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 actio
Trang 1cyclopentapeptide 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,
Japan
Fax: +81 42 367 5708
Tel: +81 42 367 5710
E-mail: hasumi@cc.tuat.ac.jp
*These authors contributed equally to this
work
(Received 30 January 2009, revised 17
February 2009, accepted 20 February 2009)
doi:10.1111/j.1742-4658.2009.06976.x
Plactin, a family of cyclopentapeptides, enhances fibrinolytic activity by elevating the activity of cellular urokinase-type plasminogen activator (u-PA), a protease involved in a variety of extracellular proteolytic events Factor(s) in the blood plasma is an absolute requirement for this plactin activity In this study, we found that plactin promoted plasma cofactor-dependent conversion of inactive single-chain u-PA to active two-chain u-PA on U937 cells Using plactin-affinity chromatography, we identified prothrombin as one of the plasma cofactors In incubations of U937 cells with prothrombin and Xa, plactin increased the formation of thrombin, which cleaved single-chain u-PA to afford the inactive two-chain form Thrombin-cleaved two-chain u-PA was alternatively activated by cellular cystatin-sensitive peptidase activity, yielding fully active two-chain u-PA In
a purified system, plactin bound to prothrombin, altered its conformation and dually modulated factor Xa-mediated proteolytic activation of pro-thrombin to a-pro-thrombin Plactin inhibited the activation catalyzed by Xa
in complex with Va, Ca2+ and phospholipids (prothrombinase), whereas the activations catalyzed by nonmembrane-associated Xa were enhanced markedly by plactin Plactin inhibited in vitro plasma coagulation, which involved prothrombinase formation Plactin did not cause prothrombin 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.
Trang 2based 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 Ca2+
-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 (propro-thrombinase complex) is
> 105 times faster than activation by free Xa [15]
Therefore, physiological coagulation is eventually
cata-lyzed by the prothrombinase complex In addition to
promoting fibrin formation, thrombin in complex with
thrombomodulin can activate protein C [16,17] and
thrombin-activated fibrinolysis inhibitor [18], which
modulate coagulation and fibrinolysis Thus, thrombin
plays multiple roles in hemostatic processes
In this study, we show that in a cultured cell system,
plactin enhances prothrombin activation to thrombin,
which cleaves cellular scu-PA to afford inactive
two-chain u-PA, which is activated by cystatin-sensitive
peptidase activity to yield fully active tcu-PA In a
purified system, plactin dually modulates prothrombin
activation, depending on the conditions of catalysis by
Xa Under conditions where membrane-associated Xa
formation is restricted, plactin enhances the formation
of a-thrombin, whereas plactin inhibits prothrombin
activation by membrane-associated Xa Plactin is
inhibitory to plasma coagulation in vitro and does not
cause prothrombin activation or thrombosis in vivo
Thus, we suggest that the dual modulation of
pro-thrombin activation by plactin leads to an
antithrom-botic state under physiological coagulating conditions
Results and Discussion
Plactin promotes cell-surface activation of scu-PA
Previous experiments have demonstrated that
plac-tin D promotes a plasma-dependent elevation in u-PA
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
No plasma
B
0 1 2 3 4
5
A
70 60 50 40 30 20 10 0
Time of second incubation (min)
1 st plactin
2 nd PM
1 st plactin
2 nd none
1 st none
2 nd none
1 st 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 with125I-labeled scu-PA in the absence or presence of 50 l M 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.
Trang 3the 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
experiment, U937 cells equilibrated with 125I-labeled
scu-PA were treated with plactin D, followed by
SDS⁄ PAGE of the labeled protein to resolve scu-PA
and tcu-PA As shown in Fig 1B, plactin D markedly
promoted conversion of scu-PA to the two-chain form
The apparent molecular masses of the resulting
poly-peptide chains were comparable with those of the
A- and B-chains of tcu-PA (an A-chain doublet was
caused by differential glycosylation) [20] The plactin
effect was specific in the presence of plasma (Fig 1B),
consistent with previous observations [2] From these results, we concluded that plactin D promoted cell-surface activation of scu-PA to tcu-PA, and that the conversion (specific proteolysis) required a cofactor in the plasma
Identification of prothrombin as a plasma factor participating in plactin activity
To identify the plasma cofactor required for plactin promotion of scu-PA proteolysis to tcu-PA, we attempted to develop affinity media to purify plactin-binding protein To immobilize plactin onto a gel matrix, we first looked for plactin derivatives with a free amino group One such candidate was plactin-14 (Fig 2A) Although plactin-14 itself had no activity, modification of its amino group with a dansyl group converted the molecule an active form (Fig 2B) This
0
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l o r t n C D n i c l P
-4 -n i t c l P e o r a p S -e o r a p S B 4 o
N
s
a
b
-4 -n i c a l P Sephaose Sepharose-4B
6 5
9 ) a D k (
5 2 6 1 4 7
R X L G K A Prothrombin
T A Q D N S
I -A -o A
V I -A -o A
Sequence not obtained
V D W S Q P D
0
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4 0
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4 1
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D n i c a l P
S D -4 -n i c a l P
4 -n i c a l P
Concentration ( μ M )
H
H 2 N
H N N O
O O O O
N O
H S e a r o s e d b
e o r a p S -4 -n i t c l P S
N -4 -n i t c l P 4
-n i t c l P D
n
i
t
c
l
P
H
H 2
N
A
B
H
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O
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u
e
P
H
H 2 N
H N N O
O O O O
H 2 s L
H
H 2 N
H N N O
O O O O
N O O N
S D -s L
0
1 0
2 0
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4 0
a X + T a X T
l o r t n C D n i c a l P
Fig 2 Identification of prothrombin as a plasma cofactor required for plactin activity (A) Structures of plactin D and its analogs DNS, dansyl (B) The activities of plactin D, plactin-14 and plactin-14–DNS to enhance cellular u-PA activity were measured by incubating each compound with U937 cells at the concentrations shown (C) Human plasma (diluted to 25% v ⁄ v in buffer D) was incubated with or without Sepharose 4B or plactin-14–Sepharose at 4 C for 20 min After centrifugation, the resulting supernatant was assayed for scu-PA activation
on U937 cells at a concentration of 10% (v ⁄ v) of original plasma in the presence or absence of plactin D (50 l M ) (D) Partially purified bovine plasma fraction E4A50 was subjected to plactin-14–Sepharose chromatography After flow-through fraction (FT) was collected and the col-umn was washed with buffer D, elution was carried out successively with buffer D containing 0.5 M NaCl or 6 M guanidine ⁄ HCl (Gnd-HCl) All fractions were dialyzed against buffer A before the assay Fractions were resolved on reduced SDS ⁄ PAGE on a 10% gel Arrowheads denote specifically enriched proteins N-terminal sequences of such proteins, and their identifications, are shown Apo-A, apolipoprotein A (E) U937 cells were incubated with the indicated protein(s) at 37 C for 30 min in the absence or presence of 50 l M plactin D The con-centrations of prothrombin and Xa were 347 n M and 50 p M, respectively After washing, cellular u-PA activity was measured Error bars represent SD from triplicate determinations In some data points, error bars are too small to be recognized.
Trang 4result was consistent with the idea that a sterically
restricted arrangement of four hydrophobic amino
acids and a basic amino acid is essential for plactin
activity [2] Therefore, we speculated that coupling of
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
thrombin Simultaneous incubation of U937 cells with
prothrombin and factor Xa produced a plactin
D-dependent increase in u-PA activity (Fig 2E)
There-fore, we suggest that prothrombin is one of the plasma
cofactors participating in plactin D-promoted scu-PA
activation on U937 cells
Mechanism of prothrombin- and plactin-mediated
enhancement of scu-PA activation
The above results suggested that prothrombin
activa-tion (thrombin formaactiva-tion) was involved in the
mecha-nism of plactin action and that plactin affected this
reaction Indeed, plactin D increased prothrombin
activation in the U937 cell system (Fig 3A), and
a-thrombin alone could produce a significant increase
in scu-PA activation on U937 cells (Fig 3B,C)
Plac-tin D affected a-thrombin-mediated scu-PA activation
only slightly (Fig 3B) Hirudin, a specific inhibitor of
thrombin, abolished the plactin D effect on scu-PA activation by prothrombin⁄ Xa (Fig 3C) Thus, it seemed likely that plactin D played a role in increasing the formation of a-thrombin in prothrombin⁄ Xa-medi-ated promotion of scu-PA activation
a-Thrombin can specifically cleave human scu-PA at Arg156–Phe157 [7], two residues proximal to the acti-vation cleavage site (Lys158–Ile159) Thrombin-cleaved tcu-PA (tcu-PA⁄ T), however, showed < 1% activity
of tcu-PA (consistent with previous reports) [7,19,21] Plactin D failed to activate tcu-PA⁄ T (data not shown) Nevertheless, incubation of tcu-PA⁄ T with U937 cells resulted in the generation of u-PA activity (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-tible to activation by Xa Under physiological coagula-tion condicoagula-tions, prothrombin activacoagula-tion is catalyzed by the prothrombinase complex (factor Xa in complex with factor Va, phospholipids and Ca2+) When pro-thrombin activation was assayed using propro-thrombinase complex, plactin D inhibited the reaction (Fig 4B) Because plactin did not affect the activity of prothrom-binase toward Spectrozyme Xa (Fig 4B, inset), it was
Trang 5also likely that plactin D altered prothrombin such
that it became resistant to the activation by Xa that
formed prothrombinase
To understand the mechanism for these conflicting
effects of plactin D on prothrombin activation, we
tested several combinations of the factors that make
prothrombinase a catalyst (Fig 4C–H) Prominent
enhancement by plactin D was observed when the
catalyst was Xa⁄ phospholipids, Xa ⁄ phospholipids ⁄ Va,
Xa⁄ Va or Xa ⁄ Ca2+ However, plactin D led to marked
inhibition when Xa⁄ phospholipids ⁄ Ca2+ was used A
marginal promotive effect of plactin D was observed
when the catalyst was Xa⁄ Va ⁄ Ca2+ 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 Ca2+was required, whereas the pro-motive effect was seen in the absence of either phospho-lipids or Ca2+, irrespective of the presence or absence of factor Va Phosphatidylserine-containing phospholipid membranes act as a scaffold for the Ca2+-dependent
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a X + T P ) n i c a l p + (
a X + T P ) l o r t n c (
l o r t n c n i c a l p + e o l a T P
A40
) n i m ( e m i T
6 5
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6 7
A -u c
n i a c -B
n i a c -A
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D n i c a l
a X + n i b m o r h t o r P
− n
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−
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hro
bin 0
1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0
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α -Th ro
bin
+pl
act D
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n i a t s C
+ –
D n i c a l P n i d r i H
+ +
) e v i c a i ( A -u
c t c u - A / T ( i a c i v e ) t c u - A ( a c i v e )
α - h r o m b i n
α - h r o m b i n
e s a t o r P
F G
I e l g i K
e s a t o r P
F G
I e l g i
F I e s a t o r P
e l g i
F G
I e s a t o r P
e l g i
K R EGF K i g l e R
e s a t o r P I F
G
E Kigle R
e s a t o r P I
e s a i p e k il -I -P
D P - I - il k e p i a s e D
n i t c l
P
n i b m o r h t o r
P
a Xa X
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 m M Spectrozyme TH to determine thrombin formation Where indicated, 0.1 n M 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 n M, respectively After washing, cellular u-PA activity was measured (C) U937 cells equilibrated with125I-labeled scu-PA were incubated with either prothrombin (347 n M ) plus factor Xa (3 n M ) or a-thrombin (10 n M ) in the absence or presence of 50 l M plactin D and 30 n M hirudin After washing, cells were lysed and subjected to reduced SDS ⁄ PAGE on a 12.5% gel, followed by autoradiography Positions of scu-PA, A- and B-chains of tcu-PA are shown (D) U937 cells (5.0 · 10 6 ) were equilibrated with tcu-PA ⁄ T (10 n M ) at 4 C for 30 min in buffer A After washing, cells were incubated with GGA-MCA in the absence or presence of 100 n M cystatin to determine u-PA activity (E) U937 cells were treated with prothrombin (347 n M ) and factor Xa (100 p M ) in the absence or presence of 25 l M plactin D After washing, cells received GGA-MCA to determine u-PA activity in the second incubation Where indicated, 30 n M hirudin or 100 n M cystatin was included both in the first and second incubations Error bars represent
SD from determinations carried out in triplicate In some data points, error bars are too small to be recognized (F) Schematic representation
of prothrombin- and plactin-mediated enhancement of scu-PA activation on U937 cells The u-PA molecule is shown schematically with each domain in a colored circle Amino acid residues involved in proteolytic cleavages are given in white circles A disulfide bond that connects A- and B-chains of tcu-PA is shown as red dashed line.
Trang 6assembly of the protein components that form
pro-thrombinase [11] Therefore, the plactin D action can be
interpreted as follows: plactin D is inhibits prothrombin
activation by membrane-associated Xa, whereas it is
promotive when free Xa is used as a catalyst
We next examined the possibility that plactin D
induced alternative proteolytic prothrombin cleavages
that resulted in increases or decreases in thrombin
activity When prothrombin activation was catalyzed
by fully assembled prothrombinase complex, plactin D
inhibited the formation of a-thrombin without
produc-ing proteolytic fragment species other than those
produced during the course of normal prothrombinase
catalysis (Fig 5A) When free Xa was used to activate
prothrombin, plactin D increased the level of
a-throm-bin (Fig 5B) Thus, the plactin D effects were
increas-ing or decreasincreas-ing 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, [14C]plactin-50
[cyclo(-d-Val-l-[14C]Leu-d-Leu-l-Phe-d-Lys-)], had two to three times
the activity of plactin D Binding of [14C]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 [14C]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 When prothrombin was incubated with plactin D in the absence of Ca2+, the intrinsic fluorescence increased by 4.6% (P < 0.01) (Fig 6B) Prothrombin has several Ca2+-binding sites, and Ca2+ binding alters its conformation Accordingly, the intrinsic fluo-rescence of prothrombin in the presence of Ca2+ was significantly lower (6.6%, P < 0.01) than in the absence of Ca2+ Plactin D increased the internal fluorescence by 7.7% (P < 0.001), even under these conditions (Fig 6B) Therefore, it was likely that plactin–prothrombin binding altered the conformation
of prothrombin and resulted in dual modulation of prothrombin activation, depending on the conditions
of factor Xa catalysis
0 0 0 0 0 0
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a
a V a
X X X a a P P L L C C a a + +
a C L P a
a V L P a
X X X a a C C a a + + V V a a
A405
3 )
Plactin D (μ M )
G
I
0 – 0 0
0 1
0 2
L P a C a V
– – – –
– – + + +
– + – + – + – +
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 n M in the other panels), PCPS (PL) (50 l M ) or CaCl 2 (2 m M ) were included The concentration of Xa was 1 p M in (B) and 0.5 n M 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 n M when added Ordinate denotes Xa activity as expressed in A 405 Æmin)1· 10 3
, and abscissa plactin D concentration in l M 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.
Trang 7Can plactin be a procoagulant or an anticoagulant?
We asked whether plactin inhibited or stimulated the
blood coagulation system, because plactin dually
modulated prothrombin activation As shown in
Fig 7A, plactin D showed anticoagulant activity in
experimental coagulation tests: plactin D significantly
prolonged both activated partial thromboplastin time
(fibrin clot formation time after Ca2+ addition to
phospholipid-supplemented, contact-phase-activated
plasma) and prothrombin time (clot formation time
after the addition of tissue factor–phospholipids
com-plex and Ca2+ to plasma) In these measurements,
the enzyme that catalyzed prothrombin activation
was in situ-generated, membrane-associated Xa
How-ever, thrombin time (clot formation time after the
addition of a-thrombin to plasma), which did not
involve prothrombin activation, was not affected by
plactin D (data not shown) These observations
appeared consistent with results obtained using
puri-fied systems
Finally, the effect of plactin D on prothrombin
activation was examined in vivo using normal mice
In one experiment, prothrombin activation was
evalu-ated as the formation of a thrombin⁄ antithrombin III
complex When mice were treated with plactin D at 0.1 and 1 mgÆkg)1, the level of the complex was not elevated significantly (Fig 7B) In another experiment, the fate of intravenously injected 125I-labeled pro-thrombin was determined Forty minutes after plac-tin D treatment, 125I-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 placplac-tin 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 These results may exclude the idea that plactin D is a procoagulant
It is possible that plactin inhibits physiological coagulation, which proceeds via membrane-associated processes [11], and that plactin does not behave as a procoagulant under normal circulation conditions
5
.
0
0
6
5
9
)
a
D
k
(
6
1
4
.
7
5 4 3 5 2 2 5 1 1 5 0 0 5 4 3 5 2 2 5 1 1
) 1 F s d ( T A -2 , 1 F B
T
2 , 1 F
A
n i m
n i t c a l P l
o r n C
A
B
n i a i c d z l a t a -e a i b m o r h t o r P
n i t c a l
P α - h r o m b i n
d r a n t s
6
5
9 ) a D k (
l o r n C
n i a i c d z l a t a -a X
Fig 5 Analysis of thrombin species formed
in the presence of plactin D (A) Human pro-thrombin was activated by propro-thrombinase complex in the absence or presence of
50 l M plactin D At the indicated times, aliquots of the incubation mixtures were withdrawn to analyze by reduced SDS ⁄ PAGE on a 10% gel Proteins were visualized by Coomassie Brilliant Blue R-250 The positions of prothrombin (PT), prothrombin(desF1) [prothrombin without fragment 1; PT(desF1)] and fragment
1 + 2 + A-chain (F1,2-A), as well as A-chain and B-chain of a-thrombin B, are shown (B) Human prothrombin was activated by free factor Xa in buffer F containing 2 m M CaCl 2
in the absence or presence of 25 l M 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.
Trang 8Our 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 domains It is postulated that substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed
by a conformational change to engage the Xa catalytic site [22] Thus, prothrombin activation is a conforma-tionally regulated process This may partly explain the plactin-mediated dual modulation of prothrombin activation The pharmacological application of dual thrombin modulation would be an intriguing approach
to intervention in thromboembolic diseases
Experimental procedures
Plactins Plactin D [cyclo(-d-Val-l-Leu-d-Leu-l-Phe-d-Arg-)] and plactin-14 [cyclo(-d-Val-l-Lys-d-Leu-l-Phe-d-Arg-)] were synthesized according to Fmoc chemistry, as described pre-viously [1,2] Dansylplactin-14 (plactin-14-DNS) was syn-thesized by mixing 1 mL of plactin-14 (1 mgÆmL)1 in water), 1 mL of dansyl chloride (4 mgÆmL)1 in acetone) and 90 mg of NaCO3 overnight at ambient temperature Plactin-14–Sepharose was prepared by reacting 35 mL of 0.7 mgÆmL)1 plactin-14 with 1.5 g of CNBr-activated Sepharose 4B (GE Healthcare Biosciences, Tokyo, Japan)
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)1of gel [14 C]Plac-tin-50 [cyclo(-d-Val-l-Leu-d-Leu-l-Phe-d-Lys-)] was synthe-sized using Fmoc-l-Leu (1-14C) (American Radiolabeled Chemicals Inc, St Louis, MO, USA) The specific radioac-tivity was 1.02 BqÆpmol)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
0
8
6
4
2
0 [14C]Plactin-50 (µM)
*
*
21
20
19
18
22
B
A
Ca2+
Control Plactin D (50 µ M )
+
− Fig 6 Interaction between plactin and prothrombin (A) The
bind-ing of [14C]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 m M ) and plactin D (50 l M ) *P < 0.01 by Student’s t-test,
com-pared with control Error bars represent SD from determinations
performed in triplicate.
Trang 9inhibitor 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-4-0 0
0 0
0 0
0 0
0 0
0 1
n i t c a l P
*
*
5 / 3
4 / 3
5 / 2
n i m s a l P l
o r n C
T
) 1 F s e ( T
α - T h r o m b i n
6
5
9
) a D k ( 4 7
1 1 0
Coagulating blood
Plactin D (mg·kg –1 ) Control
Plactin D (mg·kg–1)
1 0.1 Control
4 6
40 30 20 10
2 0
31.8
Coagu-lating blood 0
35
30 25 20 15 10 5
0 0 0 0 0 0 0
Prothrombin time
Activated partial thromboplastin time
Plactin D ( μ M )
**
**
**
**
**
**
**
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)1plactin 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 survived ⁄ total animals in each group *P < 0.01 by Fisher’s exact test, compared with control.
Trang 10methylcoumarin-7-amide (GGA-MCA) from Peptide
Institute (Osaka, Japan); Spectrozyme TH
(H-d-hexahydro-tyrosyl-Ala-Arg-p-nitroanilide), Spectrozyme Xa
(methoxy-carbonyl-d-hexahydrotyrosyl-Ala-Arg-p-nitroanilide) and
recombinant hirudin from American Diagnostica
(Green-wich, CT, USA)
Factor V (300 nm) was activated to Va by incubating
with a-thrombin (3 nm) at 37C for 10 min
Thrombin-cleaved tcu-PA (tcu-PA⁄ T) was prepared by incubating
scu-PA (1 lm) with a-thrombin (10 nm) at 37C for 22 h,
followed by the addition of 30 nm hirudin to neutralize
thrombin Phospholipid vesicles (PCPS) composed of 75%
(w⁄ w) phosphatidylcholine and 25% (w ⁄ w)
phosphatidyl-serine were prepared as described previously [23]
Radio-iodination of scu-PA and prothrombin was performed by
the IODO-GEN method [24], using carrier-free Na125I to a
specific activity of 2000–3000 cpmÆng)1of protein
Buffers used were: buffer A, 20 mm sodium phosphate,
pH 7.4, and 150 mm NaCl; buffer B, 50 mm Tris⁄ HCl,
pH 7.4, and 100 mm NaCl; buffer C, 50 mm sodium
phos-phate, pH 7.4, and 80 mm NaCl; buffer D, 50 mm sodium
phosphate, pH 7.4; buffer E, 50 mm Tris⁄ HCl, pH 7.4,
100 mm NaCl and 0.01% (w⁄ v) Tween 80; buffer F, 20 mm
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· 105
cellsÆmL)1in 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· 106cellsÆmL)1 Cells were incubated in the absence or
presence of 20% (v⁄ v) human plasma and plactin at 37 C
for 30 min, with shaking After washing with buffer B, cells
were resuspended in buffer B containing 0.1 mm
GGA-MCA, a chromogenic peptide substrate for u-PA After
incubation at 22C for 1 h, the supernatant was removed
and acetic acid was added to 10% to stop the reaction The
fluorescence of 7-amino-4-methylcoumarine liberated from
GGA-MCA by the u-PA cleavage was measured (excitation
at 380 nm and emission at 480 nm)
In the experiment shown in Fig 1A, cells treated in the
first incubation were further incubated with plasmin
(100 nm) at 22C for the indicated time in buffer B con-taining BSA (10 mgÆmL)1) After addition of aprotinin (40 kallikrein inhibitor unitsÆmL)1 to neutralize plasmin) and washing, cells were processed to determine u-PA activity as described above
In some experiments, scu-PA activation on U937 cells was also determined as the proteolytic cleavage of 125 I-labeled scu-PA In this experiment, 125I-labeled scu-PA (5.6 nm, 3000 cpmÆng)1) was included in the first incuba-tion After washing twice with buffer B, cells were lysed 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, 125I-labeled scu-PA was bound to cell surface at 4C 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 4C, 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 against buffer A before SDS⁄ PAGE and assay for plactin-dependent promotion of scu-PA activation on U937 cells
Assay for prothrombin activation The activation of prothrombin was assayed by incubating human prothrombin (20 nm) and Spectrozyme TH (0.1 mm) in the presence of factor Xa in buffer F with or without factor Va (4 pm or 2 nm), PCPS (50 lm) or CaCl2
(2 mm) The concentration of Xa was 1 pm when the incuba-tion contained Va (4 pm), PCPS and CaCl2to assemble pro-thrombinase complex In other assays, the Xa concentration