Tài liệu Báo cáo khoa học: Functional hierarchy of plasminogen kringles 1 and 4 in fibrinolysis and plasmin-induced cell detachment and apoptosis docx

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

the two-chain serine protease plasmin following clea-
vage of the Arg561–Val562 peptide bond. Plasmin for-
mation is regulated in part by conformational changes
due to interactions between plasminogen domains
(reviewed in [16]). Glu-plasminogen (Glu-Pg) consists
of an amino-terminal peptide Glu1–Lys77, five kringle
domains at the N terminus and a serine-protease
domain at the C-terminal end [17,18]. Cleavage of the
Keywords
cell detachment-induced apoptosis;
extracellular matrix proteolysis; fibrinolysis;
lysine-binding site; plasminogen activation
Correspondence
E. Angle
´
s-Cano, INSERM U698, CHU
Bichat-Claude Bernard, 46 rue Henri
Huchard, F-75877-Cdx, Paris 18, France
Fax: +33 1 40 25 86 10
Tel: +33 1 40 25 86 11
E-mail:
(Received 30 March 2005, revised 5 May
2005, accepted 9 May 2005)
doi:10.1111/j.1742-4658.2005.04754.x
Plasmin(ogen) kringles 1 and 4 are involved in anchorage of plasmin(ogen)
to fibrin and cells, an essential step in fibrinolysis and pericellular proteo-
lysis. Their contribution to these processes was investigated by selective
neutralization of their lysine-binding function. Blocking the kringle 1 lysine-
binding site with monoclonal antibody 34D3 fully abolished binding and
activation of Glu-plasminogen and prevented both fibrinolysis and plasmin-

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-

sent in K1 and K4 [19] display affinity for lysine resi-
dues, physiologically relevant for binding to fibrin
[20,21], to extracellular matrix components [22] and to
cells [15,23]. K5 has some affinity for lysine analogues,
but it is too weak to firmly anchor plasminogen to sur-
face lysines [17,24,25].
In native Glu-Pg, the LBS in K1 is exposed and
functional such that binding to targets probably occurs
through this site and not through the LBS in K4,
which is masked in Glu-Pg [26]. These differences in
LBS availability are explained by several physicochem-
ical studies indicating that native Glu-Pg is a compact
molecule with inter-domain interactions between the
N-terminal Glu1–Lys77 peptide and K5 in the C-ter-
minal region [26,27] and between K3 and K4 [28],
organized in a spiral-type domain conformation
[26,29–32]. Rupture of these interactions by proteolytic
removal of the N-terminal peptide (Glu- to Lys-Pg
conversion) or upon occupation of a weak LBS (ligan-
ded state) by 6-aminohexanoic acid (6-Ahx) results in
transition to an extended open conformation. Thus,
Lys-Pg has an open conformation similar to that of
6-Ahx-liganded Glu-Pg [33].
The compact spiral-type conformation of Glu-Pg is
resistant to activation, whereas a shift to the open
extended form increases the accessibility of its activa-
tion peptide bond to plasminogen activators and
thereby accelerates the local rate of plasmin forma-
tion [16,33]. Transitions in plasminogen conformation
can be induced by interactions with macromolecules

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.

Results
Contribution of plasminogen K1 and K4 to
surface plasminogen activation
It has been previously shown that mAb A10.2 binds
specifically to the LBS of immobilized plasminogen K4
with high affinity (K
d
¼ 0.25 nm) [35], whereas the
high affinity (K
d
¼ 1.28 nm) binding of mAb 34D3 is
localized to the K1-3 region of plasminogen [34]. In
the present study we demonstrate that this interaction
is K1-LBS-specific as indicated by its complete inhibi-
tion [50% inhibitory concentration (IC
50
) ¼ 0.75 mm]
with 6-Ahx. Because 6-Ahx also fully inhibited the
interaction of the smaller (50 kDa) Fab fragments with
plasminogen (data not shown), the possibility that
antibody binding produces steric hindrance rather than
specific LBS blockage was excluded.
Glu-Pg interactions with fibrin surfaces and Chinese
hamster ovary (CHO)-K1 cells were of similar affinity
(K
d
¼ 0.9 ± 0.2 lm, K
d
¼ 1.05 ± 0.3 lm, respect-
ively), in agreement with previous reports [37,38]. They

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

K4-LBS is permanently exposed, supports this hypo-
thesis. Blockage by mAb A10.2 of K4-LBS exposed
upon Glu-Pg binding to surfaces, may also contribute
to this inhibitory effect.
We then investigated the consequence of the inhibi-
tion of plasminogen binding on plasmin formation by
fibrin-bound tissue plasminogen activator (t-PA) or by
CHO-K1 cells. For that purpose, t-PA was first bound
to a fibrin surface, whereas no exogenous t-PA was
added to CHO-K1 cells as they constitutively express
t-PA at their membrane (detected by zymography as
described [39]). CHO-K1 cells are indeed able to acti-
vate plasminogen (K
m
¼ 43.3 ± 3.2 nm, Fig. 2A) in
an LBS-dependent manner as indicated by the com-
plete inhibition of this activation by 6-Ahx (IC
50
¼
5mm, Fig. 2A, inset). By blocking the LBS of Glu-Pg
K1, mAb 34D3 inhibits plasminogen binding (Fig. 1)
and activation on both cells (IC
50
¼ 116 nm, Fig. 2B)
and fibrin (IC
50
¼ 17 nm, Fig. 2B, inset). Interestingly,
mAb A10.2 that blocks the LBS of plasminogen K4
and was shown to partially inhibit Glu-Pg binding
(Fig. 1), does not impair its activation either on cells

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-

its both its binding to fibrin (Fig. 3A, IC
50
A10.2 ¼
290 nm,IC
50
34D3 ¼ 420 nm) and its activation
(Fig. 3B, IC
50
A10.2 ¼ 185 nm,IC
50
34D3 ¼ 106 nm).
A complete inhibition of Lys-Pg binding (Fig. 3A,
IC
50
mix ¼ 52 nm) and activation (Fig. 3B, IC
50
Fig. 1. Inhibition of Glu-Pg binding to CHO-K1 cells and fibrin by
anti-LBS A10.2 and 34D3 mAbs. CHO-K1 cells (A) or fibrin plates
(B) were incubated with Glu-Pg (100 n
M and 50 nM, respectively)
supplemented with varying concentrations of A10.2 or ⁄ and
34D3 mAbs (0–1 l
M). After two washes, bound plasminogen was
detected using HRP-conjugated CPL-15 mAb. The inhibitory effect
of the mAbs on plasminogen binding to fibrin and cells is
expressed as a percentage (mean ± SD, n ¼ 3) relative to the
amount of plasminogen bound in the absence of mAbs. The unre-
lated mAb P6E9G11used as control did not affect plasminogen
binding (data not shown). Data were fitted to a hyperbolic decay
equation allowing calculation of mAb concentrations that produce

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

¼ 5mM). (B) Kinetics of plasmin formation performed on cells (main figure)
and fibrin (inset) in the presence of varying concentrations of mAb A10.2 or mAb 34D3 (0–1 l
M for cells and 0–0.25 lM for fibrin) and fixed
concentrations of Glu-Pg (200 n
M for cells and 50 nM for fibrin) and CBS0065 (0.75 mM). Results are expressed as a percentage
(mean ± SD, n ¼ 3) relative to the rate of plasmin formation in the absence of mAbs. (C) CHO-K1 cells were incubated for 16 h with 0.5 l
M
Glu-Pg in the presence or absence of 0.5 lM of mAb A10.2 or mAb 34D3. Cell conditioned media were analysed by SDS ⁄ PAGE (7.5% acryl-
amide, nonreducing conditions) and western blotting using HRP-conjugated mAb CPL-15. Lane 1, Control Glu-Pg; lane 2, control plasmin;
lane 3, untreated control cells; lane 4, Glu-Pg-treated cells; lane 5, Glu-Pg- and mAb A10.2-treated cells; lane 6, Glu-Pg- and mAb 34D3-trea-
ted cells. Bands in lanes 1 and 2 correspond to forms I and II of plasmin(ogen). High molecular weight bands in lanes 5 and 6 correspond to
mAb A10.2- and mAb 34D3-plasminogen complexes. (D) Cells were treated as in (B), washed twice with NaCl ⁄ P
i
and residual bound plas-
min was detected by adding CBS0065 at 0.75 m
M. Results are expressed as a percentage (mean ± SD, n ¼ 3) relative to the amount of
cell-associated plasmin activity detected in the absence of mAbs. *P < 0.0001 vs. control.
Plasminogen transitions and proteolysis B. Ho-Tin-Noe
´
et al.
3390 FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS
Plasmin-induced cell detachment and apoptosis
are impaired by selectively blocking the LBS of
plasminogen K1 or K4
The incubation of CHO-K1 cells with plasminogen
leads to plasmin formation in a saturable and specific
manner (Fig. 2A). The plasmin generated in situ indu-
ces cell detachment (Fig. 4A). An inverse correlation
(r ¼ 0.89, P ¼ 0.0013) was found between the amount
of cell-associated plasmin and the percentage of resid-

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

(Fig. 5B).
Plasmin-detached cells undergo apoptosis as indica-
ted by the terminal deoxynucleotidyltransferase-medi-
ated dUTP nick end labelling (TUNEL) assay (Fig. 6).
The apoptotic index of the Glu-Pg treated cells
reached 56.6 ± 6.7% in detached cells (Fig. 6C) and
37.1 ± 9.2% in the residual adherent cells (Fig. 6B)
as compared to 0.9 ± 0.5% in untreated control cells
(Fig. 6A). In the presence of the antiplasminogen
K4-LBS mAb A10.2 the apoptotic index fall to
7.9 ± 5.7% for detached cells (Fig. 6E) and to
7.6 ± 4.3% for residual adherent cells (Fig. 6D).
Blocking of Glu-Pg K1-LBS by mAb 34D3 fully pre-
vented loss of cell adhesion and apoptosis (apoptotic
index 0.4 ± 0.2%, Fig. 6F).
The higher protective effect on cell detachment
(Fig. 4B) and apoptosis (Fig. 6F) produced by mAb
34D3 as compared to mAb A10.2 reflects its ability to
prevent both plasmin formation (Fig. 2B) and binding
Fig. 3. Effect of anti-LBS A10.2 and 34D3 mAbs on Lys-Pg binding
and activation by fibrin-bound t-PA. (A) Fibrin surfaces were incuba-
ted with a fixed concentration of Lys-Pg (100 n
M) supplemented
with varying concentrations of A10.2 or ⁄ and 34D3 mAbs (0–1 l
M).
After two washes, bound plasminogen was detected with HRP-con-
jugated CPL-15 mAb as indicated in Fig. 1. Results are expressed
as a percentage relative to the amount of Lys-Pg bound to fibrin in
the absence of mAbs. Data were fitted as indicated in Fig. 2. (B)
Fibrin surfaces with bound t-PA (10 IUÆmL

)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-

K4-LBS mAb A10.2 (Fig. 7A). However, with Lys-Pg
and plasmin, the antifibrinolytic effect of the two anti-
LBS mAbs was comparable (39 ± 5% maximum of
fibrinolysis for mAb A10.2 and 49 ± 4% for mAb
34D3) and a strong additive effect was observed when
combined doses of mAbs were used (Fig. 7B, to sim-
plify the plot only Lys-Pg data are shown).
Discussion
Cell detachment-induced apoptosis [9,10] and throm-
bus lysis [14] are distinct biological processes that
share plasmin(ogen) dependence. In vitro and ex vivo
studies have recently shown that degradation of extra-
cellular matrix proteins (fibronectin, laminin) by cell-
bound plasmin plays a key role in triggering apoptosis
following cell detachment [9,10]. These biological pro-
cesses require LBS-dependent plasminogen binding to
Fig. 4. Anti-LBS A10.2 and 34D3 mAbs protect cells from plasmino-
gen activation-induced cell detachment. CHO-K1 cells were incuba-
ted with Glu-Pg at varying concentrations (0–2 l
M) or at a fixed
concentration (0.5 l
M) supplemented with varying concentrations
of mAb A10.2 or 34D3 (0–1 l
M). After 16 h of incubation, nonad-
herent CHO-K1 cells were removed by washing with NaCl ⁄ P
i
and
residual adherent cells were detected with the colorimetric MTT
test. (A) Bars represent the percentage of residual adherent cells
as a function of Glu-Pg concentration. (B) Residual adherent cells at

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

plasmin proteolytic activity on fibrinolysis and extra-
cellular proteolysis, are lacking. Furthermore, the
relative importance of K1 and K4 in mediating inter-
actions of plasminogen with fibrin and cells remains
unsolved. This information could not be obtained with
plasminogen fragments or with lysine analogues, as
plasminogen fragments directly block binding sites on
fibrin and cells [23] and the LBS of both K1 and K4
are simultaneously occupied by lysine analogues
[41,42]. However, by nature of their conservative
amino acids substitutions at contributing aromatic sites
of the LBS [19], K1 and K4 have different abilities to
bind lysine analogues [41–43], lysine-Sepharose [40]
and most probably proteins in which lysine residues
are accessible [16,20–23]. As the LBS are not structur-
ally identical [19], we hypothesized that this problem
can be studied with the use of specific LBS-targeted
immunological probes. It has indeed been possible to
raise polyclonal antibodies [44,45] and mAbs that spe-
cifically differentiate the LBS of plasminogen K1 and
K4 [34,35,46,47].
In this study, using two mAbs that allow direct and
selective blockage of the LBS function of either K1
(mAb 34D3) [34] or K4 (mAb A10.2) [35], we were
able to evaluate their respective contribution to Glu-
and Lys-Pg binding and activation onto fibrin and
cells, as well as their effects on fibrinolysis and on plas-
min-induced cell apoptosis. The LBS specificity of
these mAbs is based on their absence of interaction
with 6-Ahx-blocked plasmin(ogen) and its isolated K4

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

i
, bound plasmin was detected by incubating the cells with
HRP-conjugated CPL-15 mAb. Results are expressed as a percent-
age (mean ± SD, n ¼ 3) relative to the amount of plasmin bound in
the absence of mAbs.
B. Ho-Tin-Noe
´
et al. Plasminogen transitions and proteolysis
FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS 3393
cell-bound plasmin (Fig. 2D). This remarkable differ-
ence in plasmin formation directly shows that in the
native compact form of Glu-Pg, the LBS of K1 is
readily exposed and plays a primary role in its initial
binding to fibrin and cells. The subsequent accessibility
of the K4-LBS is necessary to reinforce plasmin(ogen)
anchorage as its occupancy by mAb A10.2 weakly
inhibits Glu-Pg binding (Fig. 1), decreases fibrinolysis
and plasmin-induced cell detachment, thus suggesting
that this interaction may be of biological relevance.
These results are in agreement with previous
physicochemical studies [30,31,48–50]. It has thus been
established that in the native spiral-like Glu-Pg form
the K4-LBS is masked by inter-domain interactions
[27,28] and that conformational changes in Glu-Pg are
induced by ligand binding. A conformational shift to
the more expanded form, roughly similar to that of
the U-shaped open form of Lys-Pg, occurs upon occu-
pancy of an LBS with the lysine analogue 6-Ahx. It
has therefore been predicted that, as for the 6-Ahx-
liganded form, Glu-Pg would adopt, upon binding to

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
´

et al.
3394 FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS
Lys-Pg binding was obtained only when the LBS of
both K1 and K4 were blocked simultaneously
(Fig. 3A). When the binding of purified active site-
inhibited plasmin was tested directly, similar partial
and complete inhibitory effects were observed (Fig. 5)
thus indicating accessibility and participation of both
K1 and K4 in firmly anchoring plasmin to fibrin and
cell surfaces. Because the expanded Lys-form of
plasminogen may be an intermediary in plasmin for-
mation [51,52], we have also evaluated its mechanism
of activation by direct inhibition with the mAbs. The
observed effects of the mAbs on Lys-Pg binding
(Fig. 3A) resulted in the corresponding parallel
decrease in plasmin formation: it was completely abol-
ished when both antibodies were added at concentra-
tions that if used singly produce only a partial
inhibition (Fig. 3B). These results indicate that in con-
trast to the selective K1-dependent binding of Glu-Pg
to fibrin and cells, in Lys-Pg either the K1- or the
K4-LBS can mediate initial binding for its activation.
These studies provide biological support to data indi-
cating that in the Lys-Pg open conformation K1 and
K4 are equally exposed [33].
Furthermore, we add new data about their respect-
ive role in the binding ⁄ activation mechanism by link-
ing them directly to the biological functions of
plasmin. In particular, we demonstrate that these inter-
actions are determinant for the activities of plasmin

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).

*P < 0.0001 vs. control. (C) In parallel experiments, fibrin-bound
plasmin was not eluted but detected with HRP-conjugated
CPl-15 mAb as indicated. The graph represents the correlation
between the amount of fibrin-associated plasmin activity and the
degree of fibrinolysis (r ¼ 0.94, P ¼ 0.0002).
B. Ho-Tin-Noe
´
et al. Plasminogen transitions and proteolysis
FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS 3395
binding and activation of native Glu-plasminogen with
mAb 34D3 or the anchoring of plasmin with mAb
A10.2. A complete inhibition of plasmin formation
(Fig. 2B), prevention of fibrinolysis (Fig. 7), protection
from cell detachment (Fig. 4B) and apoptosis (Fig. 6F)
were readily obtained by blocking the K1-LBS of
Glu-Pg with mAb 34D3. Blockage of K4-LBS with
mAb A10.2 did not significantly affect plasmin forma-
tion (Fig. 2B) but produced a dose-dependent partial
reduction in cell-bound plasmin that progressively pro-
tected cells from detachment (Fig. 4B) and significantly
limited the rate of apoptosis (Fig. 6D,E). Furthermore,
these mAbs at doses insufficient to prevent plas-
min(ogen)-dependent cell detachment, were able to
fully preserve cell adhesion if added in combination
(Fig. 4C). Because cell detachment-induced apoptosis
may be implicated in the development of several
pathologies in which the fibrinolytic system is activated
[53–55], these mAbs represent new tools that could be
the basis for the development of pharmacological
agents that would prevent this phenomenon in vivo.

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

2
Cl), a plasmin inhibitor was from Calbiochem (La
Jolla, CA, USA). Peroxydase substrate 2,2¢-azino-bis (3-ethyl-
benzthiazoline)-6-sulphonic acid (ABTS) was from Roche
(Mannheim, Germany). Human t-PA was from Biopool
(Uppsala, Sweden). Fibrin and partially degraded fibrin
surfaces were prepared and characterized as described pre-
viously [37].
Cell culture
CHO-K1 cells (American Type Culture Collection CCL-61)
were seeded in 96-well plates and cultured at 37 °Cina
humidified atmosphere of 5% (v ⁄ v) CO
2
using Ham-F12
medium supplemented with 2 mm glutamine and 10% (v ⁄ v)
fetal bovine serum. The cells at near confluence were
starved from serum for 6 h before experiments.
Plasminogen and monoclonal antibodies
Human Glu- and Lys-Pg were purified as described [37,56]
and were considered to be > 99% pure as assessed by
SDS ⁄ PAGE and by N-terminal sequence analysis. Plasmin
was prepared by activation of Glu-plasminogen with immo-
bilized urokinase as described previously [57].
mAb A10.2 was raised in mice immunized with human
recombinant apolipoprotein(a), cloned using classical hybri-
doma procedures and purified as described [35]. mAb
A10.2 is an IgG1 that specifically binds and masks the LBS
of apolipoprotein(a) KIV-10 and of plasminogen K4, with-
out cross-reaction with the LBS of plasminogen K1 [58].
mAb 34D3 was raised in mice immunized with human

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.

3396 FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS
Binding of plasmin(ogen) to fibrin and cells
CHO-K1 cells and partially degraded fibrin surfaces were
incubated for 1 h at 37 °C with different concentrations of
plasminogen or d-Val-Phe-Lys-CH
2
Cl-inactivated plasmin
in binding buffer (for binding to fibrin) or in Ham-F12
medium supplemented with 1% (w ⁄ v) BSA (for binding to
cells). After two washes with NaCl ⁄ P
i
, bound proteins
were detected by incubation for 1 h at 37 °C with HRP-
conjugated mAb CPL-15. After washing with NaCl ⁄ P
i
,
1mgÆmL
)1
ABTS was used as a substrate for colour devel-
opment and the absorbance at 405 nm was measured in a
multiwell plate reader.
To determine the effect of 34D3 and A10.2 mAbs on
plasmin(ogen) binding to fibrin and CHO-K1 cells, fibrin
plates and cells were incubated with a fixed concentration
of plasmin(ogen) supplemented with varying concentrations
of mAbs. Bound plasmin(ogen) was detected as indicated
above. mAb P6E9G11 directed against fibrin d-dimers was
used as a plasmin(ogen)-unrelated control.
Data were fitted according to the Langmuir equation for
single site interactions and to a hyperbolic decay equation

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

vs. time and transformed into plasmin con-
centration as described [62].
Plasminogen activation on cells
CHO-K1 cells were starved of serum and then incubated
with varying concentrations of Glu-Pg (0–2 lm) and a fixed
concentration (0.75 mm final concentration) of CBS0065 in
a total volume of 100 lL of Ham-F12 medium per well.
Kinetics of plasmin formation was followed for 16 h as
indicated above.
After the activation reaction on both cells and fibrin, two
washes in NaCl ⁄ P
i
were performed and the residual bound
plasmin was detected by adding CBS0065 at 0.75 mm.
Effect of anti-LBS mAbs on plasminogen activation
To determine the effect of the mAbs 34D3 and A10.2 on
plasminogen activation, the fibrin plates and the cells were
incubated with different concentrations of these mAbs in
the presence of fixed concentrations of plasminogen and
CBS0065. mAb P6E9G11 was used as a plasmin(ogen)-
unrelated control. We previously determined that the
amidolytic activity of plasmin was not modified in the pres-
ence of the mAbs.
Inhibition of plasminogen activation by 6-Ahx
CHO-K1 cells were incubated with varying concentrations
of 6-Ahx (0–100 mm) and fixed concentrations of plasmino-
gen (200 nm) and CBS0065 (0.75 mm) in a total volume of
100 lL of Ham-F12 medium per well. Kinetics of plasmin
formation was followed for 16 h as indicated.
Western blot analysis of plasminogen activation

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

TUNEL and 4¢, 6-diamino-2-phenylindole (DAPI)
staining
After the indicated experiments, cells grown on eight-cham-
bered slides (Labtek) or detached cytospun cells were fixed
in 3.7% (v ⁄ v) paraformaldehyde for 30 min at room
B. Ho-Tin-Noe
´
et al. Plasminogen transitions and proteolysis
FEBS Journal 272 (2005) 3387–3400 ª 2005 FEBS 3397
temperature and then permeabilized in 0.1% (v ⁄ v) Triton
X-100, 0.1% (w ⁄ v) sodium citrate for 2 min on ice. TU-
NEL (Roche Applied Science) used to visualize DNA frag-
mentation and 4¢, 6-diamino-2-phenylindole (DAPI; Sigma)
nuclear counterstaining to visualize all nuclei were per-
formed according to the manufacturer’s instructions. After
washing, the slides were mounted with Fluoprep (DAKO,
Carpinteria, CA, USA) and observed under an epifluores-
cence microscope. The apoptotic index was calculated as
the percentage of TUNEL-positive nuclei relative to total
DAPI-stained nuclei.
Effect of anti-LBS mAbs on fibrinolysis
The activation of 50 nm of either Glu- or Lys-Pg by fibrin-
bound t-PA was performed as indicated above in the pres-
ence or in the absence of mAbs A10.2 or ⁄ and mAb 34D3
(0–1 lm). The generated plasmin was eluted with 100 mm
6-Ahx, 20 lmd-Val-Phe-Lys-CH
2
Cl, in Tris ⁄ HCl buffer
pH 5, and the degree of fibrinolysis was estimated by
detecting the plasmin-generated fibrin fragment E. For this

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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