From the
To describe the role of the lysyl binding site in the
interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a
forming fibrin clot, we performed binding experiments with domain
deletion mutants GK1K2P, K2P, and the corresponding point mutants
lacking the lysyl binding site in the absence and the presence of
Only in the presence of fibrin t-PA
Based on binding isotherms of t-PA to a forming fibrin clot,
two independent nonidentical binding sites on the fibrin are proposed
(Nesheim et al., 1990). The high affinity interaction is F
domain mediated, while a lower affinity interaction is K2 domain
mediated. However, this model is questioned by the observation of one
class of t-PA binding sites on a forming fibrin clot. Furthermore, the
high affinity of t-PA for a forming fibrin clot could not be fully
accounted for by the F-mediated and the K2-mediated interaction
(Horrevoets et al., 1994).
The role of the lysyl binding
site of t-PA in fibrin binding is intricate. The K2 domain is thought
to interact via an intra-chain lysyl residue of the fibrin network, and
therefore the binding site was called aminohexyl binding site. It was
shown that increasing the amount of carboxyl-terminal lysyl residues in
the fibrin network by partial degradation with plasmin results in new
binding sites for t-PA (de Vries et al., 1989). Although, the
affinity of the K2 domain for aminohexyl-Sepharose differs from the
affinity for lysyl-Sepharose, both interactions can be inhibited with
EACA (de Munk et al., 1989). Furthermore, deletion of the
lysyl binding site in the K2 domain by the substitution of one amino
acid residue (Asp
Substitution of one amino acid residue in the K2 domain
(t-PA (D236N), t-PA (D236A) results in a t-PA analogue that no longer
interacts with lysyl- or aminohexyl-Sepharose but still possesses high
affinity for fibrin (Weening-Verhoeff et al., 1990; Bennet
et al., 1991). This observation stands in clear contrast to
the large effect of EACA on the fibrin binding of t-PA (van Zonneveld
et al., 1986b, de Munk et al., 1989).
We studied
the role of the lysyl binding site of t-PA in fibrin binding by
performing fibrin binding experiments with domain deletion mutants
lacking a functional lysyl binding site in the absence and presence of
EACA. To describe the interaction site of t-PA and t-PA variants on a
forming fibrin clot, we performed competition experiments with FGK1K2P
and K2P.
We have found that for fibrin binding of t-PA, in addition
to the F and the lysyl binding site-mediated interactions, other
interactions must also exist. Furthermore, the binding sites of FGK1K2P
(D236N) and the K2P on a forming fibrin clot appear to be in close
proximity to each other. The lysyl binding site in the K2 domain
appears not to interact directly with an aminohexyl group of the fibrin
network, but it is probably involved in stabilizing a favorable
conformation of t-PA needed for fibrin binding.
Construction
of t-PA D236N del (R7-C168) was performed as follows. From the plasmid
containing the sequence coding for FGK1K2P (D236N) (Weening-Verhoeff
et al., 1990), a PstI partial fragment of 4249 bp
(missing the FGK1 fragment of 486 bp) was isolated and ligated with
itself according to Sambrook et al.(1989).
On-line formulae not verified for accuracy
880 nM plasminogen-free fibrinogen
was incubated with radiolabeled t-PA analogues (final concentration,
0.1 nM) and t-PA (final concentration, approximately 0.5
µM), K2P (final concentration, approximately 0.5
µM), or human serum albumin (final concentration,
approximately 0.3 µM) in 15 mM Veronal, 140
mM NaCl, 0.5 mM CaCl
The first models describing the interaction of t-PA with a
forming fibrin clot were based on the idea that t-PA not only consists
of structurally autonomous domains but also of functionally autonomous
domains (van Zonneveld et al., 1986a). In these first models,
there is a prominent role for the F and the K2 domain (van Zonneveld
et al., 1986b; Verheijen et al., 1986; de Vries
et al., 1990; Nesheim et al., 1990; Horrevoets et
al., 1994). However, a model in which the K1 domain plays an
important role in the interaction of t-PA to preformed fibrin has been
described (Kaczmarek et al., 1993). The F/K2 models may be
further subdivided into models in which the t-PA interaction sites on
the fibrin are in juxtaposition (van Zonneveld et al., 1986b;
de Vries et al., 1990; Horrevoets et al., 1994) and a
model in which the t-PA interaction sites are further apart (Nesheim
et al., 1990). All F/K2 models stress the importance of an
direct interaction between the lysyl binding site in the K2 domain and
a lysyl side chain of the fibrin network. Besides these models, in
which the functional autonomy of domains is stressed, a model was
presented in which the fibrin interaction sites in t-PA were spread
over many domains, except the K2 domain (Bennet et al., 1991).
Our results with the GK1K2P (D236N) a molecule that shows
considerable interaction with a forming fibrin clot, suggest that
besides the F and K2-mediated interaction, other fibrin interaction
sites in t-PA exist. Interestingly, this molecule also shows enhanced
plasminogen activation in the presence of fibrin. This indicates that
besides the finger domain and lysyl binding site in the K2 domain,
other domains of t-PA are involved in fibrin-dependent plasminogen
activation. Remarkably, the fibrin binding of FGK1K2P (D236N) a
molecule that no longer can interact via its lysyl binding site in the
K2P part can be competitively inhibited by K2P. It seems therefore
unlikely that the binding sites on fibrin for K2P and FGK1K2P (D236N)
are far apart on the fibrin surface. This result questions the model of
Nesheim (Nesheim et al., 1990) in which no such competition
would be expected.
The role of the lysyl binding site in binding to
a forming fibrin clot is more complicated than expected. In the
presence of 5 mM EACA, fibrin binding is more perturbed than
after the deletion of the lysyl binding site. Such a result could be
explained by steric hindrance. EACA binding to the lysyl binding site
blocks the fibrin binding site and so reduces fibrin binding. Deletion
of the lysyl binding site abolishes the interaction with EACA and
therefore a possible inhibition of fibrin binding by EACA should no
longer be possible. To test this steric hindrance hypothesis, we
studied the fibrin binding of K2P in more detail. Fibrin binding of
this molecule can be completely inhibited by 5 mM EACA. It is
known that the dissociation constant of EACA for K2P is approximately
100 µM (Byeon et al., 1991; de Munk et
al., 1989), 2 orders of magnitude higher than the dissociation
constant of binding of K2P to fibrin (C
A more likely explanation for
this effect is to assume additional indirect effects of occupation of
the lysyl binding site such as induction of a conformational changes in
the t-PA molecule. Conformational changes upon occupation of a lysyl
binding site in the closely related and structurally similar molecule
plasminogen have been described (Markus et al., 1978;
Christensen and Molgaard, 1992). Plasminogen can occur in a closed
conformation in the absence of EACA and an open conformation in the
presence of EACA. The most likely explanation for this behavior is the
occupancy of the lysyl binding site by an intramolecular lysyl or
arginyl residue in the closed conformation (Ponting and Marshall,
1992). Circumstantial evidence suggests that a similar mechanism could
be operating in t-PA since the solubility of t-PA increases
considerably on addition of lysine or arginine (Hasegawa and Kondo,
1985; Ichimura, 1987). Furthermore electron microscopic studies suggest
that the structure of the molecule is ellipsoidal with the domains
folded toward each other (Margossian et al., 1993). Based on
differential scanning calorimetry experiments, an interaction between
the FG domains and the P domain was predicted. Although no involvement
of the lysyl binding site could be detected (Novokhatny et
al., 1991) a strong interdomain interaction involving lysyl
binding sites has been observed in crystals of kringle 2 domain (de Vos
et al., 1992). Recently it was shown that replacement of
stretches of charged amino acid residues containing lysyl residues or
arginyl residues by alanyl residues influences the fibrin binding of
the resulting t-PA analogue (Bennet et al., 1991).
In view
of these data, we propose an alternative model for the high affinity
interaction of t-PA with fibrin and the role of the lysyl binding site
in this (Fig. 3). In analogy to plasminogen, t-PA could occur in
two conformations, an open conformation and a closed conformation. The
interaction between the lysyl binding site in the K2 domain and a lysyl
residue stabilizes the closed conformation. The addition of EACA or
mutation of the lysyl binding site would free t-PA in a more open
conformation. In the closed conformation, the affinity for fibrin is
higher than in the open conformation. It has not escaped our notice
that occupation of the lysyl binding site in t-PA by EACA could reflect
a first step in the pathway of fibrin-dependent plasminogen activation.
Occupation of the lysyl binding site by plasminogen would free the P
domain of t-PA from the fibrin surface making it available for the
hydrolysis of the Arg
Radiolabeled t-PA
analogues were applied to a 1-ml lysyl-Sepharose column. Run-through
was collected. The columns were washed with 2.5 ml of buffer and eluted
with 2.5 ml of buffer containing 50 mM lysine analogue EACA.
The radioactivity present in the flow-through, wash fraction, the
elution fraction, and remaining on the columns was determined and
expressed as a fraction of the total radioactivity. For details, see
``Experimental Procedures.''
The
amount of two-chain t-PA variant was determined using the amidolytic
substrate S-2288 as described under ``Experimental
Procedures.'' Plasminogen activator activity of t-PA and the t-PA
variants in the absence (column 2) and presence of CNBr fragments of
fibrinogen (column 3) were determined as described under
``Experimental Procedures'' and expressed as
Column 1, t-PA analogues studied. Columns
2-5, C
Radiolabeled t-PA or t-PA
analogue was incubated with 880 nM fibrin(ogen) in the absence
(column 2) or in the presence of 0.5 µM FGK1K2P (column 3)
or 0.5 µM K2P (column 4). After clotting, the fraction of
total t-PA bound to fibrin was determined. The numbers in the table
represent the fraction of total t-PA bound to the clot. The standard
deviation was calculated from three data points. NB, no binding
observed. For details, see ``Experimental Procedures.''
We thank Drs. D. C. Rijken and W. Nieuwenhuizen for
critically reading the manuscript and for helpful suggestions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amino caproic acid (EACA). Occupation of the lysyl binding site
in the K2 domain with EACA has a pronounced effect on the binding of
FGK1K2P to a fibrin clot (C
= 77 ±
11 nMversus 376 ± 45 nM with EACA).
Deleting the lysyl binding site in the K2 domain (substitution D236N)
also impairs fibrin binding but to a lesser extent (C
= 169 ± 20 nM). Although the binding of
K2P to a fibrin clot is weak (C
= 1163
± 490 nM), it still is 2 orders of magnitude stronger
than the binding of EACA to K2P. Therefore it was surprising to find
that deletion of the lysyl binding site in K2P completely abolishes
fibrin binding. Even when both the F domain and the lysyl binding site
were deleted, considerable fibrin binding is still observed
(C
= 557 ± 126 nM),
suggesting other than F and K2-mediated interactions. The binding of
FGK1K2P, FGK1K2P (D236N), GK1K2P, and GK1K2P (D236N) to fibrin could be
competitively inhibited by FGK1K2P and K2P, indicating that all
molecules recognize the same interaction sites on a fibrin clot. Based
on these results, a new model for the interaction of t-PA with a
forming fibrin clot is proposed. The fibrin binding sites in t-PA are
not confined to the F and K2 domain. The main role of the lysyl binding
site in the K2 domain of t-PA appears indirect rather than direct, most
likely stabilizing a conformation favorable for fibrin binding.
(
)
efficiently converts its substrate plasminogen into the
fibrin-degrading enzyme plasmin. The enzyme appears to play an
essential role in dissolving fibrin rich clots in the bloodstream
(Thorsen et al., 1972; Collen, 1980; Carmeliet et
al., 1994). Fibrin binding of t-PA is thought to be a prerequisite
for this enhanced plasminogen activation (Hoylaerts et al.,
1982; R, 1982; Nieuwenhuizen et al., 1985). This
fibrin binding is localized in the heavy chain of t-PA (Rijken et
al., 1986). After the elucidation of the cDNA structure, it became
apparent that t-PA is composed of several domains (Pennica et
al., 1983; Ny et al., 1984). From the amino terminus,
t-PA consists of a finger domain (F), an epidermal growth factor domain
(G), two kringle domains (K1, K2), and a serine protease domain (P).
Both the F and K2 domain were found to be involved in fibrin binding to
a forming fibrin clot (Verheijen et al., 1986; van Zonneveld
et al., 1986a). It was further shown that t-PA interacts with
lysyl-Sepharose and arginyl-Sepharose (Radcliffe and Heinze, 1978;
Wallen et al., 1981). The interaction with lysyl-Sepharose can
be disturbed by L-lysine, L-arginine, or the lysine
analogue
-amino caproic acid (EACA) (Radcliffe and Heinze, 1978;
Allen and Pepper, 1981; de Munk et al., 1989). The binding of
t-PA with fibrin can be partially blocked with the lysine analogue EACA
(van Zonneveld et al., 1986b, de Munk et al., 1989).
Subsequently, isolated kringle 2 domains were shown to interact with
EACA and lysyl-Sepharose (Byeon et al., 1991; de Serrano and
Castellino, 1993; de Vos et al., 1992). This led to the view
that t-PA binds to a forming fibrin clot via two modes: a lysyl binding
site-mediated interaction and a non-lysyl-dependent interaction that
requires the presence of the F domain (van Zonneveld et al.,
1986b).
replaced by Asn
)
abolishes binding to aminohexyl- and lysyl-Sepharose (Weening-Verhoeff
et al., 1990). Therefore, the structures in the K2 domain
mediating lysyl binding and aminohexyl binding must be considered
equivalent.
Proteins Used in this Report
Nomenclature and
numbering of t-PA mutant proteins is according to Pannekoek et al. (1990). Recombinant t-PA (referred to as FGK1K2P), t-PA del
(I5-H44) (referred to as GK1K2P), t-PA del (R7-C168) (referred to as
K2P), and the point mutant t-PA D236N (referred to as FGK1K2P (D236N))
have been described before (Verheijen et al., 1986;
Weening-Verhoeff et al., 1990). The construction of the
corresponding domain deletion mutants t-PA D236N del (I5-H44) (referred
to as GK1K2P (D236N)) and t-PA D236N del (R7- C168) (referred to as K2P
(D236N)) are described below.
Construction of Mutant Proteins
The construction
of the t-PA D236N del (I5-H44) was performed as follows. From the
plasmids containing the reading frame for GK1K2P (peV2t-PA4) (Verheijen
et al., 1986), a 3835-bp NarI-SacI
restriction fragment lacking the K1K2 and part of the P domain was
isolated. From the plasmid containing the sequence coding for FGK1K2P
(D236N) (Weening-Verhoeff et al., 1990) a 900-bp
NarI-SacI restriction fragment containing K1K2 (with
the D236N substitution) and part of the P domain was isolated. This
fragment was ligated into the above mentioned 3835-bp fragment
according to Sambrook (Sambrook et al. 1989).
LB6 Cell Transfections
t-PA expression plasmids
were used to transfect mouse L cells (LB6) by calcium phosphate
co-precipitation with peV2/Neo, which contains the gene for
aminoglycoside phosphotransferase 3` (Graham and van der Eb, 1973).
Cells that incorporated the plasmids and thus were Neo-resistant were
selected in Dulbecco's modified Eagle's medium supplemented
with 10% (v/v) fetal calf serum (Boehringer Mannheim),
L-glutamine (Life Technologies, Inc.), 100 units of
penicillin/ml, 100 µg/ml streptomycin (Life Technologies, Inc.),
and 1.2 mg/ml of the neomycin analogue geneticin (Life Technologies,
Inc.). For purification of the recombinant proteins, cells were
cultured in Dulbecco's modified Eagle's medium supplemented
with 100 KIU/ml Trasylol (Bayer, Leverkusen, Germany) and 10
mM -amino caproic acid (Merck, Darmstadt, Germany) to
prevent plasmin activity, 0.3 g/liter human serum albumin (CLB,
Amsterdam, The Netherlands) L-glutamine, 100 units of
penicillin/ml, 100 µg/ml streptomycin, and 1.2 mg/ml geneticin.
Recombinant t-PA mutants were purified by immunoaffinity chromatography
using a monoclonal antibody ESP-2 (Campro Scientific, The Netherlands)
against the protease domain of t-PA coupled to agarose. A 0.5-ml
aliquot of anti-t-PA-Sepharose suspension was placed on a disposable
PD-10 gel filtration column (Pharmacia Biotech Inc.). The tandem column
was equilibrated with 0.1 M Tris-HCl, 0.01% (v/v) Tween 80, pH
7.5. Approximately 50 ml of conditioned medium was loaded onto the
column followed by washing with the buffer mentioned above. The column
was then washed with 2 column volumes of a buffer containing 0.1
M Tris-HCl, pH 7.5, 1.0 M NaCl, and 0.01% (v/v) Tween
80. Subsequently, the column was reequilibrated with the same buffer
without NaCl. The t-PA mutant was eluted from the column with a buffer
containing 0.1 M Tris-HCl, 0.01% (v/v) Tween 80, and 3.0
M KSCN, pH 7.5 (Merck). Column fractions containing
plasminogen activator activity were pooled for further
characterization.
Gel Electrophoresis and Zymography
Polyacrylamide
gel electrophoresis in the presence of SDS was performed under
nonreducing conditions on 10% acrylamide gels with 5% stacking gels
using the Laemmli system (Laemmli, 1970). After electrophoresis, gels
were washed in 2.5% (v/v) Triton X-100 to remove SDS and placed on
plasminogen-containing fibrin agarose layers (Granelli-Piperno and
Reich, 1978). Upon incubation, the positions of plasminogen activators
appear as clear lysis zones on an opaque background.
Conversion of the Single-chain to the Two-chain Form of
t-PA and t-PA Analogues
Conversion of the single-chain form t-PA
analogues to the two-chain form was performed as described previously
(Wallén et al., 1981). In short, plasmin-Sepharose
slurry (200 µg plasmin/g of wet Sepharose-4B or Sepharose-4B
(Pharmacia) was washed with 10 mM Tris-HCl, pH 7.5 and 0.01%
(v/v) Tween 80. 50 µl of a 50% plasmin-Sepharose suspension or 50%
Sepharose-4B suspension was added to 450 µl of t-PA analogue (10
pmol) in the same buffer. The reaction was carried out with constant
mixing at 37 °C. Samples were removed from the incubation mixture
at time intervals (t = 0, t = 10, t = 20, t = 30, t = 60, t = 90 min) centrifuged down, and 20 µl was transferred
to wells of a microtiterplate containing 130 µl of 100 mM
Tris-HCl, pH 7.4, 0.1% (v/v) Tween 80 and 20 KIU/ml Trasylol.
Conversion of t-PA analogues from the single-chain to the two-chain
form was confirmed by spectrophotometric activity determination with
S-2288
(H-D-Ile-L-Pro-L-Arg-p-nitroanilide
dihydrochloride, Chromogenix, Mölndal, Sweden). Spectrophotometric
assays were performed as described previously (Verheijen et
al., 1985). Briefly, the reaction mixture (250 µl total
volume) contained plasmin-Sepharose or Sepharose-treated t-PA
analogues, 100 mM Tris-HCl, pH 7.4, 0.1% (v/v) Tween 80,
Trasylol 20 KIU/ml, and 1 mM S-2288. The absorbance of the
reaction mixtures was measured at 405 nm in an eight-channel microtiter
plate reader against suitable blanks without termination of the
reaction. The absorbance at 405 nm was plotted against time
(A/
t) for six time points.
Labeling of t-PA and t-PA Analogues
For labeling
of two-chain t-PA and t-PA analogues, an active site-directed inhibitor
of t-PA was used. The inhibitor 4-aminobenzoyl-Gly-Arg-CHCl
(a kind gift of Dr. E. Shaw) was iodinated with
I and
purified as described before (Rauber et al., 1988). 1.5 pmol
of t-PA analogues in 100 µl of 0.1 M Tris-HCl, pH 7.5, and
0.1% (v/v) Tween 80 were incubated with 2 µM iodinated
inhibitor for 4 h at room temperature. Radiolabeled t-PA and t-PA
analogues were bound on a 1-ml column of zinc chelate-Sepharose and
extensively washed with 0.02 M Tris-HCl, pH 7.4, 1 M
NaCl, and 0.01% (v/v) Tween 80, and then eluted with the same buffer
containing 100 mM imidazole (Merck, Darmstadt, Germany).
Specific activities of the labeled t-PA or t-PA analogues (final
concentration, 5 nM) were approximately 2.6 10
cpm/pmol.
Binding of Two-chain t-PA or t-PA Analogues to
Lysyl-Sepharose
Binding to a lysyl-Sepharose column was
performed as described previously (de Munk et al., 1989). In
short, radiolabeled two-chain t-PA analogues (approximately 100 fmol in
500 µl) in buffer (0.1 M Tris-HCl, pH 7.4, 0.4 M
NaCl, 0.01% (v/v) Tween 80) were applied to 1-ml lysyl-Sepharose
columns (Pharmacia) equilibrated in the same buffer (flow rate, 50 ml/h
at room temperature). These columns were washed with 2.5 ml of buffer.
Specifically bound analogues (see below) were eluted with 2.5 ml of
buffer containing 50 mM EACA. Radioactivity was assessed in
the run-through, in washing fluid, in eluate, and in the column. The
fraction of the total counts/min is given.
Binding of t-PA or t-PA Analogues to a Forming Fibrin
Clot
Fibrin binding was performed as described previously (de
Munk et al., 1989). Radiolabeled t-PA analogues (0.06
nM, final concentration) were mixed with fibrinogen
(Chromogenix, Mölndal, Sweden), which was depleted of plasminogen
and plasmin as described before (de Munk et al., 1989) in a
buffer containing 15 mM Veronal, 140 mM NaCl, 0.5
mM CaCl, 0.2 mM MgCl
, 5
mM Tris-HCl, 0.005% Tween 80, and 500 Trasylol KIU/ml, pH
7.75. After 1 h of incubation at 37 °C, clots were centrifuged, and
radioactivity in the supernatant was determined. t-PA bound was
expressed as the fraction of the total amount of t-PA analogue added to
the fibrinogen solution (F). The data were fitted to the
Equation 1.
Determination of the t-PA and t-PA Analogue
Concentration
Spectrophotometric assays were performed as
described previously (Verheijen et al., 1985). Briefly, the
reaction mixture (250 µl total volume) contained plasmin-treated
t-PA analogues in 100 mM Tris-HCl, pH 7.4, 0.1% (v/v) Tween
80, and 1.0 mM S-2288. The two-chain t-PA or t-PA analogue
sample was tested at four different concentrations. The absorbance
change at 405 nm (A/
t) for each
concentration was determined. These were plotted against the four
different concentrations of t-PA or t-PA analogues, representing the
absorbance change/concentration t-PA analogue. The absorbance
change/concentration for a known amount of t-PA standard was compared
with the absorbance change/concentration of the t-PA analogues. Since
the amidolytic activity for the P domain of the t-PA standard is
similar to the amidolytic activity of the P domain of the t-PA
analogues, the concentration of t-PA was calculated (Bakker et
al., 1993).
Determination of the Stimulation
Factor
Spectrophotometric assays were performed as described
previously (Verheijen et al., 1982). Briefly, the reaction
mixture (250 µl of total volume) contained various amounts of
plasmin-treated t-PA analogues, 100 mM Tris-HCl, pH 7.4, 0.1%
(v/v) Tween 80, 0.12 µM Glu-plasminogen, and 0.7
mM S-2251. In certain cases, 120 µg/ml of CNBr-digested
fibrinogen were included. The absorbance of the reaction mixtures was
measured at 405 nm in an eight-channel microtiter plate reader against
suitable blanks without termination of the reaction. The t-PA analogue
sample was tested at 10, 20, and 40 pM (final concentration)
of active enzyme both for reaction mixtures containing fibrinogen
fragments and for reactions mixtures without fragments. Fibrinogen
fragments were prepared as described previously (Verheijen et
al., 1982). The enhancement factors were determined as follows.
The change in absorbance was monitored over time for each t-PA analogue
in the presence and in the absence of CNBr digest of fibrinogen. For
each enzyme concentration, a slope was calculated, representing the
absorbance change over time squared
(A/
t
). These slopes, in turn,
were plotted against enzyme concentration, representing the absorbance
change/time squared/molar concentration of enzyme, and finally
expressed as
A h
pmol
. The ratio of the slope in the presence
of fibrinogen fragments to the slope in the absence of fragments is the
enhancement factor. This ratio reflects the extent to which fibrinogen
fragments enhance the activity of the particular t-PA analogue
preparation.
Competition Experiments
t-PA (final concentration,
approximately 4 µM) and K2P (final concentration,
approximately 8 µM) were inactivated with 50
µM PPACK in 0.1 M Tris-HCl, pH 7.5, 0.01% (v/v)
Tween 80, and 1 M NaCl for 6 h at room temperature.
PPACK-treated t-PA and K2P were separated from PPACK with a Sephadex
G-50 fine column equilibrated in 0.1 M Tris-HCl, pH 7.5, 0.01%
(v/v) Tween 80, and 1 M NaCl. The inhibition of t-PA and K2P
was confirmed using a spectrophotometric assay (see ``Determination of the t-PA Analogue
Concentration.'')
, 0.2 mM
MgCl
, 5 mM Tris-HCl, and 0.005% (v/v) Tween pH
7.75. Clotting was performed with 2 NIH units of thrombin/ml. After 1 h
of incubation at 37 °C, clots were disrupted by vortexing. After
centrifugation, radioactivity in the supernatant was determined. t-PA
bound is expressed as the fraction of total added amount of t-PA
analogue.
Characterization of the Recombinant Proteins
To
study the role of the lysyl binding site in the K2 domain of t-PA in
the interaction of t-PA with a forming fibrin clot, we constructed t-PA
domain deletion analogues in which the lysyl binding site is impaired
by a single amino acid substitution, FGK1K2P (D236N), GK1K2P (D236N),
and K2P (D236N). The recombinant proteins show the expected molecular
weight on a zymogram (Fig. 1).
Figure 1:
Fibrin zymography of t-PA analogues.
FGK1K2P (laneA), GK1K2P (laneB),
K2P (laneC), FGK1K2P (D236N) (laneD), GK1K2P (D236N) (laneE), and K2P
(D236N) (laneF) were isolated from culture media by
affinity chromatography. Gel electrophoresis and zymography were
performed as described under ``Experimental Procedures.''
High molecular weight standards were run in a separate lane (not
shown).
All of the domain deletion
variants specifically interact with lysyl-Sepharose. The mutation D236N
in the different domain deletion mutants results in a loss of lysyl
binding capacity (). All molecules convert plasminogen to
plasmin (). In comparison with the t-PA variants, which
show interaction with lysyl-Sepharose, the t-PA variants lacking the
lysyl binding site activate plasminogen with lower efficiency
(, column 2). In the presence of CNBr-digested fibrinogen
as a fibrin mimic, the plasminogen activation of all molecules was
enhanced (, column 3). The FGK1K2P and GK1K2P molecules
showed a higher enhancement of plasminogen activation than the
corresponding lysyl binding site mutants FGK1K2P (D236N) and GK1K2P
(D236N). Interestingly K2P (D236N), which shows no interaction with a
forming fibrin clot (see below), still activates plasminogen in the
presence of fibrin mimic as efficiently as K2P.
Fibrin Binding Sites within t-PA
Fibrin binding
experiments were performed with low concentrations of t-PA or t-PA
analogues (<0.1 nM), and the fraction of total t-PA or t-PA
analogues bound at different fibrin(ogen) concentrations was assessed
(Fig. 2). For t-PA, a high affinity interaction with a forming
fibrin clot is found (Fig.2A). When the
C of this interaction was determined, a value of
77 ± 11 nM was found (I). Fibrin binding
of t-PA in the presence of 5 mM EACA is markedly reduced
(Fig. 2A). Determination of the C
for this
interaction resulted in a value of 376 ± 46 nM
(I). Deletion of the lysyl binding site (FGK1K2P (D236N))
also effects the binding to a forming fibrin clot
(Fig. 2B), resulting in a C
value
of 169 ± 20 nM (I). The presence of 5
mM EACA had no influence on the fibrin binding of FGK1K2P
(D236N), confirming the absence of a functional lysyl binding site in
this molecule. Deletion of the F domain in t-PA (GK1K2P) reduces fibrin
binding (Fig. 2C). Blocking the lysyl binding site in
GK1K2P with 5 mM of EACA resulted in a lowered fibrin binding.
Surprisingly, even when the F domain and the lysyl binding site in t-PA
are lacking (GK1K2P (D236N)), considerable fibrin binding is still
observed (Fig. 2D, C
GK1K2P
(D236N); 557 ± 126 nM (I)). As noticed
with t-PA, occupying the lysyl binding site with EACA has a greater
effect on fibrin binding than deleting the lysyl binding site. The K2P
binding to fibrin is weak (Fig. 2E,
C
1163 ± 490 nM (I))
and is completely inhibited in the presence of 5 mM EACA.
Deleting the lysyl binding site in this molecule also abolishes fibrin
binding (Fig. 2F).
Figure 2:
Experimental determination of the fraction
of total t-PA bound (F) at various fibrin(ogen)
concentrations. Radiolabeled t-PA was incubated with various amounts of
fibrinogen (0-3.4 µM) in the absence and presence of
EACA. After clotting, the amount of radiolabeled t-PA or t-PA analogue
bound to the fibrin clot was determined. On the y axis, F = the fraction of total t-PA bound to the fibrin clot; on
the x axis is the amount of fibrin(ogen) present in the clot.
A, FGK1K2P; B, FGK1K2P (D236N); C, GK1K2P;
D, GK1K2P (D236N); E, K2P; F, K2P (D236N);
, no addition;
, in the presence of 5 mM EACA
(see ``Experimental Procedures'' and for further explanation
see text).
t-PA Binding Sites on a Forming Fibrin Clot
To
study the t-PA binding site on a forming fibrin clot, we performed
competition experiments. shows the result of such a
competition experiment. The binding of radiolabeled FGK1K2P is
partially competed by FGK1K2P and K2P. The fibrin binding of FGK1K2P
(D236N) lacking the lysyl binding site was only partially inhibited by
FGK1K2P but also by K2P. This result suggests that the t-PA binding
site in fibrin is also recognized by K2P. Binding of GK1K2P lacking the
F domain is competed by K2P but also by FGK1K2P. Fibrin binding of
GK1K2P (D236N) lacking both the F domain and the lysyl binding site in
the K2 domain could be completely inhibited by FGK1K2P and K2P,
indicating that the molecule GKK
P (D236N) still
recognizes the same t-PA binding site on fibrin. Competition with
bovine serum albumin at competitor concentrations comparable with K2P
and FGK1K2P did not occur (not shown).
1163
± 490 nM). Therefore, it seems reasonable to assume
that fibrin binding is not solely aminohexyl-mediated and that the
fibrin binding site comprises more than the lysyl binding site. However
deletion of the lysyl binding site in the K2P molecule not only
abolishes binding to EACA but also to fibrin. Therefore, steric
hindrance does not seem to be a satisfying explanation for the observed
difference in fibrin binding between t-PA in the presence of EACA and
t-PA without a lysyl binding site.
-Val
bond of
plasminogen. However, the enhancement of the plasminogen to plasmin
conversion by K2P (D236N), that does not interact with a forming fibrin
clot suggests that yet another mechanism of plasminogen activation
exists, independent of the t-PA binding to fibrin.
Figure 3:
Presentation of a model to explain the
effect of EACA and the deletion of the lysyl binding site in t-PA on
the fibrin binding to a forming fibrin clot. Within the t-PA molecule
( = FGK1 part; &cjs0540; = K2P part;
= lysyl binding site;
= lysyl/arginyl residue
from another t-PA domain or EACA) an intramolecular interaction
probably between the lysyl binding site and a lysyl/arginyl residue of
the P domain, results in an equilibrium between a closed and an open
conformation. The closed conformation is hypothesized to possess the
structural requirements for a high affinity interaction with a forming
fibrin clot (upperpart). In the presence of EACA the
interaction between the lysyl binding site in K2 and the lysyl/arginyl
residue is disturbed resulting in a shift in equilibrium toward the
open conformation (middlepart). The open
conformation is hypothesized to possess a fibrin interaction site of
lower affinity. Deletion of the lysyl binding site also results,
although to a lesser extent, in a shift in equilibrium toward a more
open conformation (lowerpart).
Table:
Binding to lysyl-Sepharose
Table:
Fibrin-dependent plasminogen activation
A h
pmol
. Stimulation factors
(column 4) were calculated as follows. The
A h
pmol
(in the presence of
CNBr fragments of fibrinogen) divided by
A h
pmol
(in the absence of CNBr fragments of
fibrinogen).
Table:
Determination of C values of
binding of t-PA and t-PA analogues to fibrin in the absence and
presence of EACA
values for binding were determined
from binding experiments of t-PA or t-PA analogues to a forming fibrin
clot, comparable with the ones described (see Fig. 2). Using nonlinear
regression analysis, the C
of t-PA and t-PA
domain deletion analogues to a forming fibrin clot in the absence or
presence of 5 mM EACA and before and after deletion of the
lysyl binding site (D236N) were determined. C
values and the standard deviation are presented. NB, no binding
observed. For details, see ``Experimental Procedures.''
Table:
Competition experiments of radiolabeled t-PA
and t-PA analogues with t-PA and K2P
-amino caproic acid; bp, base
pair(s); S-2288,
H-D-Ile-L-Pro-L-Arg-p-nitroanilide
dihydrochloride; C
, concentration of fibrin(ogen)
at which binding is half-maximal; S-2251,
H-D-Val-L-Leu-L-Lys-p-nitroanilide
dihydrochloride; PPACK, Phe-Pro-Arg-CH
Cl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.