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 Depart
Trang 1Identification 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 antiplasplas-min 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]
Antiplasmin in plasma is slowly converted into a
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: bjorn.wiman@ks.se
Enzyme: Human plasmin (EC 3.4.21.7).
(Received 12 February 2003, accepted 17 March 2003)
Trang 2Drosophilametallothionein (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 (DESsystem, 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)1streptomycin 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 CuSO4was added
to the medium (final concentration 500 lM) 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-philaSchneider S2 cells (3· 106mL)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
methylated and hemimethylated DNA It is used to digest 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)1streptomycin sulfate After the volume had been increased to 500 mL with a cell concen-tration of about 3· 106mL)1, the cells were transferred to the same volume of Drosophila serum-free medium con-tainingL-glutamine and penicillin/streptomycin at the same concentrations as above Pluronic F68 (final concentration 0.05%) and CuSO4at a final concentration of 500 lMwere 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
Fig 1 Schematic presentation of the antiplasmin structure and sites where mutations were introduced.
Trang 3Determination 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
0.1M NaHCO3 buffer, pH 9.6 The plates were then
incubated at room temperature for 30 min with 0.04M
sodium phosphate buffer, pH 7.3, containing 0.1M 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 H2O2was added After another incubation for
10 min, 50 lL stop solution (3MH2SO4) was added to each
well, and A492 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
0.05MTris/HCl buffer, pH 8.0) with slow stirring for 2 h at
4C Each suspension was then filtered through a Bu¨chner
funnel and washed with about 1 L 0.05MTris/HCl buffer,
pH 8.0 (until the A280was less than 0.1), and then with the
equilibration buffer, containing 0.2M NaCl
Anti-plasmin was then eluted from the Bu¨chner funnel using about
100 mL equilibration buffer containing 0.4M NaCl The
fractions containing antiplasmin (detected by ELISA) were
dialysed overnight at 4C against 0.05MTris/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 0.05MTris/HCl buffer, pH 8.0 After a wash
with this buffer, the column was eluted with a linear gradient
from 0 to 0.4M 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 0.04M sodium phosphate
buffer, pH 7.5, containing 0.1MNaCl 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
A280 was less than 0.1 Elution was performed with the
equilibration buffer, containing 0.3M arginine Fractions
containing antiplasmin were dialysed against 0.04Msodium
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 0.1Msodium phosphate buffer, pH 7.3, or the same buffer containing 1.0 mM6-aminohexanoic acid The final plasmin concentration (active site titrated) used in these experiments was 0.6 nM, whereas the antiplasmin concen-tration varied between 1 and 5 nM After specified times
of reaction (0–300 s), samples were withdrawn into tubes containing high concentration of a plasmin substrate (0.6 mM Flavigen Pli, final concentration), 20 mM
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
A405recorded A405is 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 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 CSCHEM3D ULTRA, 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
Trang 4K452T 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 nM) was mixed with antiplasmin (final concentration 1.3 nM) 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,
A405was 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 mM
6-aminohexanoic acid The plasmin concentration was the same, but the concentrations of the antiplasmin variants were higher (5.0 nM) 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
107M )1Æs)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)
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 shown in lane 9.
Trang 5binding-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 antiplasplas-min 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 106M 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.
Trang 6All mutations were performed in the C-terminal 23 residues.
After expression and purification of the different
antiplas-min variants, they were characterized with regard to their
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 mM 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 mM 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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