The absorbance value at a certain time,
compared with the absorbance value at zero time, was used
to calculate residual plasmin activity at that time. The
prerequisite was that less than 50% of the added plasmin
had been inhibited. The rate constants were calculated from
the classical formula of second-order reactions [1]. Similar
experiments were performed in the presence of 1.0 m
M
6-aminohexanoic acid. The plasmin concentration was the
same, but the concentrations of the antiplasmin variants
were higher (5.0 n
M
) and the incubation time prolonged
(0–300 s). Residual plasmin activity was measured, and rate
constants were calculated assuming pseudo-first-order kine-
tics. The rate constants for the reactions between plasmin
and the antiplasmin variants are shown in Table 3 (in both

the presence and absence of 6-aminohexanoic acid). The
reactions between ‘native’ human antiplasmin and plasmin
in the presence or absence of 6-aminohexanoic acid were
also studied for comparison. All variants of antiplasmin
except for K436E had a rate constant higher than
10
7
M
)1
Æs
)1
. This is not far from the rate constant obtained
with native antiplasmin. In addition, the method used here
gave very similar results to those reported in earlier studies
[1,2]. Interestingly, the two mutants K452E and K452T did
not differ in activity from wt-antiplasmin, suggesting that
the C-terminal lysine is of little importance in the lysine-
Table 2. Concentration of antiplasmin in the conditioned media from
the various S2 cells expressing the different variants of antiplasmin.
About 500 mL was harvested from each cell line.
Antiplasmin variant
Concentration
(lgÆmL
)1
)
wt-antiplasmin 12.8
K429E 13.6
K436E 72.0
E442G 4.5
E443G 7.7

D444G 6.8
K452E 42.0
K452T 23.3
Fig. 2. SDS/PAGE of some of the antiplasmin
variants in the presence (lanes 1–4) or absence
(lanes 5–8) of plasmin. The antiplasmin vari-
ants shown are: wt (lanes 4 and 8); K436E
(lanes 3 and 7); K452E (lanes 2 and 6); K452T
(lanes 1 and 5). In addition pure plasmin is
showninlane9.
2026 H. Wang et al.(Eur. J. Biochem. 270) Ó FEBS 2003
binding-site-mediated interaction between plasmin and
antiplasmin. On the other hand, the variant K436E reacts
much more slowly (about fivefold) than the other variants,
suggesting that this residue is important in this interaction.
In the presence of 6-aminohexanoic acid, the reaction rate
decreased 10-fold or more for most variants. Also in this
case the results with wt-antiplasmin and the two mutants of
the C-terminal lysine did not differ. Only K436E was less
affected by 6-aminohexanoic acid (2.5-fold decrease in
reaction rate), again suggesting that this residue is involved
in the lysine-binding-site-mediated interaction between
plasmin and antiplasmin.
Molecular modelling of the C-terminal portion
of antiplasmin
The amino-acid sequence of the C-terminal 40 residues in
antiplasmin is GNKDFLQSLKGFPRGDKLFGPDLKL
VPPMEEDYPQFGSPK-OH [11]. The C-terminal lysine is
residue 452 in the antiplasmin molecule. Molecular model-
ling resulted in the structure shown in Fig. 3. We construc-

ted a number of models with different lengths, ranging from
30 to 50 residues from the C-terminal Lys452. All models
were similar around the two sites Lys452 and Lys436. In
these models, the side chain of the C-terminal lysine residue
(K452) seems to be in the near vicinity of the side chain of
Phe448. Lys436, on the other hand, is found at the surface
of the molecule with a protruding side chain.
Discussion
Antiplasmin belongs to the serpin superfamily of proteins [6],
from which many individual members have been crystallised.
The general structures of these proteins are therefore well
established [6]. However, the C-terminal portion of antiplas-
min is unique to this inhibitor [11,12] with no known
similarities to the other members of this protein family.
The lysine-binding-site-mediated interaction between plas-
min and antiplasmin is of great importance in regulating the
fibrinolytic process and keeping the plasmin active in the right
place and at the correct time, as fibrin and antiplasmin
compete for active plasmin molecules [7,8]. In addition, it is
known that the lysine-binding sites in plasminogen are
important for binding to other proteins, such as receptor
proteins at the cellular surface, in both mammalian cells
[13,23] and bacteria [14,24,25]. It has been reported that
plasminogen bound to such receptor proteins is more readily
activated to plasmin [26,27], providing the cells with a
proteolytic shield, and thereby enhancing processes such as
invasive growth and cell migration. Increasing our knowledge
about structures involved in the interaction with the lysine-
binding sites in plasmin(ogen) may be important for finding
new agents that can effectively interfere with these processes.

We have here studied the lysine-binding-site-dependent
interaction between plasmin and antiplasmin in detail
by constructing seven single-site mutants of antiplasmin.
Fig. 3. Computer model of the C-terminal 40 amino acids in antiplasmin. Some of the residues are labelled to facilitate viewing.
Table 3. Rate constants (in 10
6
M
–1
Æs
–1
) in the reactions between plasmin
and the different antiplasmin variants in the absence (No 6-AHA) or
presence (6-AHA) of 1.0 m
M
6-aminohexanoic acid.
Antiplasmin variant No 6-AHA 6-AHA
Native antiplasmin 25.3 ± 1.7 2.5
wt-antiplasmin 10.9 ± 0.3 1.1
K429E 27.3 ± 2.5 2.7
K436E 2.1 ± 0.3 0.8
E442G 19.5 ± 1.0 1.5
E443G 24.3 ± 1.2 1.6
D444G 21.6 ± 0.9 1.6
K452E 11.5 ± 0.7 0.8
K452T 12.7 ± 1.0 0.9
Ó FEBS 2003 Antiplasmin–plasmin interaction (Eur. J. Biochem. 270) 2027
All mutations were performed in the C-terminal 23 residues.
After expression and purification of the different antiplas-
minvariants,theywerecharacterizedwithregardtotheir
reactions with plasmin, both structurally (SDS/PAGE) and

kinetically. The results were compared with results obtained
for ‘native’ human antiplasmin. All antiplasmin variants
were found to be very active, forming stable complexes with
plasmin (Fig. 2) in a comparable way to ‘native’ human
antiplasmin [1,2,4]. The complexes formed were completely
stable during analysis by SDS/PAGE. Also, the rate
constant determined for the reaction between plasmin
and wt-antiplasmin was only slightly lower than that found
for ‘native’ antiplasmin using an identical experimental
system. The latter constant is also very similar to that pre-
viously reported [1,2]. Furthermore, the reaction between
wt-antiplasmin and plasmin is decreased by about one order
of magnitude in the presence of 1 m
M
6-aminohexanoic
acid, which is almost identical with the results with ‘native’
antiplasmin using the same experimental set up (Table 3).
This clearly demonstrates that the overall structure and
main functions of wt-antiplasmin were not altered by the
expression in S2 cells or during purification. As already
pointed out, all the constructed antiplasmin variants formed
SDS-stable complexes with plasmin. In addition, the
reaction rate with plasmin for most of these variants was
comparable to that of the ‘native’ antiplasmin, again
demonstrating the reliability of our techniques.
A major finding in this report is that replacement of the
C-terminal amino acid in antiplasmin, Lys452, with an
acidic residue (Glu) or a neutral hydrophilic residue (Thr)
did not significantly change the activity or kinetic properties.
This is interesting, as it has been generally accepted that this

residue was responsible for the lysine-binding-site-mediated
interaction between antiplasmin and plasmin [15–18]. In
fact, many other proteins with a C-terminal lysine seem to
bind to plasmin(ogen) quite efficiently [13,14]. However, in
antiplasmin the C-terminal lysine does not seem to be
important in this respect.
In the presence of 1.0 m
M
6-aminohexanoic acid, the
reaction rates between plasmin and the antiplasmin variants
typically decreased about one order of magnitude. The only
exception was the variant K436E, for which the reaction
rate decreased by only a factor of 2–3. On the other hand, in
the absence of 6-aminohexanoic acid, it reacted about
fivefold more slowly than the wild-type with plasmin. Both
these findings clearly suggest that Lys436 is important for
the interaction of antiplasmin with the lysine-binding sites in
plasmin. The relatively small (fivefold) difference in reaction
rate suggests that other structures in the vicinity of Lys436
are probably involved, but the positive charge of Lys436
most certainly has a key function. It was previously shown
that Lys436 may be involved in the lysine-binding-site-
mediated interaction between plasmin and antiplasmin
[17]. Replacement of several other charged residues in this
portion of the molecule did not significantly affect the
reaction rate with plasmin.
To shed more light on possible mechanisms explaining
the behaviour of our mutants, we constructed a computer
model of the C-terminal part (40 residues) of antiplasmin
(Fig. 3). We cannot claim that this model shows an

absolutely correct picture of the structure of the C-terminal
portion of antiplasmin. However, there are a large number
of proline residues in this part (10 of the C-terminal 55
residues), increasing the possibility of obtaining a model
that at least partly mimics the true structure. In fact, the
computer model supports our experimental data. The side
chain of Lys452 in this model is found in close vicinity to the
side chain of Phe448. If this is true, it may explain possible
restrictions in the interaction between this residue and the
lysine-binding sites in the intact plasmin molecule. In
addition, the side chain of Lys436 in our computer model
is found at the surface of the molecule and may definitely be
involved in interactions with a lysine-binding site in plasmin.
Some of the mutants, especially K429E and D443G, reacted
slightly more rapidly than wt-antiplasmin with plasmin. The
reason for this is not known.
Since submission of the original version of this paper,
a study on the interaction of a recombinant C-terminal
55-residue peptide from antiplasmin, expressed in Escheri-
chia coli, and isolated ‘kringle’ 1 or ‘kringle’ 4 structures
from plasminogen has been published [28]. The authors
concluded that Lys452 is important in these interactions,
but that other structures may also be involved. In view of
our data with complete molecules, it is indeed possible that
Lys452 may be more involved in interactions with smaller
molecules, such as isolated ‘kringles’, but to a much lesser
extent with the complete plasmin(ogen) molecule. More
work is certainly needed to resolve this question.
Acknowledgements
Skilful technical assistance by Anette Dahlin and Marie Haegerstrand-

Bjo
¨
rkman is gratefully acknowledged. We thank Dr Roger Lijnen,
Center for Vascular Research, University of Leuven, Belgium, for
providing us with antiplasmin cDNA. Financial support was obtained
from the Swedish Medical Research Council (project no. 05193), the
Swedish Cancer Foundation, the Heart and Lung Foundation and
funds from Karolinska Institute.
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