Mutations in Autolytic Loop-2 and at Asp554
of Human Prothrombin That Enhance Protein C Activation by
Meizothrombin*
Hisashi
Koike,
Daiju
Okuda, and
Takashi
Morita
From the Department of Biochemistry, Meiji Pharmaceutical
University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
Received for publication, August 12, 2002, and in revised form, February 13, 2003
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ABSTRACT |
Thrombin acts on many protein substrates during
the hemostatic process. Its specificity for these substrates is
modulated through interactions at regions remote from the active site
of the thrombin molecule, designated exosites. Exosite interactions can
be with the substrate, cofactors such as thrombomodulin, or fragments
from prothrombin. The relative activity of
-thrombin for
fibrinogen is 10 times greater than that for protein C. However, the
relative activity of meizothrombin for protein C is 14 times greater
than that for fibrinogen. Modulation of thrombin specificity is linked
to its Na+-binding site and residues in autolytic
loop-2 that interact with the Na+-binding site. Recombinant
prothrombins that yield recombinant meizothrombin (rMT) and rMT
des-fragment 1 (rMT(desF1)) enable comparisons of the effects of
mutations at the Na+-binding residue (Asp554)
and deletion of loop-2 (Glu466-Thr469) on the
relative activity of meizothrombin for several substrates. Hydrolysis
of t-butoxycarbonyl-VPR-p-nitroanilide by
-thrombin, recombinant
-thrombin, or rMT(desF1) was almost
identical, but that by rMT was only 40% of that by
-thrombin.
Clotting of fibrinogen by rMT and rMT(desF1) was 12-16% of that by
-thrombin, as already known. Strikingly, however, although
meizothrombins modified by substitution of Asp554 with
either Ala or Leu or by deletion of loop-2 had 6-8 and <1%, respectively, of the clotting activity of
-thrombin, the activity of
these meizothrombins for protein C was increased to >10 times that of
-thrombin. It is proposed that interactions within thrombin that
involve autolytic loop-2 and the Na+-binding site primarily
enhance thrombin action on fibrinogen, but impair thrombin action on
protein C.
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INTRODUCTION |
In the procoagulant reactions of blood coagulation,
-thrombin
plays a pivotal role in activating coagulation factors (factors V,
VIII, XI, and XIII and fibrinogen) and in stimulating a
variety of cells such as platelets, leukocytes, and
endothelial cells (1-7). In anticoagulant reactions,
-thrombin
binds to thrombomodulin, and the thrombin-thrombomodulin complex
activates protein C, the proteinase that is key to shutting down the
procoagulant processes (8). Meizothrombin and meizothrombin
des-fragment 1 are short-lived intermediates in the activation process
of prothrombin to
-thrombin; both have enzymatic activity (9-17).
Fig. 1 illustrates the cleavage sites in prothrombin and the structures
of
-thrombin, meizothrombin, and meizothrombin des-fragment 1. Meizothrombin in its normal interaction with thrombomodulin efficiently
activates protein C in vitro, but it cleaves fibrinogen
inefficiently (18). Moreover, meizothrombin exhibits only 2% of the
platelet aggregation activity of thrombin, and its rate of inhibition
by antithrombin III is 43% if that of
-thrombin (18, 19).
In the
-thrombin molecule, both autolytic loop-2
(Glu146-Lys149E, chymotrypsin
numbering1; residues
Glu466-Lys474 of prothrombin) and the
Na+-binding region (Asp221-Tyr225,
chymotrypsin numbering; residues Asp552-Tyr557
of prothrombin) are involved in determining thrombin specificity for
fibrinogen clotting and protein C (20-24). When these regions are
deleted or substituted in
-thrombin to create a
non-Na+-binding thrombin form, fibrinogen-clotting activity
is remarkably low, but the protein C activator activity decreases only
slightly (22-24). No systematic examination of the role of the
Na+-binding region and autolytic loop-2 in meizothrombin
has been made. If mutations in these regions are constructed in
meizothrombin, investigation of the relative activities of the mutants
will enable the relationships between Na+ binding and the
functional differences between
-thrombin and meizothrombin to be
determined. Determination of the specificity differences between
-thrombin and meizothrombin is made difficult by the autolytic
cleavages that occur in prothrombin and meizothrombin. We have
constructed recombinant prothrombin molecules with substitutions of Ala
for Arg at the sites of autolysis to eliminate the confounding cleavages, as reported previously (18), that permit definitive testing
of their specificity for two substrates, protein C and fibrinogen.
We then constructed novel meizothrombin mutants with alterations in the
Na+-binding site or with a deletion of autolytic loop-2. We
report here that these loop-2- and Na+-binding
site-modified recombinant meizothrombin derivatives show moderate to
large reductions in fibrinogen-clotting activity, but large enhancement
of protein C activator activity.
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EXPERIMENTAL PROCEDURES |
Materials--
Human prothrombin was purified following a
published method (25). Human protein C was purified using the following
modifications of the prothrombin purification method. The
Ba2+-protein precipitate, after
(NH4)2SO4 precipitation (25), was applied to a DEAE-Sepharose column and eluted with a linear gradient of
0-0.5 M NaCl. Fractions that contained factors IX and X
and protein C were applied to heparin-Sepharose and eluted with 0.25 M NaCl. The protein C-containing fractions were applied to
a Mono Q column and eluted with a linear gradient of 0-0.5
M NaCl. The purification of human protein C was analyzed by
SDS-PAGE at each step. The prothrombin activator ecarin, from
Echis carinatus venom, was purified as reported previously
(26, 27). Human recombinant thrombomodulin
(rTM)2 was a kind gift from
Dr. Yasuo Sasaki (Asahi Kasei Corp., Shizuoka, Japan). The
chromogenic substrate for thrombin,
t-butoxycarbonyl-Val-Pro-Arg-p-nitroanilide (Boc-VPR-pNA), and the substrate for activated protein C,
Boc-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide (Boc-LSTR-MCA), were from
Seikagaku Corp. (Tokyo, Japan) and Peptide Institute, Inc.
(Osaka, Japan), respectively.
Construction of Prothrombin Mutants--
A full-length human
prothrombin cDNA was isolated from a human liver cDNA library
using two oligonucleotide primers from human prothrombin to which
restriction enzyme sites (underlined in the following sequences) were
added. The 5'-primer sequence was
5'-TTTGAATTCACCATGGCGCACGTCCGAGGC-3', and the
3'-primer sequence was
5'-CATTGATCAGTTTGGAGAGCGGCCGCGGTT-3'. An amplified cDNA
of 1.9 kb was cloned into pUC19, and the resulting pUC/hPT was
sequenced using a DSQ-2000L DNA sequencer (Shimadzu Corp., Kyoto,
Japan). pUC/hPT was digested with EcoRI and NotI, and the 1.9-kb cDNA fragment was separated by agarose gel
electrophoresis and purified using a QIAquick DNA purification kit
(QIAGEN GmbH, Hilden, Germany). This fragment was cloned into a
mammalian expression vector (pSecTag, Invitrogen) that contains
Myc and His tags at the C-terminal end. The protein product of
this construct, i.e. human prothrombin produced with
pSecTag/hPT, is designated PT-tag. Prothrombin mutants were constructed
from pUC/hPT by a PCR-based site-directed mutagenesis method (28). The
sequences of all prothrombin mutants were determined by DNA sequencing.
In the mutant PT-RA155, Arg155 is replaced by Ala
(the site between fragments 1 and 2) (see Fig. 1); and in the mutant
PT-RA271/284, Arg271 and Arg284 are replaced by
Ala (see Fig. 1). In the mutant PT-RA155/271/284, Arg155,
Arg271, and Arg284 are replaced by Ala (29).
The Na+-binding site mutants PT-DA554 and PT-DL554 have
Asp554 replaced by Ala or Leu, respectively. In
PT
466-469, Glu466-Thr469 was deleted from
PT-RA155/271/284. All PT constructs contain Myc and His tag sequences
at their C-terminal ends, adding 3.4 kDa to these proteins compared
with their hypothetical plasma prothrombin-derived homologs. The Myc
tag was used to detect the cloned products by anti-Myc immunoblotting.
Expression and Purification of Recombinant Prothrombin
Mutants--
Expression constructs were transfected into COS-7 cells
using FuGENE 6 (Roche Molecular Biochemicals). The transfected cells were cultured in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum for 24 h. Two cultures were grown: one with 10 µg/ml vitamin K and one without. The transfected cells were
subsequently transferred to serum-free Dulbecco's modified Eagle's
medium, again either with or without 10 µg/ml vitamin K, and further
incubated for 48 h. Expressed PT-tag secreted into the cultured
medium was recovered after centrifugation at 2000 × g
for 10 min at 4 °C to remove the cells.
The supernatant culture medium was applied to an
Ni2+-nitrilotriacetic acid SF column (8-ml volume; QIAGEN
GmbH) equilibrated with 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl to capture the His-tagged protein. The effluent was
directly connected to ÄKTA Explorer 10S. The column was washed
with 5 column volumes of the equilibrating buffer and eluted by a
two-step gradient (0-40 and 40-250 mM imidazole in the
equilibrating buffer; flow rate of 2 ml/min). One-ml fractions were collected.
The fractions were dialyzed overnight against Tris-buffered saline
(TBS; 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and
5 mM CaCl2), and the protein concentrations of
all fractions were determined with a DC protein assay kit
(Bio-Rad). Aliquots of the fractions were diluted in 2× SDS buffer
(125 mM Tris-HCl (pH 6.8), 30% glycerol, 2% SDS, 0.2%
bromphenol blue, and 5%
-mercaptoethanol (for disulfide reduction
where noted)). Samples were subjected to electrophoresis on 10%
SDS-polyacrylamide gels. The proteins were transferred onto a
polyvinylidene difluoride membrane (Finetrap NT-32, Nihon Eido,
Tokyo, Japan) and incubated with anti-Myc (Invitrogen) and anti-human PT antibodies overnight at 4 °C. The proteins were visualized with a VECTASTAIN Elite ABC kit (Vector Labs, Inc., Burlingame, CA) and a POD immunostain set (Wako, Osaka,
Japan). All prothrombin mutants were expressed and purified by the same method as PT-tag.
Amino Acid Sequence Determination--
N-terminal amino acid
sequences of the recombinant prothrombins were determined in
Ni2+-nitrilotriacetic acid-purified fractions. Aliquots of
the column fractions were diluted in 2× SDS buffer, subjected to
electrophoresis on 10% SDS-polyacrylamide gels, transferred to a
polyvinylidene difluoride membrane (ProBlottTM, PerkinElmer
Life Sciences), and stained with 0.1% Amido Black 10B in 50% methanol
and 10% acetic acid. Protein bands to be sequenced were cut out and
washed with deionized water (MilliQ, Millipore Corp). The amino acid
sequences were determined with an Applied Biosystems Model 477A-120A
amino acid sequencer.
Thrombin Assay of Recombinant Prothrombin--
All prothrombin
mutants were activated by ecarin, from E. carinatus venom
(26, 27). In a typical assay, the plasma-derived or recombinant
prothrombins (50 nM final concentration) were incubated with ecarin (0.5 nM final concentration) in TBS and 0.1%
bovine serum albumin at 37 °C in a 96-well microtiter plate. After
15 min, 0.1 volume of 2.5 mM Boc-VPR-pNA was
added to the activation mixture, and the initial rate of
p-nitroaniline formation was monitored at 405 nm with a
kinetic plate reader (Wellreader, Seikagaku Corp.). Products of
prothrombin activation (after a 30-min incubation) were analyzed by
SDS-PAGE and immunoblotting with anti-Myc antibody.
Clotting Assay--
Plasma and recombinant prothrombins were
adjusted to yield protein concentrations with equal rates of hydrolysis
of Boc-VPR-pNA (~0.5 absorbance unit/min) after
complete activation with ecarin. Prothrombin samples adjusted to these
protein concentrations were incubated with ecarin (0.5 nM
final concentration) in TBS and 0.1% bovine serum albumin at 37 °C
in a coagulometer cup (Amelung KC4A, Baxter). After 30 min, 0.1 volume of 20 mg/ml fibrinogen was added to the activation
mixture, and the clotting time was measured. The clotting times were
converted to units of thrombin activity using a standard curve
constructed from various concentrations of
-thrombin from plasma prothrombin.
Protein C Activator Activity--
Plasma or recombinant
prothrombin (2 nM final concentration) was activated by
ecarin (0.2 nM final concentration) in TBS and 0.1% bovine
serum albumin at 37 °C in a 96-well microtiter plate. After 30 min,
equal volumes of human protein C (80 nM final
concentration), rTM (either 40 nM final or varying
concentrations), and 3:1 (mol/mol) phosphatidylcholine/phosphatidylserine vesicles (100 µM
final or various concentrations) in TBS containing 0.1% bovine serum albumin were added to the reaction mixture and incubated for 60 min at
37 °C to activate protein C. Activated protein C was determined from
the initial rate of hydrolysis of Boc-LSTR-MCA (1 mM final concentration), measured by 7-amino-4-methylcoumarin liberation (
ex = 380 nm,
em = 465 nm) with a GENios
fluorometer (Tecan).
Platelet Aggregation Assay--
Platelet aggregation was
measured by determining the changes in light transmission of platelet
suspensions with an optical aggregometer (Niko Bioscience Hema Tracer
601). Maximal aggregation induced by 100 nM r-
-thrombin
was defined as 100%. Washed platelets were prepared from blood from
healthy volunteers with informed consent. All volunteers denied taking
any medications that would alter the response of platelets to thrombin.
Blood was collected into 0.1 volume of 3.8% sodium citrate by
venipuncture and centrifuged at 150 × g for 20 min at
room temperature to prepare platelet-rich plasma. Washed platelets were
prepared by further centrifugation (300 × g, 3 min) of
platelet-rich plasma to separate the platelets, which were then washed
twice with 15% acid/citrate/dextrose and 100 nM
prostacyclin. Platelet pellets were suspended in HEPES/Tyrode's buffer (20 mM HEPES (pH 7.4), 138 mM NaCl, 3.3 mM NaH2PO4, 2.9 mM KCl,
1.0 mM MgCl2, 1.0 mM
CaCl2, and 1 mg/ml glucose). Resuspended platelets were
incubated for 2 min at 37 °C, after which r-
-thrombin, rMT,
rMT-DL554, or rMT
466-469 (0.1-100 nM final
concentrations) was added to initiate aggregation.
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RESULTS |
To compare the specificity of meizothrombin and meizothrombin
des-fragment 1 with that of
-thrombin, recombinant prothrombin molecules with a substitution of Ala for Arg at the sites of autolysis were constructed. Fig. 1 shows the
cleavage sites and the sites of the mutations; the substitutions and
deletions are defined in Table I. PT-tag
is an recombinant prothrombin with Myc and His tags at the C terminus,
but without other mutations. PT-tag thus serves as the control for
recombinant prothrombin and r-
-thrombin. PT-RA155, with
Arg155 substituted with Ala, is not cleaved to form
fragments 1 and 2, but produces r-
-thrombin that is structurally
identical to that derived from PT-tag. PT-RA271/284 and
PT-RA155/271/284 produce rMT(desF1) and rMT, respectively, and have
been reported previously (29). The three mutants PT-DA554, PT-DL554,
and PT
466-469 are new constructs designed to produce rMT-DA554,
rMT-DL554, and rMT
466-469, respectively. These products permit the
evaluation of the contributions of the Na+-binding site Asp
residue and autolytic loop-2 to the specificity of meizothrombins for
fibrinogen and protein C.

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Fig. 1.
Activation products of human recombinant
prothrombins. Human plasma prothrombin is converted to
-thrombin by the cleavage of Arg271 and
Arg320 by coagulation factor Xa. -Thrombin cleaves
between fragments 1 (F1) and 2 (F2) at
Arg155 and in the -thrombin A chain at
Arg284 to produce fragments 1 and 2 and a small N-terminal
fragment of the A chain (residues 272-284). The arrows
indicate the sites of proteolytic cleavage or the sites of amino acid
mutagenesis. The gray shading shows the activated forms of
meizothrombin, thrombin, and the recombinant proteins after activation.
A and B are the thrombin A and B chains, which
are linked by a disulfide bond.
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Expression in COS Cells and Purification of Recombinant
Prothrombins--
The seven recombinant prothrombin constructs were
transfected into COS cells. All prothrombin mutants were efficiently
expressed (Fig. 2A). The
importance of
-carboxylation for prothrombin secretion by COS cells
was also demonstrated.
-Carboxylation, which modifies a glutamate in
the
-carboxyglutamic acid domain, depends on vitamin K as a
cofactor. Recombinant prothrombin derivatives in COS cells were
efficiently secreted into the culture medium only in the presence of
vitamin K (Fig. 2B). Thus, prothrombin secretion in this
cell system depends on
-carboxylation.

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Fig. 2.
Expression and secretion of recombinant
prothrombin mutants. Cell lysates and culture media from COS cells
transfected with various constructs were subjected to SDS-PAGE and
immunoblotted with anti-Myc antibody. A, analysis of the
prothrombin mutants expressed by COS cells. Cell lysates were analyzed
on a 10% SDS-polyacrylamide gel (10 µg of protein/lane).
B, analysis of the prothrombin mutants secreted into the
cultured medium with or without vitamin K (Vit. K; 10 µg/ml). Samples were analyzed on a 10% SDS-polyacrylamide gel (10 µg of protein/lane).
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The mutants PT-tag and PT-RA271/284 had the molecular
masses expected for wild-type prothrombin plus the mass of the tag
(Fig. 2, A and B). For recombinant prothrombins
with the Arg155-to-Ala mutation, the molecular masses
appeared higher upon SDS-PAGE by ~3-4 kDa (Fig. 2A). The
N-terminal amino acid sequences of all expressed recombinant
prothrombins were identical to that of wild-type prothrombin
(Ala-Asn-Thr-Phe-Leu-). All mutants bound to an
Ni2+-nitrilotriacetic acid column
(Ni2+-chelating bead), implying that the His tag at the C
terminus was correctly constructed and intact. Because both the N and C termini appeared to be correct, the mobility shift of the
Arg155 mutants was not investigated further.
The prothrombin precursor mutants with Myc and His tag sequences at
their C termini were purified using metal-chelating column chromatography and detected by anti-Myc antibody. The purity of the
PT-tag-containing fractions eluted from the column was demonstrated by
SDS-PAGE and by immunoblot analysis (Fig.
3). No autolysis products could be
detected in the PT-tag-containing fractions. All mutants were purified
by identical metal-chelating column chromatographic procedures (data
not shown).

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Fig. 3.
Purification of recombinant prothrombin from
the culture medium. The flow-through (F) and eluted
fractions from Ni2+-nitrilotriacetic acid column
chromatography (10 µl of each fraction) were immunostained with
anti-human PT antibody ( -hPT; upper panel) and
anti-Myc antibody ( -myc; middle panel). The
fractions were also electrophoresed and stained with Coomassie
Brilliant Blue R-250 (CBB; lower panel). The
arrowheads indicate the position of PT-tag.
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Activation of Prothrombin Mutants and Functional Characterization
of Activated Forms--
As anticipated, PT-tag and PT-RA155 were
readily converted to r-
-thrombin by the prothrombin activator
ecarin, whereas PT-RA271/284 and PT-RA155/271/284 were activated to
rMT(desF1) and rMT, respectively (Fig.
4). PT
466-469, PT-DA554, and PT-DL554
were similarly converted to the mutant meizothrombin derivatives.
All recombinant prothrombins were rapidly activated to their cleaved
forms by ecarin, implying that the structure around the relevant
scissile bond, Arg320, was not grossly perturbed.

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Fig. 4.
Activation products of recombinant
prothrombin mutants. Left panel, after disulfide
reduction; right panel, without disulfide reduction. Each
recombinant prothrombin was incubated in the presence and absence
of ecarin for 30 min at 37 °C. The reaction mixtures were subjected
to SDS-PAGE and visualized by immunoblotting with anti- Myc
antibody. MT, meizothrombin; MT(desF1),
meizothrombin des-fragment 1; -T, -thrombin.
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We assessed the activity of each recombinant thrombin and meizothrombin
derivative by measuring Boc-VPR-pNA hydrolysis rates and by
fibrinogen clotting (Table II). PT-RA155
and r-
-thrombin produced from PT-tag had activities for both
substrates experimentally indistinguishable from those of
-thrombin
derived from native prothrombin. The Boc-VPR-pNA hydrolytic
activity of rMT(desF1) was 94% of that of r-
-thrombin, but rMT
activity was decreased to 38%. This is consistent with a published
report that the prothrombin fragments, which remain covalently linked
in rMT, reduce
-thrombin hydrolysis of peptide pNA (30).
rMT-DA554, rMT-DL554, and rMT
466-469 had ~20%
Boc-VPR-pNA hydrolytic activity compared with r-
-thrombin (Table II). These reductions in activity are similar to those cited in
Ref. 31 for different Na+-binding site mutants of
-thrombin. Fig. 5 shows the
fibrinogen-clotting activities of various activated forms of
recombinant prothrombin derivatives.
-Thrombin and r-
-thrombin
derived from PT-tag had essentially the same clotting activity, but the
activity of rMT(desF1) derived from PT-RA271/284 decreased to 16% and
that of rMT derived from PT-RA155/271/284 decreased to 12% of the
r-
-thrombin activity. rMT-DA554 and rMT-DL554 had 8 and 6% of the
r-
-thrombin activity, respectively. rMT
466-469 had no
significant fibrinogen-clotting activity (<1%). Thus, deletion of
autolytic loop-2 abolishes meizothrombin activity for fibrinogen, and
mutation of the Na+-binding residue reduces this
activity.

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Fig. 5.
Fibrinogen-clotting activity of activated
recombinant mutants. The concentrations of each of the recombinant
prothrombin products were adjusted to have the same rate of hydrolysis
of Boc-VPR-pNA. The recombinant prothrombin samples were
activated by ecarin. Fibrinogen (2.0 mg/ml) was added to the activated
mixtures, and the clotting time was measured. Relative clotting
activity is the ratio of thrombin activity to r- -thrombin activity
as described under "Experimental Procedures."
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Protein C Activator Activity--
We examined each of the forms of
thrombin and meizothrombin for their ability to activate protein C in
the presence of phospholipids and thrombomodulin (Fig.
6A). The significance of
-carboxyglutamic acid domain-containing fragment 1 can be assessed
by comparing the activity of rMT(desF1), which lacks fragment 1, with
that of the fragment 1-containing rMT derivatives (Fig. 6A,
black bars). The activities of rMT and its derivatives were
dramatically higher for protein C than those of both rMT(desF1) and
r-
-thrombin, in contrast to the lower activities of rMT and its
derivatives for fibrinogen. In the absence of covalently attached
fragment 1, no enhancement of protein C activation was observed,
i.e. with rMT(desF1) or r-
-thrombin (Fig. 6A).
Fig. 6 (B and C) shows the effects of rTM and
phospholipid concentration on protein C activation by r-
-thrombin,
rMT, and rMT derivatives and verifies that the observations are not
unique to a limited set of reactant concentrations.

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Fig. 6.
Protein C activator activities of recombinant
mutants under various conditions. Each recombinant prothrombin (2 nM) was activated by ecarin and then incubated with protein
C (80 nM) and phospholipid (100 µM) in the
presence or absence of rTM (40 nM). Protein C activation
was assayed by liberation of 7-amino-4-methylcoumarin from LSTR-MCA.
A, relative protein C (PC) activator activities
of thrombin and meizothrombin derivatives. Black and
white bars show the activities in the presence and absence
of rTM, respectively. B, effect of rTM on activation of
protein C by r- -thrombin and activated forms of prothrombin
derivatives. Each mutant was assayed for protein C activation in the
presence of various concentrations of rTM and a fixed phospholipid
concentration (100 µM). , thrombin from wild-type
prothrombin; , rMT(desF1); , rMT; , rMT-DA554; , rMT-DL554;
, rMT 466-469. C, effect of phospholipid on activation
of protein C. Various concentrations of phospholipid were tested in the
presence of a fixed rTM concentration (40 nM). Symbols are
as defined for B. PCPS, 3:1 (mol/mol)
phosphatidylcholine/phosphatidylserine vesicles.
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Platelet Aggregation Activity--
-Thrombin is a potent
physiological platelet activator. The effects of r-
-thrombin and rMT
derivatives on platelet aggregation are shown in Fig.
7. Whereas r-
-thrombin at
3 nM induced 95% platelet aggregation, rMT at 100 nM induced only ~60% aggregation. Both rMT-DL554 and
rMT
466-469 had poor platelet aggregation-inducing activity (<5%),
even at the highest concentration tested (100 nM). This
result shows that both meizothrombin derivatives rMT-DL554 and
rMT
466-469 lack significant platelet aggregation-inducing activity.
Ecarin, the snake venom-derived prothrombin activator present in these
reaction mixtures, did not initiate platelet aggregation (data not
shown).

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Fig. 7.
Effect of
r- -thrombin and rMT derivatives on platelet
aggregation. The percentages of platelet aggregation were
calculated by comparing the extent of change relative to a control
platelet aggregation induced by 100 nM r- -thrombin. ,
r- -thrombin; , rMT; , rMT-DL554; , rMT 466-469.
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DISCUSSION |
Meizothrombin is known to have enhanced protein C activator
activity and diminished fibrinogen-clotting activity compared with
-thrombin (11, 18, 32). In fibrinogen clotting, prothrombin fragment 2 in meizothrombin and meizothrombin(desF1) inhibits interaction with fibrinogen (32). The marked stimulation of meizothrombin protein C activator activity by phospholipids clearly indicates the importance of fragment 1, which includes the
Ca2+- and phospholipid-binding
-carboxyglutamic acid
domain (33, 34), for enhancement of the reaction by phospholipid
vesicles (18). Côté et al. (18) observed no
enhancement of meizothrombin activity for protein C when
soluble thrombomodulin was used rather than the
lipid-binding native form of thrombomodulin. One plausible explanation
for this difference is the contribution that binding to phospholipid
membranes makes to the reaction rates by promoting the formation of
thrombin- or meizothrombin-thrombomodulin complexes. More
complex alterations in the conformation of meizothrombin can also be
speculated to occur, e.g. as a result of an intramolecular interaction involving the fragment 1 region and a thrombin exosite. Such conformational changes might be similar to the conformational changes that are caused by thrombomodulin in
-thrombin that convert it from a fibrinogen-preferring to a protein C-preferring proteinase.
In
-thrombin, deletion of autolytic loop-2
(Glu146-Lys149E, chymotrypsin numbering;
Glu466-Lys474 of prothrombin) or the
substitution of the Na+-binding site residues
(Asp221-Tyr225, chymotrypsin numbering;
Asp552-Tyr557 of prothrombin) decreases its
fibrinogen-clotting activity, but affects only slightly its protein C
activator activity in the presence of thrombomodulin and phospholipids
(20, 23, 35). In particular, deletion of the entire loop-2 of
-thrombin nearly eliminates clotting activity, but protein C
activator activity is reduced by only 2-fold (23). Most of the effect
must originate from direct perturbation of fibrinogen binding due to
the loss of critical interactions between loop-2 and fibrinogen in the autolytic loop-2 deletion mutant.
In this study, Ala or Leu substitution at the Na+-binding
site (Asp554) or deletion of autolytic loop-2 residues
(Glu466-Thr469) (Fig. 1) in
meizothrombin was used to test the importance of the
Na+-binding site and autolytic loop-2, known "specificity
determinants" for
-thrombin, in meizothrombins. The results
demonstrate that these regions are also specificity determinants for
meizothrombin, but with opposite effects in meizothrombin and
-thrombin. Whereas autolytic loop-2 and the Na+-binding
residue Asp554 are crucial for
-thrombin action on
fibrinogen, mutation of the Na+-binding site in
meizothrombin causes only a modest decrease in action on fibrinogen.
The differences in the relative specific activity of the
activated meizothrombin mutants for fibrinogen and protein C are more
dramatic; in fact, they change from reduced to enhanced relative activity for these two substrates. Specifically, in fibrinogen clotting, rMT is substantially decreased to 12% and rMT(desF1) to 16%
relative to r-
-thrombin (Fig. 5 and Table II). rMT-DA554 (8%) and
rMT-DL554 (6%) had even lower clotting activity; rMT
466-469 had
<1% of the r-
-thrombin clotting activity. Because these rMT derivatives (rMT-DA554, rMT-DA554, and rMT
466-469) have no
Na+-binding site, their conformation is possibly similar to
the non-Na+-binding form of thrombin (35).
The control mutants (r-
-thrombin from PT-tag and rMT(desF1)) were
indistinguishable from native
-thrombin in their ability to catalyze
protein C activation in the presence of thrombomodulin and phospholipid
vesicles (Fig. 6A). The protein C activator activities of
both rMT and its derivatives were increased by 10-fold or more compared
with those of
-thrombin (Fig. 6A). These four highly active proteins are distinguished by having the fragment 1 region attached, whereas
-thrombin, r-
-thrombin, and rMT(desF1) do not.
Both rTM and phospholipids increased the protein C activator activities
of the meizothrombin mutants with fragment 1 (Fig. 6, B and
C). Interestingly, even in the absence of thrombomodulin, the fragment 1-containing proteins showed enhanced protein C activator activity compared with
-thrombin (Fig. 6A). Table II
summarizes the relative chromogenic, fibrinogen-clotting, and protein C
activator activities of the activated forms of recombinant
prothrombins. Most significant is the fact that, although rMT-DA554,
rMT-DL554, and rMT
466-469 lack an Na+-binding function,
these activated recombinant meizothrombins have very high protein C
activator activities, indicating that the Na+-binding site
has an insignificant role (if any) in protein C activation by meizothrombin.
The other procoagulant process recognized as a key function of
-thrombin is activation of platelets. In this process, similar to
the action of these various forms of thrombin on fibrinogen, the
mutations resulted in reduction in relative activity. Fig. 7 shows the
effects of r-
-thrombin, rMT, and meizothrombin derivatives on the
aggregation of washed platelets. Both rMT-DL554 and rMT
466-469 had
essentially no platelet aggregation activity. These results imply that
rMT
466-469 has little or no protease activities for fibrinogen or a
physiological substrate such as PAR-1 (protease-activated receptor-1), the thrombin receptor on platelets. Further studies are
required to determine whether this is due to decreased binding or
decreased ability to catalyze proteolytic cleavage.
It is useful to attempt to relate these observations to the crystal
structure of thrombin, although it must be noted that surface residues
can be subject to crystal packing variations that prevent detailed
interpretations from being made. Fig.
8A shows a model of
-thrombin derived from one of the thrombin crystal structures (36).
Active-site residues are shown in red. The Na+-binding site (Asp552-Asp554)
and the autolytic loop-2 region
(Glu466-Thr469) are yellow and
orange, respectively. Other side chains of amino acids in dark green are the interaction sites of fibrinogen,
which are located throughout the areas surrounding both the
Na+-binding site and the loop-2 region of thrombin (24,
37-39). rMT-DA554, rMT-DL554, and rMT
466-469 lack a functional
Na+-binding site in the thrombin region shown in Fig.
8A. Moreover, rMT
466-469, which is defective in
autolytic loop-2, is missing Glu466 (Glu146,
chymotrypsin numbering), which ion pairs with Arg553
(Arg221A, chymotrypsin numbering) of the
Na+-binding site (31, 40, 41), and had essentially no
fibrinogen-clotting activity, suggesting that the side chains of loop-2
might have a significant role in the interaction between meizothrombin
and fibrinogen (Fig. 8B). The mutation of Asp554
(Asp222, chymotrypsin numbering), adjacent to the
salt-bridging Arg553 (Arg221A, chymotrypsin
numbering), could similarly be related to the conformational changes
that are responsible for the differences between fibrinogen and protein
C cleavage by the mutant meizothrombins. In conclusion, it is proposed
that interactions within thrombin that involve autolytic loop-2 and the
Na+-binding site primarily enhance thrombin action on
fibrinogen, but impair thrombin action on protein C.

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|
Fig. 8.
Model of thrombin and schematic
representation of the activities of -thrombin
and meizothrombin derivatives. A, Swiss Protein
Database plot of thrombin derived from the crystal structure of
the thrombin-haemadin complex (1E0F; Ref. 36). Active-site
residues are shown in red. The Na+-binding site
(Asp552-Asp554) and the autolytic loop-2
region (Glu466-Thr469) are in
yellow and orange, respectively. Side
chains in dark green indicate the fibrinogen interaction
sites (37), which are widely located in the areas surrounding both the
Na+-binding site and the loop-2 region. -Helixes
(blue) and -sheets (light green) are
indicated. B, schematic representation of the significance
of prothrombin fragment 1, the autolytic loop-2 region, and the
Na+-binding site in meizothrombin (MT) for
fibrinogen (Fbg)-clotting and protein C (PC)
activator activities. -T, -thrombin; TM,
thrombomodulin; APC, activated protein C. The
orange and black bars indicate fragments 1 and 2, respectively.
|
|
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Craig M. Jackson for
reading the manuscript, critical comments, and useful suggestions. The
human recombinant soluble thrombomodulin was a gift from Dr. Yasuo
Sasaki. We are grateful to Yasuo Yamazaki and Yusuke Sugiyama for
purifying experimental proteins.
 |
FOOTNOTES |
*
This work was supported in part by Health Sciences
Research Grants on Advanced Medical Technology from the Ministry of
Health, Labor, and Welfare of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel. and Fax:
81-424-95-8479; E-mail: tmorita@my-pharm.ac.jp.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M208220200
1
The numbering of the prothrombin mutants
reported in this study begins at the amino terminus of human
prothrombin. Glu146 in autolytic loop-2 of
-thrombin
(chymotrypsin numbering) is equivalent to Glu466 in
prothrombin, and the Na+-binding residue Asp222
in
-thrombin (chymotrypsin numbering) is Asp554 in
prothrombin (see Ref. 7).
 |
ABBREVIATIONS |
The abbreviations used are:
rTM, recombinant
thrombomodulin;
Boc, t-butoxycarbonyl;
pNA, p-nitroanilide;
MCA, 4-methylcoumaryl-7-amide;
PT, prothrombin;
TBS, Tris-buffered saline;
r-
-thrombin, recombinant
-thrombin;
rMT, recombinant meizothrombin;
rMT(desF1), recombinant
meizothrombin des-fragment 1.
 |
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.