Functional hierarchy of plasminogen kringles 1 and 4 in
fibrinolysis and plasmin-induced cell detachment and
apoptosis
Benoı
ˆ
t Ho-Tin-Noe
´
1
, Gertrudis Rojas
2
, Roger Vranckx
1
, H. Roger Lijnen
3
and Eduardo Angle
´
s-Cano
1
1 INSERM U698, Centre Hospitalier Universitaire Bichat-Claude Bernard, Paris, France
2 Center for Genetic Engineering and Biotechnology, Havana, Cuba
3 Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Leuven, Belgium
Plasminogen, a 92-kDa single-chain zymogen, present
in blood and most extravascular fluids, has a broad
physiological and pathophysiological role; it is essen-
tial for efficient fibrinolysis [1–3], facilitates cell migra-
tion [4–7] and participates in vascular remodelling [8].
Recent in vitro studies indicate that it may be implica-
ted in cell detachment-induced apoptosis [9–13]. These
activities depend upon the ability of plasminogen and
its activators to assemble onto macromolecules (such
as fibrin) or cellular receptors [14,15], where it becomes

induced cell detachment-induced apoptosis. In contrast, blocking the kringle
4 lysine-binding site with monoclonal antibody A10.2 did not impair its
activation although it partially inhibited plasmin(ogen) binding, fibrinolysis
and cell detachment. This remarkable, biologically relevant, distinctive
response was not observed for plasmin or Lys-plasminogen; each antibody
inhibited their binding and activation of Lys-plasminogen to a limited
extent, and full inhibition of fibrinolysis required simultaneous neutraliza-
tion of both kringles. Thus, in Lys-plasminogen and plasmin, kringles 1 and
4 act as independent and complementary domains, both able to support
binding and activation. We conclude that Glu- ⁄ Lys-plasminogen and plas-
min conformations are associated with transitions in the lysine-binding
function of kringles 1 and 4 that modulate fibrinolysis and pericellular
proteolysis and may be of biological relevance during athero-thrombosis
and inflammatory states. These findings constitute the first biological link
between plasmin(ogen) transitions and functions.
Abbreviations
6-Ahx, 6-aminohexanoic acid; ABTS, 2,2¢-azino-bis (3-ethylbenzthiazoline)-6-sulphonic acid; CBS0065, (methylmalonyl)-hydroxyprolylarginine-
p-nitroanilide; CHO, Chinese hamster ovary; DAPI, 4¢, 6-diamino-2-phenylindole;
D-Val-Phe-Lys-CH
2
Cl, D-valyl-L-phenylalanyl-L-lysine
chloromethylketone; Glu-Pg, Glu-plasminogen; HRP, horseradish peroxydase; K, kringle; LBS, lysine-binding site; Lys-Pg, Lys-plasminogen;
mAb, monoclonal antibody; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; t-PA, tissue-type plasminogen activator;
TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling.
FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS 3387
N-terminal peptide at either Lys62, Arg68 or Lys77 by
plasmin yields a truncated form collectively known
as Lys-Pg. Kringles (K) are triple-loop structures of
about 80 amino acids constrained by three disulfide
bridges. Lysine-binding site (LBS) substructures pre-

such as fibrin [30]. It has been suggested that the
open form can be induced only if the LBS in K1 and
K4 are both occupied in the same molecule [33].
However, the relative contribution of the LBS func-
tion in each of these kringles to the regulation of
plasminogen activation on fibrin and cell surfaces as
well as its consequences on fibrinolysis and cell beha-
viour (migration, survival) remains unknown. This
information is important to assess the relevance of a
functional hierarchy of plasminogen kringle inter-
actions with cells in cell detachment-induced apoptosis.
We have developed monoclonal antibodies (mAbs)
that specifically inhibit the LBS function of either K1
[34] or K4 [35] and have used them to analyse, in
functional isolation, the role of these LBS on plas-
min(ogen) binding and activation. The effect of these
mAbs on plasmin formation and function was further
analysed by using well established models for fibrino-
lysis [36,37] and cell detachment-induced apoptosis
[9,10].
We demonstrate that initial binding and activation
of native Glu-Pg onto fibrin and cells is governed by
K1-LBS, whereas K4-LBS contributes to reinforce
plasmin(ogen) anchorage and functioning. Both
K1- and K4-LBS support Lys-plasmin(ogen) binding
and activation. These structural–functional transitions
are determinant in the initiation and development of
both fibrinolysis and cell detachment-induced apopto-
sis and may therefore be of relevance during athero-
thrombosis and inflammatory states.

were specifically inhibited by 6-Ahx that blocks the
LBS of plasminogen K1 and K4 (IC
50
¼ 0.9 mm on
fibrin, IC
50
¼ 1.7 mm on cells). Single LBS-targeted
blockage of either K1 by mAb 34D3 or of K4 by mAb
A10.2 prevented binding of Glu-Pg to both CHO-K1
cells (Fig. 1A) and fibrin (Fig. 1B) in a concentration-
dependent manner. However, at saturating mAb con-
centrations, mAb 34D3 fully inhibited the binding of
Glu-Pg, whereas mAb A10.2 produced a partial inhibi-
tory effect as indicated by the residual amount (< 5%
vs. % 60%, respectively) of plasminogen bound to cells
and fibrin (Fig. 1). The partial inhibitory effect of
mAb A10.2 is most probably related to the dynamic
equilibrium between the open and compact conforma-
tions of Glu-Pg in the unliganded state [16]. The lower
apparent affinity of immobilized mAb A10.2 for
Glu-Pg (K
d
¼ 1.64 nm ± 0.2 nm) as compare to its
affinity for Lys-Pg (K
d
¼ 0.16 nm ± 0.06 nm) in which
Plasminogen transitions and proteolysis B. Ho-Tin-Noe
´
et al.
3388 FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS

or on fibrin (Fig. 2B). These results were confirmed by
Western blot analysis of cell-conditioned media from
CHO-K1 cells treated with 0.5 lm Glu-Pg in the pres-
ence or absence of 0.5 lm of mAb A10.2 or 34D3
(Fig. 2C). In the absence of mAbs, plasminogen was
fully converted into plasmin by CHO-K1 cells (lane 4).
Plasmin formation was not affected by mAb A10.2
(lane 5). However, the amount of plasmin activity that
remains associated to cells and fibrin was progressively
decreased by mAb A10.2 in a concentration-dependent
manner, to 30% ± 1.4% cell-associated plasmin activ-
ity at the highest mAb concentration used (1 lm)
(Fig. 2D, to simplify the plot only results on cells are
shown). In contrast, the conversion of plasminogen
into plasmin was fully inhibited by mAb 34D3
(Fig. 2C, lane 6): the rate of plasmin formation
(Fig. 2B) and the amount of cell-associated plasmin
activity (Fig. 2D) decreased in parallel, reaching back-
ground level (6.7% ± 0.9% maximum of cell-associ-
ated plasmin activity) at 125 nm mAb concentration.
This fully inhibitory dose of anti-K1-LBS mAb 34D3
is eightfold lower than the dose of anti-K4-LBS mAb
A10.2 required to produce only a partial reduction in
cell-associated plasmin. Because Glu-Pg is in a closed
conformation, we further analysed the effect of mAb
A10.2 on the activation of Lys-Pg that is known to be
in an open conformation. In contrast to the full inhibi-
tion of Glu-Pg activation by mAb 34D3 (Fig. 2B),
blocking either the K4-LBS by mAb A10.2 or the
K1-LBS by mAb 34D3 on Lys-Pg only partially inhib-

50% of the inhibitory effect, IC
50
, on, respectively, cells and fibrin:
A10.2 ¼ 9.2 n
M and 3 nM; 34D3 ¼ 13.1 nM and 5 nM; mAb mix ¼
11.8 n
M and 7 nM.
B. Ho-Tin-Noe
´
et al. Plasminogen transitions and proteolysis
FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS 3389
mix ¼ 15 nm) was obtained only when the two mAbs
were combined, thus indicating an additive effect.
Experiments were performed to exclude nonspecific
effects of intact mAbs, including the reproduction of
similar results with their Fab fragments and the
absence of effect on plasminogen binding and activa-
tion in the presence of the unrelated anti-D-dimers
mAb P6E9G11 (data not shown).
AB
CD
Fig. 2. Effect of anti-LBS A10.2 and 34D3 mAbs on Glu-plasminogen activation by CHO-K1 cells and fibrin-bound t-PA. (A) CHO-K1 cells
were incubated with varying concentrations of Glu-Pg (0–2 l
M) and 0.75 mM CBS0065. Kinetics of plasmin formation was followed for 16 h.
Data were fitted according to the Michaelis–Menten equation. Inset: inhibition of Glu-Pg activation (200 n
M) by varying concentrations of
6-Ahx (0–100 m
M). Results are expressed as a percentage (mean ± SD, n ¼ 3) relative to the maximal rate of plasmin formation in the
absence of 6-Ahx. Data were fitted as indicated in Fig. 1 (IC
50

ual adherent cells as assessed by the 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
test. Plasmin(ogen)-induced cell detachment was pre-
vented in a dose-dependent manner by mAb 34D3 or
mAb A10.2; however, at similar concentrations, the
effect of mAb 34D3 was more pronounced (Fig. 4B).
Interestingly, a synergistic effect was observed by
combining single doses of mAb 34D3 and mAb A10.2
that have little or no protective effect on cell detach-
ment induced by 500 nm of plasminogen (Fig. 4C).
Cell anchorage was indeed fully preserved by a com-
bination of 75 nm doses of these mAbs (Fig. 4C). In
a similar fashion, the concomitant blocking of plasmi-
nogen K1-LBS and K4-LBS by 6-Ahx fully prevented
cell detachment (data not shown). This protective
effect of the two anti-LBS mAbs 34D3 and A10.2
(Fig. 4B,C) was parallel to a decrease in cell-associ-
ated plasmin activity (Fig. 2D). An inverse correlation
(r ¼ 0.94, P ¼ 0.005 for mAb A10.2, r ¼ 0.94, P ¼
0.017 for mAb 34D3) was indeed found between the
amount of cell-associated plasmin and the percentage
of residual adherent cells. The decrease in cell-associ-
ated plasmin (Fig. 2D) can be due to a decrease in
plasmin formation, to a decrease in plasmin binding
or to a combination of the two. We demonstrate that
both mAb 34D3 and mAb A10.2 inhibit plasmin
binding to cells (Fig. 5A) and fibrin (Fig. 5B) in a
concentration-dependent manner. When combined,
these mAbs produced an additive effect for the inhibi-
tion of plasmin binding to cells (Fig. 5A) and fibrin

)1
) were incubated with
different concentrations of A10.2 or ⁄ and 34D3 mAbs (0–1 l
M)in
the presence of fixed concentrations of Lys-Pg (50 n
M)and
CBS0065 (0.75 m
M). Kinetics of plasmin formation was followed for
3 h. Results are expressed as a percentage (mean ± SD, n ¼ 3) rel-
ative to the rate of plasmin formation in the absence of mAbs.
B. Ho-Tin-Noe
´
et al. Plasminogen transitions and proteolysis
FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS 3391
to cells (Fig. 5A) whereas mAb A10.2 prevents only
plasmin binding to cells (Figs 2D and 5A).
Anti-LBS mAbs 34D3 and A10.2 inhibit
fibrinolysis
As fibrinolysis is the main physiological function of
fibrin-bound plasmin, we explored the effect of the two
anti-LBS mAbs on this process. Both anti-LBS mAbs
decreased fibrinolysis in a dose-dependent manner
(Fig. 7A). The decrease in fibrinolysis was directly rela-
ted to the amount of fibrin-associated plasmin (r ¼
0.94, P ¼ 0.0002, Fig. 7C). No effect on fibrinolysis
could be detected in the presence of the unrelated anti-
D-dimers mAb P6E9G11 (data not shown). As for
Glu-Pg binding (Fig. 1) and activation (Fig. 2B), the
inhibitory effect on fibrinolysis was more pronounced
with the anti-K1-LBS mAb 34D3 than with the anti-

varying concentrations of mAb A10.2 or mAb 34D3 (0–1 l
M) and
a fixed concentration of Glu-Pg (0.5 l
M) (mean ± SD, n ¼ 3). (C)
Residual adherent cells at single 75 n
M dose of mAb A10.2 or mAb
34D3 and their combination (mix) for a fixed concentration of
Glu-Pg (0.5 l
M) (mean ± SD, n ¼ 3). Results are expressed as a
percentage relative to the amount of adherent untreated-control
cells incubated with Ham-F12 medium alone. *P < 0.0001 vs.
control.
A
B
C
Plasminogen transitions and proteolysis B. Ho-Tin-Noe
´
et al.
3392 FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS
fibrin and cell surfaces [21,37,38]. Functional LBS in
plasminogen have been identified in K1 and K4. As
K4 in native Glu-Pg is most probably masked by
inter-domain interactions [28,40], previous studies
using lysine analogues or plasminogen fragments have
indirectly suggested the role of K1 in mediating inter-
actions with cells [23] and fibrin [21] for its transforma-
tion into plasmin in situ. However, data linking
plasminogen conformations and LBS accessibility to
its functional properties such as binding to fibrin and
cells, activation on these surfaces and the effect of

and K1-3 fragments [35]. As expected, simultaneous
inhibition with both mAbs resulted in a comple-
mentary blocking effect of plasmin biological activities
(Figs 4C and 7). These data indicate that the observed
biological effects of these mAbs are strictly dependent
on LBS interactions and reasonably exclude the possi-
bility of steric hindrance. The reproduction of similar
results with Fab fragments is in agreement with this
concept.
We demonstrate that the selective neutralization of
the K1-LBS with mAb 34D3 completely abolished the
specific binding of Glu-Pg to fibrin and cells (Fig. 1).
As a consequence, a dose-dependent inhibition of plas-
min formation was observed (Fig. 2B), fibrinolysis was
impaired (Fig. 7), and plasmin-induced cell detachment
(Fig. 4B,C) and apoptosis (Fig. 6) were fully preven-
ted. In contrast, the anti-K4-LBS mAb A10.2 did
not significantly affect the generation of plasmin
(Fig. 2B,C), although it decreased the amount of
Fig. 5. Inhibition of plasmin binding to CHO-K1 cells and fibrin by
the anti-LBS A10.2 and 34D3 mAbs. CHO-K1 cells (A) or fibrin (B)
were incubated with a constant concentration of
D-Val-Phe-Lys-
CH
2
Cl-inactivated plasmin (50 nM for cells, 5 nM for fibrin) supple-
mented with varying concentrations of A10.2 or ⁄ and 34D3 mAbs
(0–0.5 l
M for cells, 0–50 nM for fibrin). After two washes with
NaCl ⁄ P

its lysine targets on fibrin and cells, an open conforma-
tion in which K4 is exposed [16]. An increase in affin-
ity of K4 for 6-Ahx upon transformation of Glu-Pg
(K
d
¼ 5mm) to Lys-Pg (K
d
¼ 36 lm) has indeed been
documented [40,41].
This mechanism was further explored by measuring
the effect of the mAbs on the interaction of purified
plasmin and Lys-Pg with fibrin and cells. The binding
of Lys-Pg was partially inhibited by both 34D3 and
A10.2 mAbs to a similar degree (Fig. 3A). The anti-
K1-LBS mAb 34D3 was indeed unable to induce an
inhibition of Lys-Pg binding (Fig. 3A) as marked as
for the Glu-Pg form (Fig. 1). A complete inhibition of
Fig. 6. Anti-LBS A10.2 and 34D3 mAbs protect cells from plasminogen activation-induced apoptosis. CHO-K1 cells were incubated for 36 h
without (control) or with 0.5 l
M of Glu-Pg in the presence or absence of 0.5 lM mAb A10.2 or mAb 34D3. The TUNEL reaction was then
used to visualize DNA fragmentation (green fluorescence) in adherent cells and detached cytospun cells. Counterstaining with DAPI (blue
fluorescence) was performed to visualize all nuclei. The apoptotic index (bars graph) was calculated as the percentage of TUNEL-positive
nuclei relative to total DAPI-stained nuclei (bars represent mean ± SD, n ¼ 5). (A) Untreated control cells. (B, C) Cells incubated with Glu-Pg:
(B) residual adherent cells; (C) detached cytospun cells. (D, E) Cells incubated with Glu-Pg and 0.5 l
M of mAb A10.2: (D) residual adherent
cells; (E) detached cytospun cells. (F) Cells incubated with Glu-Pg and 0.5 l
M of mAb 34D3. No cells were recovered after cytospining the
supernatant of control and Glu-Pg ⁄ mAb 34D3-treated cells. *P < 0.0001 vs. control.
Plasminogen transitions and proteolysis B. Ho-Tin-Noe
´

that we have evaluated, namely: fibrinolysis, cell
detachment and apoptosis. We show, in agreement
with previous work [9,10], that uncontrolled plasmin
formation by adherent cells leads to cell detachment
(Fig. 4A) and subsequent apoptosis (Fig. 6B,C). We
demonstrate that this plasmin(ogen)-dependent
sequence of events can be prevented to different
degrees by selectively inhibiting either the initial
A
B
C
Fig. 7. Anti-LBS A10.2 and 34D3 mAbs decrease fibrinolysis. Fibrin
surfaces were incubated with 10 IUÆmL
)1
human t-PA. The plates
were then washed to eliminate unbound proteins and the reaction
was started by adding 50 n
M of either (A) Glu-plasminogen or (B)
Lys-Pg in the presence or in the absence of varying concentrations
(0–1 l
M) of A10-2 and ⁄ or 34D3 mAbs. The generated plasmin was
eluted with 100 m
M 6-Ahx containing 20 lMD-Val-Phe-Lys-CH
2
Cl in
Tris ⁄ HCl pH 5 and the degree of fibrinolysis was estimated by
detecting the plasmin-generated fibrin fragment E with mAb
FDP-14 and an HRP-conjugated goat anti-mouse IgG. Results are
expressed as a percentage relative to the extent of fibrin degrada-
tion in the absence of mAbs (bars represent mean ± SD, n ¼ 3).

In conclusion, we demonstrate that in Glu-Pg,
K1-LBS mediates initial binding and activation
whereas K4-LBS is secondarily required to reinforce
plasmin(ogen) anchorage. In contrast, in Lys-Pg as in
plasmin, K1- and K4-LBS act as independent and
complementary domains both able to support plasmi-
nogen binding and activation, and both required to
ensure plasmin anchorage and subsequent proteolytic
activity. These structural and functional transitions
are determinant in the initiation and acceleration of
fibrinolysis and cell detachment and may be of biologi-
cal relevance in inflammatory states. As plasminogen
activation is involved in several physiological and
pathological conditions, the effects of the anti-LBS
mAbs on fibrinolysis and plasmin-induced apoptosis
may also have a pharmacological interest; they may
serve for future strategies in the development of poten-
tial therapeutic agents in cardiovascular diseases where
uncontrolled plasmin formation results in unwanted
cell death.
Experimental procedures
Reagents
The chromogenic substrate selective for plasmin (methyl-
malonyl)-hydroxyprolylarginine-p-nitroanilide (CBS0065)
was from Stago (Asnie
`
res, France). The lysine analogue
6-Ahx was from Sigma (St-Louis, MO, USA). d-Valyl-l-
phenylalanyl-l-lysine chloromethylketone (d-Val-Phe-Lys-
CH

plasmin-a2-antiplasmin complex, cloned and purified as
described [34]. This mAb reacts with plasminogen frag-
ment K1+2+3 and shows no cross-reaction with plasmi-
nogen K4 [34]. The specificity of this mAb for the LBS of
plasminogen K1 was determined as follows. Glutaralde-
hyde-immobilized mAb 34D3 was incubated with a fixed
concentration of plasminogen (25 nm) supplemented with
varying concentrations of 6-Ahx (0–200 mm) in binding
buffer (50 mm sodium phosphate pH 6.8, 80 mm NaCl,
4mgÆmL
)1
BSA, 2 mm EDTA, 0.01% Tween 20, 0.01%
azide). Bound plasminogen was detected as indicated in
the following section.
mAb CPL-15 is an IgG1 specifically directed against an
epitope of plasminogen K1 not overlapping with the LBS.
It was produced and characterized previously [59]. Horse-
radish peroxidase (HRP)-conjugated CPL-15 mAb was
obtained using a peroxydase labelling kit according to the
manufacturer’s instructions (Roche, Mannheim, Germany).
mAb FDP-14, an IgG1 that reacts with a neo-epitope
exposed in the Bb chain stretch 54–118 of the fibrin degra-
dation products X, Y and E but not in fibrin [60], was
kindly provided by W. Nieuwenhuizen (Gaubius Laborat-
ory, Leiden, the Netherlands).
mAb P6E9G11 directed against fibrin d-dimers (provided
by Biomerieux France) was used as a control.
Plasminogen transitions and proteolysis B. Ho-Tin-Noe
´
et al.

that allows calculation of the concentration of mAb that
produces 50% of the inhibitory effect (IC
50
) [61].
Effect of anti-LBS mAbs on the activation of
plasminogen by fibrin- and cell-bound t-PA
Plasminogen activation on fibrin
The activation of plasminogen by fibrin-bound t-PA was
studied as described elsewhere [36,62]. Briefly, a 96-well
plate coated with fibrin was incubated for 1 h at 37 °C
with 10 IUÆmL
)1
human t-PA in binding buffer (50 lLÆ
well
)1
). The plate was then washed twice to eliminate
unbound proteins, and the reaction was started by adding
50 lL per well of plasminogen and of CBS0065 in the
same buffer. Kinetics of plasmin formation was followed
for 5 h by measuring the release of p-nitroaniline, detected
as a change in absorbance (DA
405
Á
À1
min
), using a multiwell
plate reader (MX5000, Dynex) held at 37 °C. Rates of
plasmin production were calculated from the slopes of
curves of A
405

After incubating CHO-K1 cells for 16 h with 0.5 lm
Glu-Pg in the presence or absence of 0.5 lm of mAb A10.2
or 34D3, cell conditioned media were analysed by
SDS ⁄ PAGE (7.5% acrylamide, nonreducing conditions)
and western blotting using HRP-conjugated mAb CPL-15.
Effects of anti-LBS mAbs on matrix degradation
and cell survival
After the plasminogen activation experiments described
above, nonadherent CHO-K1 cells were removed by wash-
ing with NaCl ⁄ P
i
and the following analyses were per-
formed.
Cell detachment assay
The residual adherent cells were incubated for 1 h at 37 °C
with 0.5 mgÆmL
)1
of the tetrazolium salt MTT in NaCl ⁄ P
i
.
Remaining living adherent cells form formazan crystals that
are dissolved in dimethylsulfoxide and colorimetrically
detected at A
550
using a multiwell plate reader. Absorbance
readings are proportional to the number of living cells.
Results are expressed as a percentage relative to the amount
of residual adherent cells obtained for control cells incuba-
ted with Ham-F12 medium.
Terminal deoxynucleotidyltransferase-mediated

purpose the specific antifragment E mAb FDP-14 was used
at 200 ngÆmL
)1
followed by an HRP-conjugated goat anti-
mouse secondary antibody (DAKO AS, Glostrup,
Denmark). After washing with binding buffer, 1 mgÆmL
)1
ABTS was used as a substrate for colour development and
the absorbance at 405 nm was measured in a multiwell
plate reader. In parallel experiments, fibrin-bound plasmin
was not eluted but detected with the HRP-conjugated
CPL-15 at 250 ngÆmL
)1
as indicated above.
Statistical analysis
Statistical analysis was performed using statview 5.0 soft-
ware. Results are expressed as means ± SD (at least three
independent experiments performed in triplicate). Compari-
sons used one-way analysis of variance with Scheffe’s
F-test. Statistical significance was set at P<0.05.
Acknowledgements
This study was founded by the INSERM and grants
Adrienne et Pierre Sommer from the Fondation de
France to E. AC. and Interuniversity Attraction Poles
(P5 ⁄ o2) to H.R.L.
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