Dual modulation of prothrombin activation by the
cyclopentapeptide plactin
Tomotaka Harada*, Tomoko Tsuruta*, Kumi Yamagata, Toshiki Inoue and Keiji Hasumi
Department of Applied Biological Science, Tokyo Noko University, Tokyo, Japan
Plactin is a family of cyclic pentapeptides that enhance
fibrinolytic activity both in vitro and in vivo [1,2].
Structure–activity relationship studies using 50 plactin
congeners revealed that a sterically restricted arrange-
ment of four hydrophobic amino acids and one basic
amino acid is essential for their activity. The plactin-
mediated increase in fibrinolytic activity accompanies
an elevation in cellular urokinase-type plasminogen
activator (u-PA) activity [2]. In this mechanism, the
presence of plasma is an absolute requirement.
u-PA, as well as tissue-type plasminogen activator, is
a physiologically relevant protease that catalyzes the
limited proteolysis of plasminogen to afford the fibri-
nolytic enzyme plasmin [3,4]. u-PA is produced as an
inactive, single-chain proenzyme (scu-PA) that binds to
a cell-surface receptor in an autocrine fashion follow-
ing secretion [5]. Activation of scu-PA is catalyzed by
plasmin [4] and some other proteases, such as cathep-
sin B [6], plasma kallikrein [7] and mast cell tryptase
[8], involves cleavage at Lys158–Ile159 (numbering
Keywords
blood coagulation; fibrinolysis; proteolysis;
prothrombin; urokinase
Correspondence
K. Hasumi, Department of Applied Biological
Science, Tokyo Noko University, 3-5-8
Saiwaicho, Fuchu-shi, Tokyo 183 8509,

activation or thrombosis in normal mice at doses that produced a protec-
tive effect in a thrombin-induced pulmonary embolism mouse model.
Therefore, the dual modulation of prothrombin activation by plactin may
be interpreted as leading to anticoagulation under physiological coagulat-
ing conditions.
Abbreviations
DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide; DPP-I, dipeptidyl peptidase I; GGA-MCA, glutaryl-Gly-Arg-4-methylcoumarin-7-amide;
PCPS, phospholipid vesicles composed of 75% (w ⁄ w) phosphatidylcholine and 25% (w ⁄ w) phosphatidylserine; scu-PA, single-chain u-PA;
tcu-PA, two-chain u-PA; tcu-PA ⁄ T, thrombin-cleaved two-chain u-PA; u-PA, urokinase-type plasminogen activator.
2516 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS
based on the human scu-PA sequence), and yields an
active two-chain form of the enzyme (tcu-PA). u-PA
establishes a localized cell-surface proteolytic system
through activation of plasminogen and some matrix-
degrading metalloproteinases [9,10].
In this study, we investigated the plasma-dependent
mechanism by which plactin increases cellular u-PA
activity and identified prothrombin as one plasma
component that supported the action of plactin. Pro-
thrombin is a zymogen of the blood coagulation
enzyme thrombin that proteolytically forms fibrin from
fibrinogen [11]. At the site of vascular injury,
prothrombin is rapidly activated to thrombin by
coagulation factor Xa, which is assembled in a Ca
2+
-
dependent manner with factor Va on acidic phospho-
lipid membranes of damaged vascular endothelium or
activated platelet aggregates [12–14]. Activation of pro-
thrombin by the complex (prothrombinase complex) is

activity in U937 cells. The increase in u-PA activity
was not associated with an increase in the total
amount of u-PA [2]. Therefore, we tested whether plac-
tin D increased the conversion of inactive scu-PA to
active tcu-PA on cell surfaces in the plasma milieu.
First, we determined the levels of total and active
u-PA on U937 cells. Total u-PA activity was obtained
by treating U937 cells with plasmin, which could acti-
vate scu-PA to tcu-PA by cleaving at Lys158–Ile159.
Taking this value as 100%, the level of cellular active
u-PA, obtained without plasmin pretreatment, was as
low as  1% (Fig. 1A). This implied that  99% of
the total u-PA on U937 cells was in the inactive single-
chain form. Treatment of U937 cells with 50 lm plac-
tin D increased the level of active u-PA to  35% of
scu-PA
B-chain
A-chain
66
45
29
(kDa)
20% plasma
Plactin D
+
−
+
−
No plasma
B

none
2
nd
PM
Fig. 1. Promotion of scu-PA activation on U937 cells by plactin. (A)
U937 cells were first incubated with or without plactin D in the
presence of 20% (v ⁄ v) human plasma. After washing, cells were
incubated in the absence or presence of 100 n
M plasmin (PM) for
the indicated time (second incubation). After incubation, cellular
uPA activity was determined using a chromogenic u-PA substrate
in the presence of aprotinin, an inhibitor of plasmin. Line indicates
the average of duplicate determinations. (B) U937 cells were incu-
bated with
125
I-labeled scu-PA in the absence or presence of 50 lM
plactin D and 20% plasma. Aliquots of cell lysates were resolved
on reduced SDS ⁄ PAGE on a 12.5% gel. The positions of molecular
mass standards, as well as scu-PA, A- and B-chains of tcu-PA, are
shown.
T. Harada et al. Dual modulation of prothrombin activation
FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2517
the total u-PA level (Fig. 1A). The finding that  60%
of u-PA in plactin-treated cells was not activated by
plasmin might be partly explained by the observation
that thrombin-cleaved tcu-PA (see below for the
involvement of thrombin-cleaved tcu-PA) was 500
times less sensitive to activation by plasmin when com-
pared with scu-PA [19]. Next, we determined the con-
version of scu-PA to tcu-PA on the cell surface. In this

0
4
.
0
6
.0
8.
0
lortnoC
D nitc
a
l
P
u-PA activity (fluorescence intensity)
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hpeS
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4.0
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.0
8.0
1
2.1
4.1
05040302010
D nitcalP
SND-41-nitcalP
41-nitcalP
Concentration (μ
M
)
u
-PA activity (fluorescence intensity)
HN
HN

HN
HN
2
N
H
A
C
D
E
B
N
H
HN
HN
N
H
NH
O
O
O
O
O
-
D
g
r
A
-D
laV
ueL

NH
HN
HN
N
H
N
H
O
O
O
O
O
N
H
S
O
O
N
S
N
D
-
s
yL
0
1.0
2.0
3.0
4.0
u-

plactin-14 to CNBr-activated Sepharose gels via its
amino group should afford an active affinity matrix
(Fig. 2A). Indeed, plactin cofactor activity in human
plasma was successfully adsorbed to plactin-14–Sepha-
rose affinity gel (Fig. 2C). Similar results were
obtained when partially purified bovine plasma (frac-
tion E4A50; see Experimental Procedures) was used
for plactin-14–Sepharose chromatography, and cofac-
tor activity was recovered in fractions eluted with
0.5 m NaCl or 6 m guanidine ⁄ HCl. Some proteins
were specifically enriched in these fractions, although
many protein bands were detected on reduced
SDS ⁄ PAGE (Fig. 2D). No significantly adsorbed pro-
tein was detected when Sepharose 4B alone was used,
suggesting that the nonspecific protein binding in the
plactin-14–Sepharose chromatography was caused by
the hydrophobic surface provided by the coupled
plactin-14. The N-terminal amino acid sequences of
three specifically enriched proteins suggested that these
were prothrombin, apolipoprotein A-IV and apolipo-
protein A-I (Fig. 2D).
We chose prothrombin for further analysis because
prothrombin, but not apolipoproteins, might participate
in the proteolytic cleavage of scu-PA. When prothrom-
bin was used in place of plasma to determine plactin
cofactor activity, it did not support plactin D-dependent
enhancement of u-PA activity in U937 cells (Fig. 2E).
This was not unexpected, as prothrombin itself is an
inactive protease zymogen. Specific proteolysis by the
coagulation factor Xa activates prothrombin to

(Fig. 3D). Thus, there was an additional, cell-associ-
ated mechanism to achieve the generation of fully
active u-PA. One possible candidate is dipeptidyl pep-
tidase I (DPP-I), a thiol protease that could activate
tcu-PA ⁄ T [19], and is expressed at high levels in cyto-
toxic lymphocytes and myeloid cells, including U937
cells. Therefore, we examined the effects of cystatin, an
inhibitor of DPP-I, on tcu-PA ⁄ T activation by U937
cells. Cystatin effectively inhibited tcu-PA activation
by U937 cells (Fig. 3D) and prothrombin ⁄ Xa-mediated
scu-PA activation on U937 cells (Fig. 3E). These
results were consistent with the observation that DPP-I
was able to activate tcu-PA ⁄ T by removing two amino
acids (Phe157–Lys158) from the N-terminus of its
B-chain [19]. The sequential mechanism leading to
enhancement of scu-PA activation is shown in Fig. 3F.
Dual modulation of prothrombin activation
by plactin
The above results suggested that plactin D affected
Xa-catalyzed activation of prothrombin not only in
the U937 system, but also under other conditions. To
characterize the plactin action, prothrombin activation
was assayed using a purified system. Consistent with
the results obtained with the U937 system, prothrom-
bin activation was markedly increased by plactin D
when prothrombin was incubated with Xa (Fig. 4A).
Xa activity, measured using a chromogenic peptide
substrate (Spectrozyme Xa), was minimally affected by
plactin D (Fig. 4A, inset). Thus, it appeared likely that
plactin D altered prothrombin such that it was suscep-

. Under all these
conditions, plactin D did not affect Xa activity (Fig. 4,
insets) or the activity of isolated a-thrombin (data not
shown). In summary, the data demonstrated that plac-
tin D could promote or inhibit prothrombin activation,
depending on the conditions of activation (Fig. 4I). For
the inhibitory plactin D effect, the presence of both
phospholipids and Ca
2+
was required, whereas the pro-
motive effect was seen in the absence of either phospho-
lipids or Ca
2+
, irrespective of the presence or absence of
factor Va. Phosphatidylserine-containing phospholipid
membranes act as a scaffold for the Ca
2+
-dependent
0
20.0
40.0
60.0
0.08
0.1
A
DE F
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C
080604020
aX

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D nitc
a
lP
+
−
+
−
aX + nibmorhtorP
−
ni
duriH
−−
−
+++
−
u-PA activity (fluorescence intensity)
α
-
T
h
r
o
m
b
i
n
0
1.0
2.0

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c
t
i
n
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0
2
.0
4
.0
6
.
0
8.0
1
2
.1
4.1
6
.
1
u-PA activity (fluorescence intensity)
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ort
n
o
C
ni
t

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H
++
–––+–––+
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–
––+
–
–
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a
n
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ucs
)
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a
ni(
T
/
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t

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elgnir
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K
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e
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orP
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es
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e
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orP
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R
FGE
elgnirK
R
es
aetorP

p

e
k
i
l
-
I
-PPD
nitc
a
lPnitc
a
lP
nibmorhtorPnibmorhtorP
aXaX
Fig. 3. Mechanism of plactin promotion of prothrombin-mediated scu-PA activation in U937 cells. (A) U937 cells were incubated with human
prothrombin in the presence of 2 m
M CaCl
2
and 0.1 mM Spectrozyme TH to determine thrombin formation. Where indicated, 0.1 nM human
Xa and 25 l
M plactin D were included in the incubation. (B) U937 cells were incubated with the indicated protein in the absence or presence
of 50 l
M plactin D. The concentrations of prothrombin and a-thrombin were 347 and 27 nM, respectively. After washing, cellular u-PA activity
was measured. (C) U937 cells equilibrated with
125
I-labeled scu-PA were incubated with either prothrombin (347 nM) plus factor Xa (3 nM)or
a-thrombin (10 n
M) in the absence or presence of 50 lM plactin D and 30 nM hirudin. After washing, cells were lysed and subjected to

prothrombin, plactin D increased the level of a-throm-
bin (Fig. 5B). Thus, the plactin D effects were increas-
ing or decreasing the formation of a-thrombin without
the accompanying conversion of prothrombin to highly
active or inactive thrombin species.
Interaction between plactin and prothrombin
To investigate the interaction between plactin and
prothrombin, we synthesized a radiolabeled plactin
analog. The analog, [
14
C]plactin-50 [cyclo(-d-Val-l-
[
14
C]Leu-d-Leu-l-Phe-d-Lys-)], had two to three times
the activity of plactin D. Binding of [
14
C]plactin-50 to
prothrombin gave a curve that appeared to become
sigmoidal (Fig. 6A), although maximum binding was
not obtained because of the low solubility of
[
14
C]plactin-50. This observation was consistent with
the promotion and inhibition of prothrombin activa-
tion by plactin, which gave sigmoidal or bell-shaped
dose–response curves (Fig. 4). These properties of the
plactin–prothrombin interaction suggested a change in
the conformation of prothrombin after plactin bind-
ing. We measured the intrinsic fluorescence of
prothrombin to assess any conformational change.

0504
0
3020
1
0
0
002
004
0
0
6
0
0
8
0001
0
02
1
0
5
0
40302010
0
0
2
04
06
08
0
01

0
01
051
0
02
0
52
003
0
5
0
4
0302010
0
001
0
02
003
004
005
05040302010
0
05
001
051
002
052
003
05040302010
0

0
10
Xa activity
0
1
2
3
0
504
0
3
0
2
010
Xa activity
0
1
2
3
0
50
4
030
2
010
Xa activity
0
1
2
3

/
aX
a
V
/
a
X
aC / LP /aX
+
2
aC

/ LP
/
aX
+2
aC / LP /aX
+2
aV /
aC /
L
P /aX
+
2
aV /
aV / LP
/a
X
aV


0
001
4121%
0002
LP
aC
aV
––––++++
––++++––
–+–+–+–+
Fig. 4. Dual modulation of prothrombin activation by plactin D. (A–H) Factor Xa-catalyzed activation of human prothrombin was determined
by measuring the generation of thrombin using the chromogenic substrate Spectrozyme TH, in the presence of the indicated concentrations
of plactin D. Where indicated, factor Va (4 p
M in panel B and 2 nM in the other panels), PCPS (PL) (50 lM) or CaCl
2
(2 mM) were included.
The concentration of Xa was 1 p
M in (B) and 0.5 nM in the other panels. Inset shows the effect of plactin D on factor Xa activity in each con-
dition. The Xa concentration was 0.5 n
M for all incubations and the Va concentration was 2 nM when added. Ordinate denotes Xa activity as
expressed in A
405
Æmin
)1
· 10
3
, and abscissa plactin D concentration in lM. Each value represents the mean ± SD from determinations
performed in triplicate. (I) Summary of the plactin D effects on prothrombin activation. Maximal response values are plotted.
T. Harada et al. Dual modulation of prothrombin activation
FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2521

)1
, the level of the complex was not
elevated significantly (Fig. 7B). In another experiment,
the fate of intravenously injected
125
I-labeled pro-
thrombin was determined. Forty minutes after plac-
tin D treatment,
125
I-labeled prothrombin ⁄ thrombin
species in the blood were immunopurified and resolved
by SDS ⁄ PAGE. We did not detect the formation of
thrombin or its complex with antithrombin III in plac-
tin D-treated mice (Fig. 7C). The dose of plactin D
used in these experiments (0.1 or 1 mgÆkg
)1
) was suffi-
cient for plactin D to produce a protective effect in a
thrombin-induced pulmonary embolism model. In this
model, plactin D improved the survival of thrombin-
treated mice. A plactin D dose of 0.1 mgÆkg
)1
increased the survival rate to levels comparable with
that produced by 0.01 UÆkg
)1
of the fibrinolytic
enzyme plasmin (Fig. 7D). Furthermore, plactin D did
not show acute toxicity after intravenous injection at
25 mgÆkg
)1

P
A-2,1F
B
T
P
2,
1
F
A
nim
ni
tcalPl
ort
noC
A
B
noitavitca dezylatac-esanibmorhtorP
nitcalP
α nibm
or
hT
-
dr
a
d
n
a
ts
66
5

plactin D. Proteolytically active molecular
species were visualized by casein zymo-
graphy after resolving on nonreduced
SDS ⁄ PAGE on a 10% gel. Human
a-thrombin (0.3 lg) was used as a
standard.
Dual modulation of prothrombin activation T. Harada et al.
2522 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS
Conclusion
Our studies demonstrate plactin-mediated modulation
of prothrombin activation. Plactin binds to prothrom-
bin and dually modulates its activation, depending on
the form of the catalyst, factor Xa. Under physiologi-
cal conditions, the coagulation reaction proceeds via
membrane-associated processes. Plactin inhibits pro-
thrombin activation catalyzed by membrane-associated
Xa. This is consistent with the observation that plactin
inhibits the coagulation of plasma in activated partial
thromboplastin time tests and prothrombin time tests.
However, plactin enhances prothrombin activation
when the catalyst is nonmembrane-bound Xa. This
mechanism may participate in the enhancement of
fibrinolytic activity in the U937 cell system, in which
plactin enhances prothrombin activation and the for-
mation of inactive tcu-PA ⁄ T, which is subsequently
converted to fully active tcu-PA by cellular cystatin-
sensitive, DPP-I-like peptidase.
The specificity of prothrombinase for prothrombin is
mediated by exosites, which are physically separated
from the catalytic site, on the surfaces of the catalytic

in 0.1 m sodium bicarbonate, pH 9.0, and 0.5 m NaCl, fol-
lowed by blocking with 1 m ethanolamine. The amount of
plactin-14 immobilized was 7.0 lmolÆmL
)1
of gel. [
14
C]Plac-
tin-50 [cyclo(-d-Val-l-Leu-d-Leu-l-Phe-d-Lys-)] was synthe-
sized using Fmoc-l-Leu (1-
14
C) (American Radiolabeled
Chemicals Inc, St Louis, MO, USA). The specific radioac-
tivity was 1.02 BqÆ pmol
)1
. For assays, plactins dissolved in
dimethylsulfoxide were used at a solvent concentration of
1% (v ⁄ v).
Other materials
Human scu-PA was provided by Mitsubishi Tanabe
Pharma Corporation (Osaka, Japan). Other proteins and
chemicals were from the following sources: human tcu-PA
from JCR Pharmaceutical (Kobe, Japan); human plasmin
and aprotinin from Wako (Osaka, Japan); human pro-
thrombin, human coagulation factor Xa, the thrombin
1.8
1.5
1.2
0.9
0.6
0.3

Fig. 6. Interaction between plactin and prothrombin. (A) The bind-
ing of [
14
C]plactin-50 to human prothrombin was determined in the
presence of the indicated concentrations of [
14
C]plactin-50. Specific
binding data are shown. (B) The intrinsic fluorescence of human
prothrombin was measured in the absence or presence of CaCl
2
(2 mM) and plactin D (50 lM). *P < 0.01 by Student’s t-test, com-
pared with control. Error bars represent SD from determinations
performed in triplicate.
T. Harada et al. Dual modulation of prothrombin activation
FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2523
inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide
(DAPA) and polyclonal anti-(human thrombin) sheep IgG
from Haematologic Technologies (Essex Junction, VT,
USA); human coagulation factor V from Serbio (Paris,
France); human a-thrombin, BSA, cystatin and l-a-phos-
phatidylcholine (egg yolk) from Sigma (St Louis, MO,
USA); l-a-phosphatidylserine (porcine brain) from Avanti
Polar Lipids (Alabaster, AL, USA); glutaryl-Gly-Arg-4-
00.0
02.0
04.0
06.0
08
.
0

92
)a
D
k
(
4.
7
9
11.0
Coagulating
blood
Plactin D
(mg·kg
–1
)
Control
Plactin D
(mg·kg
–1
)
10.1
Control
Plasma TAT level (ng·mL
–1
)
4
6
40
30
20

**
**
**
**
**
Fig. 7. Effects of plactin D on plasma coagulation in vitro and prothrombin activation in vivo. (A) Activated partial thromboplastin time and
prothrombin time were measured using normal human plasma. Plactin D was added 5 min before the initiation of each reaction. The clotting
times in the absence of plactin D were 26.9 ± 0.2 s for activated partial thromboplastin time and 15.1 ± 0.7 s for prothrombin time. Error bars
represent SD from triplicate determinations. *P < 0.05 and **P < 0.01 by Dunnett’s test, compared with control. (B) Plactin D, at the indicated
dose, was given intravenously to mice (n = 5 for each group), and blood was drawn in a mixture of protease inhibitors, 40 min after the treat-
ment. The level of thrombin ⁄ antithrombin III complex in the resulting plasma was determined by enzyme immunoassay. Serum obtained from
blood drawn without anticoagulants from normal mice (Coagulating blood) was used as a standard. There were no statistical differences among
control, 0.1 mgÆkg
)1
plactin D and 1 mgÆkg
)1
plactin D groups by Dunnett’s test. (C) Plactin D and human
125
I-labeled prothrombin were
successively given intravenously to mice (n = 3 for each group). Blood was drawn in a mixture of protease inhibitors, 40 min after treatment.
Labeled proteins were purified from plasma with anti-(human thrombin) IgG–Sepharose and resolved on nonreduced SDS ⁄ PAGE on a 10% gel.
Serum from control mouse blood was similarly processed as a standard to detect prothrombin activation (Coagulating blood). Data shown are
representative. Essentially the same results were obtained in each group. The positions of prothrombin (PT), and prothrombin(desF1)
[PT(desF1)] and a-thrombin are shown. (D) Effect of plactin D on thrombin-induced pulmonary embolism in mice. Mice received intravenous
injection with saline (Control) plactin D (0.1 mgÆkg
)1
) or plasmin (0.01 UÆkg
)1
). After 15 min, human a-thrombin was injected intravenously to
induce pulmonary thromboembolism. Next day, the number of surviving animals was counted. Numbers above bars denote the number of

Tris ⁄ HCl, pH 7.4, 150 mm NaCl and 0.1% (w ⁄ v)
Tween 80; buffer G, 62.5 mm Tris ⁄ HCl, pH 6.8, 2% SDS,
10% glycerol, 5% 2-mercapthoethanol and 0.002% bromo-
phenol blue.
Cell culture
Human monocytoid line U937 cells (obtained from the
Japanese Cancer Research Resources Bank, Tokyo) were
maintained in RPMI-1640 medium supplemented with 10%
fetal bovine serum (JRH Biosciences, Lenexa, KS, USA),
100 UÆmL
)1
penicillin G and 100 lgÆmL
)1
streptomycin.
For assays, cells were seeded at 2 · 10
5
cellsÆmL
)1
in 15 mL
of the medium and grown for 2 days. Prior to use in experi-
ments, exponentially growing cells were harvested, washed
twice and suspended with buffer A.
Assay for cellular scu-PA activation
U937 cells were suspended with buffer A at a density of
5.0 · 10
6
cellsÆmL
)1
. Cells were incubated in the absence or
presence of 20% (v ⁄ v) human plasma and plactin at 37 °C

with buffer G. An aliquot of the lysate was subjected to
SDS ⁄ PAGE on a 12.5% gel. After fixing and drying, the
gel was exposed to an X-ray film at )80 °C for 16 h. In the
experiment shown in Fig. 3C,
125
I-labeled scu-PA was
bound to cell surface at 4 °C for 30 min in RPMI-1640
medium supplemented with 10% fetal bovine serum and
20 mm Hepes, pH 7.4. The labeled cells were used for
incubations, as described in the legend to Fig. 3.
Partial purification of plactin cofactor from
bovine plasma
Citrated bovine platelet-poor plasma (490 mL) was frac-
tionated using the method described by Cohn et al. [25].
Most of the cofactor activity to support plactin-dependent
activation of cellular scu-PA was recovered in the ‘precipi-
tate IV-1¢ fraction. The fraction was subjected to ammo-
nium sulfate fractionation at 4 °C, and precipitates
obtained from 25–50% saturation were dialyzed against
buffer C, yielding 2.3 g of partially purified cofactor
preparation (fraction E4A50). The specific activity of the
preparation was 24 times that of the original plasma.
Plactin-14–Sepharose chromatography
A column containing 0.5 mL of plactin-14–Sepharose was
equilibrated with buffer D at room temperature, and
0.6 mL of fraction E4A50 (11 mg protein) was applied to
the column. After washing with 2.5 mL of buffer D, the
column was developed with 2.5 mL of buffer D containing
0.5 m NaCl, followed by 2.5 mL of buffer D containing
6 m guanidine ⁄ HCl. Each eluate was dialyzed overnight

5nm Va at 37 °C for the indicated times in buffer F
(DAPA was included to inhibit the feedback proteolysis of
prothrombin by the generated thrombin). The reaction was
stopped by the addition of an equal volume of acetic acid,
and the resulting mixture was dialyzed against 0.2 m acetic
acid. After lyophilization, followed by dissolving in
buffer G, samples were resolved on reduced SDS ⁄ PAGE.
In the zymography assay for the free Xa-catalyzed reac-
tion, a reaction mixture (20 lL) containing 4 lm prothrom-
bin, 2 mm CaCl
2
,3nm Xa and 10 lm DAPA was
incubated at 37 °C for 1 h in buffer F. After incubation,
aliquots of the mixture were subjected to nonreduced
SDS ⁄ PAGE on a 10% gel containing 0.5 mgÆmL
)1
casein.
The gel was washed twice for 30 min with 2.5% (w ⁄ v)
Triton X-100 to remove SDS, followed by overnight incu-
bation at 37 °C with buffer F containing 2 mm CaCl
2
.
After staining with Coomassie Brilliant Blue R-250 the pro-
teolytically active position appeared as a colorless band on
dark blue background.
The activation of prothrombin in the presence of U937
cells was assayed by incubating 1.0 · 10
6
cellsÆmL
)1

guidelines for animal experiments at Tokyo Noko Univer-
sity. We took adequate steps to ensure that animals did not
suffer unnecessarily at any stage of an experiment. The
protocol was approved by the Animal Experiment Commit-
tee of Tokyo Noko University.
To determine the level of thrombin ⁄ antithrombin III
complex in plasma, male ICR mice ( 30 g; Japan SLC,
Hamamatsu) were anesthetized with intraperitoneal
urethane ⁄ a-chlorarose (750 and 60 mgÆkg
)1
, respectively).
Plactin D dissolved in saline was given to the mice intrave-
nously from caudal vein. After 40 min, blood collected by
cardiac puncture (720 lL) was immediately mixed with
80 lL of 3.8% (w ⁄ v) sodium citrate containing an inhibitor
cocktail (300 mm benzamidine, 50 lm leupeptin, 10 lm
antipain, 5 mm EDTA, 28 lm E64, 1 lm pepstatin and
0.2 mm FUT-175) to obtain plasma. The level of throm-
bin ⁄ antithrombin III complex was determined by enzyme
immunoassay at SRL (Tokyo, Japan).
To assay for
125
I-labeled prothrombin activation in vivo ,
male ICR mice (anesthetized with urethane ⁄ a-chlorarose)
received intravenous plactin D. Immediately after the plactin
injection, mice received an intravenous injection of human
125
I-labeled prothrombin (3.8 · 10
7
cpmÆkg

(50 lm)orVa(2nm) using 0.5 nm human Xa and Spectro-
zyme Xa. The intrinsic fluorescence of human prothrombin
was measured in buffer E with or without 2 mm CaCl
2
after incubation of 100 nm prothrombin and 50 lm plac-
tin D for 5 min at room temperature. Excitation and emis-
sion wave lengths were 290 and 340 nm, respectively. The
N-terminal amino acid sequence was determined after
transferring to poly(vinylidene difluoride) membrane using
an Applied Biosystems model 476A protein sequencer.
Dual modulation of prothrombin activation T. Harada et al.
2526 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS
Activated partial thromboplastin time and prothrombin
time were measured using commercial kits (Sysmex Interna-
tional Reagents, Kobe, Japan) according to the manufac-
turer’s instructions.
Acknowledgements
We thank Akira Endo for encouragement and Hiro-
yuki Yoshii and Emiko Iwao for technical assistance.
This work was supported by a grant from the Japan
Society for the Promotion of Science.
References
1 Inoue T, Hasumi K, Kuniyasu T & Endo A (1996)
Isolation of plactins A, B, C and D, novel cyclic
pentapeptides that stimulate cellular fibrinolytic activity.
J Antibiot (Tokyo) 49, 45–49.
2 Inoue T, Hasumi K, Sugimoto M & Endo A (1998)
Enhancement of fibrinolysis by plactins: structure–activ-
ity relationship and effects in human U937 cells and in
mice. Thromb Haemost 79, 591–596.

coagulation. Thromb Haemost 82, 165–174.
12 Krishnaswamy S, Mann KG & Nesheim ME (1986)
The prothrombinase-catalyzed activation of prothrom-
bin proceeds through the intermediate meizothrombin
in an ordered, sequential reaction. J Biol Chem 261,
8977–8984.
13 Krishnaswamy S, Church WR, Nesheim ME & Mann
KG (1987) Activation of human prothrombin by human
prothrombinase. Influence of factor Va on the reaction
mechanism. J Biol Chem 262, 3291–3299.
14 Mann KG, Nesheim ME, Church WR, Haley P &
Krishnaswamy S (1990) Surface-dependent reactions of
the vitamin K-dependent enzyme complexes. Blood 76,
1–16.
15 Rosing J, Tans G, Govers-Riemslag JW, Zwaal RF &
Hemker HC (1980) The role of phospholipids and fac-
tor Va in the prothrombinase complex. J Biol Chem
255, 274–283.
16 Esmon CT (1989) The roles of protein C and thrombo-
modulin in the regulation of blood coagulation. J Biol
Chem 264, 4743–4746.
17 Esmon CT (1995) Thrombomodulin as a model of
molecular mechanisms that modulate protease
specificity and function at the vessel surface. FASEB J
9, 946–955.
18 Von dem Borne PAK, Bajzar L, Meijers JCM, Nesheim
ME & Bouma BN (1997) Thrombin-mediated activation
of factor XI results in a thrombin-activatable fibrinoly-
sis inhibitor-dependent inhibition of fibrinolysis. J Clin
Invest 99, 2323–2327.

25 Cohn EJ, Strog LE, Hughes WL, Mulford DJ, Ash-
worth JN, Melin M & Taylor HL (1946) Preparation
and properties of serum and plasma proteins. IV. A
system for the separation into fractions of the protein
and lipoprotein components of biological tissues and
fluid. J Am Chem Soc 68, 459–475.
26 Verheijen JH, Mullaart E, Chang GTG, Kluft C &
Wijngaards J (1982) A simple, sensitive spectrophoto-
metric assay for extrinsic (tissue-type) plasminogen
activator applicable to measurements in plasma.
Thromb Haemost 48, 266–269.
27 Gresele P, Momi S, Berrettini M, Nenci GG, Schwarz
HP, Semeraro N & Colucci M (1998) Activated human
protein C prevents thrombin-induced thromboembolism
in mice. Evidence that activated protein c reduces intra-
vascular fibrin accumulation through the inhibition of
additional thrombin generation. J Clin Invest 101, 667–
676.
Dual modulation of prothrombin activation T. Harada et al.
2528 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS