From the Division of Molecular, Cellular, and Microbial Biology,
Department of Biological Sciences, University of Calgary, Calgary,
Alberta T2N 1N4, Canada and Department of Chemistry and
Biochemistry and W. M. Keck Center for Transgene Research,
University of Notre Dame, Notre Dame, Indiana 46556
Received for publication, October 25, 2002, and in revised form, March 5, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To develop a fast-acting clot dissolving agent, a
clot-targeting domain derived from the Kringle-1 domain in human
plasminogen was fused to the C-terminal end of staphylokinase with a
linker sequence in between. Production of this fusion protein in
Bacillus subtilis and Pichia pastoris was
examined. The Kringle domain in the fusion protein produced from
B. subtilis was improperly folded because of its
complicated disulfide-bond profile, whereas the staphylokinase domain
produced from P. pastoris was only partially active because
of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was
functional, and this fusion showed fibrin binding ability by binding
directly to plasmin-digested clots. In vitro fibrin clot
lysis in a static environment and plasma clot lysis in a flow-cell
system demonstrated that the engineered fusion outperformed the
non-fused staphylokinase. The time required for 50% clot lysis was
reduced by 20 to 500% under different conditions. Faster clot lysis
can potentially reduce the degree of damage to occluded heart tissues.
Thrombolysis (1-4) is one of the well established treatments for
patients with acute myocardial infarction (commonly known as heart
attack). Blood clot-dissolving agents currently approved for
thrombolytic therapy include tissue plasminogen activator (tPA),1 urokinase,
streptokinase, and their derivatives (3, 4). Although the treatment can
reduce mortality, several large scale clinical trials indicate that
these blood clot-dissolving agents are far from ideal (4, 5). Even with
tPAs, only about 60% of the treated patients have their blood flow
restored 90 min after the onset of treatment (5). Some of the treated
patients will suffer from reocclusion or bleeding complications such as hemorrhagic strokes (2, 3, 6). Furthermore, the reoccluded clots are
usually platelet-rich and are more resistant to tPA-mediated clot lysis
(7, 8). Hence, choices of more potent blood clot dissolving agents,
which provide a rapid, complete, and sustained reperfusion with minimal
side effects, are needed.
Staphylokinase (SAK), a 136-amino acid protein from certain lysogenic
Staphylococcus aureus strains, is a plasminogen
activator and a promising blood clot-dissolving agent with clinical
potency that is at least as good as tPA (9, 10). In addition, it has
some desirable features that are superior to tPA (11). Notably, SAK
mediates the lysis of platelet-rich and retracted clots efficiently (12, 13) and shows exceptional fibrin specificity (9, 10, 14, 15).
These properties can help minimize reocclusion and bleeding complications.
Much has been studied on the action of SAK in vivo. To
function as the plasminogen activator, SAK first forms a complex with plasmin(ogen) (16). Complex formation is followed by SAK processing in
which the N-terminal peptide containing the first 10 amino acids from
SAK is removed by cleaving at a twin lysine site between residues 10 and 11 (Fig. 1A). This processing step is essential for
attaining an active form of SAK (17-19). The SAK-plasminogen complex
then forms a ternary complex with another molecule of plasminogen and
converts this plasminogen to plasmin. When the SAK-plasmin complex is
not fibrin-bound, it can be inhibited by the natural plasmin inhibitor,
Although SAK is a fibrin-specific thrombolytic agent, it has no fibrin
binding ability by itself. It binds to fibrin clots only indirectly
through the interaction with any clot-bound plasmin(ogen). It would be
of interest to determine whether the clot lysis efficacy of SAK can be
improved by engineering it with direct fibrin binding ability. Faster
clot lysis will restore blood flow in a more timely manner and reduce
damage to heart tissues. Furthermore, if less SAK is required to
achieve the same degree of clot lysis, the risk for side effects can be minimized.
Plasminogen binds to fibrin clots via its Kringle domains at the
N-terminal regions (20). These Kringle domains are ~80 amino acids
long; some of them possess the lysine binding capability (21-23).
Thus, plasminogen binds abundantly to fibrin clots during clot lysis
since more C-terminal lysine residues are generated through the action
of plasmin, a trypsin-like protease that cuts after lysine and arginine
residues. Among the characterized Kringles from human plasminogen
(24-28) and tPA (29), Kringle-1 from human plasminogen has the highest
lysine binding affinity. Therefore, Kringle-1 was selected as the
fibrin targeting domain and fused to the C-terminal end of SAK (Fig.
1). To ensure that each domain within the fusion can fold independently
and that each has sufficient space to interact with its target, a
20-amino acid linker is inserted between these domains. The resulting
fusion (designated SAK-L-K1) and its three site-specific muteins
(SAKM1-L-K1, SAKM2-L-K1, and SAKM3-L-K1) were produced via secretion
from Bacillus subtilis and Pichia pastoris.
Biochemical properties, clot targeting ability, and clot lysis activity
of the purified SAK-L-K1 and its derivatives were determined. In
comparison with SAK, the engineered SAKM3-L-K1 consistently mediated
faster lysis of both fibrin and plasma clots in vitro.
Construction of B. subtilis Expression Vectors for SAK-L-K1 and
Its Derivatives--
pSAKLK1 is a pUB18-based B. subtilis
plasmid (30) for secretory production of SAK-L-K1 under control of the
B. subtilis P43 promoter for transcription and the
levansucrase signal sequence (SacB SP) for secretion. To introduce the
linker sequence between SAK and the Kringle-1 domain from human
plasminogen, the nucleotide sequence of the expression cassette
(P43-SacBSP-SAK-L-K1) was split into two portions. The first
portion covered from the P43 promoter to the middle half of the linker
sequence. The sequence was generated by PCR using pSAKP (31) as
template. The primers used for this amplification were P43F
(5'-GGGAATTCGAGCTCAGCATTATTG-3') and SAKLB
(5'-CTTGTCGACCCACCAGAAGTACTTCCTTTCTTTTCTATAACAACCTTTG-3'). The
resulting product was an 895-bp EcoRI-SalI
fragment. After digestion with both EcoRI and
SalI, the fragment was inserted into pUB18 (30) at the
corresponding sites located in the polylinker region of the vector to
generate pUB18-SAKLF. The second half of the expression cassette
carried a sequence encoding the C-terminal half of the linker and
the Kringle-1 domain. This sequence was generated by PCR using a
cDNA clone of human plasminogen as template. This cDNA clone
was kindly provided by Dr. Ross T. A. MacGillivray at the
Department of Biochemistry and Molecular Biology, University of British
Columbia. The forward primer LK1F
(5'-GGGTCGACAAGTGGTGGATCTACTAGTGGCTCTGGATCCGGAATTTGCAAGACTGGGAATGG-3') encoded the sequence for both the C-terminal half of the linker and the
beginning of the Kringle-1 domain. The backward primer LK1B
(5'-CTTCTAGATTATGATCAACACTCAAGAATGTCGCAGTAG-3') introduced a
translation termination codon (TGA) at the end of the Kringle-1 sequence and an XbaI site at the 3' end of the fragment. The
resulting 310-bp SalI-XbaI PCR product was
digested with SalI and XbaI and inserted into the
corresponding sites in the polylinker region of the pUB18-SAKLF to
generate pSAKLK1.
To eliminate the N-linked glycosylation site in SAK-L-K1,
Asn-28 was changed to Asp and Ala to generate SAKM1-L-K1 and
SAKM2-L-K1, respectively. The change of Thr-30 to Ala resulted in the
generation of SAKM3-L-K1. These mutations were generated by inverse PCR
(32) using the primer pairs shown in Table I with pSAKLK1 as the
template. The entire plasmid was amplified, and the PCR product was
treated with T4 polynucleotide kinase, self-ligated, and transformed to B. subtilis WB600 (33). The resulting clones were screened
for the appropriate mutation by direct sequencing of the expression cassette (P43-SacBSP-SAK-L-K1). Each of the mutated
cassettes was then inserted as an EcoRI-XbaI
fragment into pUB18 to generate pSAKM1LK1, pSAKM2LK1, and pSAKM3LK1.
These vectors allowed the rapid examination of the effects of mutation
on SAK activity in B. subtilis.
Cloning of Structural Gene Encoding SAK-L-K1, SAKM2-L-K1, and
SAKM3-L-K1 into P. pastoris Expression Vector--
Two primers
(AFACSAKF and SAKK1CPICB) were designed to facilitate cloning of these
structural genes immediately downstream of the Transformation of P. pastoris--
pPICSAKM2LK1 or
pPICSAKM3LK1 was linearized with PmeI and transformed to
P. pastoris X-33 (a wild-type strain, Invitrogen) using
EasyComp method according to the manufacturer's protocol. Transformants were selected on zeocin (600 and 1000 µg/ml) and screened for secretory production of SAKM2-L-K1 or SAKM3-L-K1. X-33
cells with pPICZ Production and Affinity Purification of SAKM3-L-K1--
For
protein purification, a Pichia transformant showing the
highest expression level of SAKM3-L-K1 was cultured for 16-18 h at
28-30 °C in buffered glycerol-complex medium (34). Cells were
pelleted at 3,000 × g for 5 min and resuspended to 100 Klett units using a Klett-Summerson photoelectric colorimeter (Klett Mfg. Co.) in buffered methanol-complex medium (34). Growth continued at
28-30 °C in a shake flask. Production of SAKM3-L-K1 was induced by
methanol (0.5% final concentration) administered every 12 h during the entire culture period.
After 28-30 h of culture, the culture supernatant was collected by
centrifuging the cells at 3,000 × g for 5 min and
applied to a lysine-agarose column (Sigma) equilibrated with column
binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH
7.5). After washing the column with 3-5 bed volumes of the binding
buffer, SAKM3-L-K1 was eluted with 0.15 M
Matrix-assisted Laser Desorption Ionization-Time of Flight
(MALDI-TOF) Mass Spectrometry--
Purified SAKM3-L-K1 in deionized
water was mixed with the matrix solution of sinapinic acid and analyzed
on an Applied Biosystems Voyager-DE PRO mass spectrometer calibrated
with trypsinogen (m/z 23,981). The instrument
operated in the linear mode with an acquisition range 10,000-35,000
Da. This analysis was performed at the Alberta Peptide Institute,
Edmonton, Alberta, Canada.
Isothermal Titration Calorimetry--
Purified SAKM3-L-K1 was
extensively dialyzed against 150 mM sodium phosphate, pH
7.4, and quantified spectrophotometrically at 280 nm as previously
described. Titrations were performed using a Microcal VP-ITC
calorimeter at 25 °C in 150 mM sodium phosphate, pH 7.4, following the manufacturer's guidelines and using Microcal Origin for
data analysis. EACA (Sigma) was used as the ligand. The incremental
heat change accompanying binding was corrected for the corresponding
heat of dilution of EACA into buffer that was obtained in a separate
experiment by titrating EACA into the sample cell containing buffer only.
Fibrin Binding Study--
An ELISA method was used to assess the
fibrin binding ability of SAKM3-L-K1 and SAK. Cross-linked fibrin was
formed on the wells of a Nunc-Immuno MaxiSorp module (Nalge Nunc
International Corp.) using the procedure described by Wu et
al. (36). Formation of cross-linked fibrin was confirmed by
SDS-PAGE (36). The fibrin was digested with plasmin (Roche
Applied Science) at 4 milliunits/well (~1 pmol/well) at room
temperature. At different time points, the unbound plasmin was removed
immediately by washing 4 times with PBST (0.1 M sodium
phosphate, 0.15 M sodium chloride, pH 7.2, 0.1% Tween 20).
Purified SAKM3-L-K1 or SAK in PBST containing 3% bovine serum albumin
was added to the wells at a final concentration of 200 nM (20 pmol/well). After 2 h at room temperature,
unbound materials were removed by washing. SAKM3-L-K1 or SAK retained on the well was probed with polyclonal antibodies against SAK (31)
followed by horseradish peroxidase-conjugated anti-mouse secondary
antibodies. The amount of horseradish peroxidase retained was assessed
using 1-Step Turbo TMB (Pierce) as the horseradish peroxidase
substrate according to the manufacturer's instructions. Color
development at end point was determined at 450 nm using a microplate
reader (CERES 900, Bio-Tek Instruments, Inc.). The experiment was
repeated three times.
Fibrin Clot Lysis Study--
Fibrin clots were formed by adding
human thrombin (to 0.6 NIH units/ml) (1NIH unit = 0.324 ± 0.073 µg thrombin) and CaCl2 (to 20 mM) to human
fibrinogen (1 mg/ml, final concentration) in HEPES-buffered saline
(HBS; 0.01 M HEPES, 0.13 M NaCl, pH 7.4). Both
thrombin and fibrinogen were highly purified materials from Sigma.
Immediately after mixing, 100-µl aliquots of the polymerizing fibrin
solution were pipetted to the wells of a microtiter plate (Falcon 3912 flat-bottom polyvinyl chloride plate, BD Biosciences). Clot
formation was allowed to proceed for 3 h at room temperature. The
surface of the clots was washed with HBS, and excess fluid was
carefully removed. A 100-µl solution containing freshly mixed human
plasminogen (1.5 µM, Sigma) and varied concentrations of purified SAKM3-L-K1 or SAK in HBS was layered on each clot. The changes
in clot turbidity with time were monitored by measuring changes in the
absorbance at 405 nm at 25 °C using the CERES 900 microtiter plate
reader operated in the kinetic mode. After 30 min the surface of each
clot was gently washed with HBS three times to remove any unbound
SAKM3-L-K1 or SAK. 100-µl aliquots of plasminogen (1.5 µM) in HBS were layered on the clots, and measurement was
immediately resumed until all readings reached the low plateau, which
was taken as completion of clot lysis. Duplicate wells were prepared
for each concentration of SAKM3-L-K1 or SAK in each experiment, and the
experiment was repeated three times. As a control, some clots were
layered only with HBS but treated otherwise the same.
Plasma Clot Lysis Study (Clot Perfusion Model)--
Blood was
drawn by venipuncture from healthy adult donors (who had taken no
aspirin in the preceding 2 weeks) into 1/10 volume of buffered
sodium citrate (129 mM). Platelet-poor plasma (PPP) was
prepared from the pooled citrated blood by centrifugation at 1,500 × g for 15 min and frozen immediately at Other Methods--
Vent DNA polymerase (New England
BioLabs) was used for all DNA amplification reactions. The
sequence of all PCR products was confirmed to be free of PCR errors by
nucleotide sequencing performed at the University Core DNA and Protein
Services, University of Calgary, Calgary, Alberta, Canada. Purification
of SAK and specific activity determination of SAK and SAKM3-L-K1
followed the procedure described by Szarka et al. (19).
Plasminogen activation assay with the radial caseinolysis method was
performed as described by Wong (38).
Production and Characterization of the SAK-L-K1 Fusion and Its
Derivatives from B. subtilis WB800--
SAK-L-K1 (Fig.
1B) was produced as a
secretory protein in the culture supernatant using B. subtilis WB800, an eight extracellular protease deficient strain
(36), as the host. Radial caseinolysis study showed that SAK-L-K1 and
SAK had similar activities for plasminogen activation. However, more
than 95% of SAK-L-K1 was found in the flow-through fractions with a
lysine-agarose column even though the sample had been extensively
dialyzed to ensure that the Kringle domain in the fusion protein was
not saturated with free lysine molecules present in the culture medium
(data not shown). The observation suggested a defective Kringle-1
domain in the fusion protein. This result is not unexpected since the Kringle-1 domain contains three pairs of disulfide bonds arranged in a
1-6, 2-4, 3-5 pattern (i.e. cysteine residues forming
disulfide bonds are not arranged in a sequential manner, Fig.
1B). Inability to form disulfide bonds or mis-pairing of
cysteine residues can result in the formation of defective Kringle-1
domains (39). Because functional human plasminogen Kringle-1 domain has
been shown to be produced efficiently from P. pastoris (40),
it would be a logical approach to produce SAK-L-K1 in P. pastoris. However, using P. pastoris as the production
host of the SAK-L-K1 fusion protein has another concern. P. pastoris has been shown to produce SAK only in a partially active
form because of an N-linked glycosylation at Asn-28 of the
mature SAK (41). To eliminate glycosylation of SAK-L-K1 in P. pastoris, two key residues (Table I,
shown in bold) that constitute part of the consensus
N-linked glycosylation site (Asn-28-Val-29-Thr-30,
numbering according to the mature SAK sequence) were changed (Table I).
The first two muteins, SAKM1-L-K1 and SAKM2-L-K1, have Asn-28 changed
to Asp and Ala, respectively, whereas the third mutein SAKM3-L-K1 has
Thr-30 changed to Ala. To quickly examine the effects of these
mutations on SAK activity, the muteins were first produced in B. subtilis via secretion. Although the production yields of these
muteins and the wild-type control, SAK-L-K1, from B. subtilis were comparable, radial caseinolysis study indicated that
SAKM1-L-K1, SAKM2-L-K1, and SAKM3-L-K1 retained 1 (or less), 40, and
90% of the wild type SAK activity, respectively (data not shown).
Structural genes encoding SAKM2-L-K1 and SAKM3-L-K1 were then
transferred to the P. pastoris expression vector for further
characterization.
Production and Characterization of SAKM2-L-K1 and SAKM3-L-K1 in P. pastoris--
Secretory production of SAKM2-L-K1 or SAKM3-L-K1 from 30 zeocin-resistant clones of P. pastoris transformants was
analyzed in a time course study ranging from 15 to 60 h of
culture. For all the clones, optimal conditions (high secretion yield
with little degradation) were achieved with 28-30 h of cultures. On the other hand, the level of expression varied considerably across the
clones. Clones showing the highest level of expression of SAKM2-L-K1
and SAKM3-L-K1 were designated PM2 and PM3, respectively, and were used
to produce these muteins for all subsequent experiments. Using known
amounts of pure SAKM3-L-K1 as the standard, the secretory levels of
these muteins from PM2 and PM3 were estimated to be around 100 and 150 mg/liter of culture, respectively. These amounts exceed those secreted
by B. subtilis (Fig.
2A, lanes 3 and
4 versus lane 5). SAK-L-K1 produced from P. pastoris migrated as a duplex on the SDS-polyacrylamide gel. More
than 80% of this protein had a slower mobility and represented the
glycosylated form. In contrast, both muteins (SAKM2-L-K1 and
SAKM3-L-K1) produced from P. pastoris showed a faster
migration (Fig. 2A, lanes 3 and 4)
with mobility similar to the non-glycosylated SAK-L-K1 from P. pastoris and SAKM3-L-K1 produced from B. subtilis. The
absence of N-linked glycosylation with these muteins was
further confirmed by Western blotting probed with concanavalin
A-conjugated peroxidase. Strong concanavalin A binding activity
was observed only with the P. pastoris-produced SAK-L-K1 but
not with SAKM2-L-K1 or SAKM3-L-K1 (data not shown). Thus, changing
either Asn-28 or Thr-30 to Ala did successfully eliminate
N-linked glycosylation of SAK in P. pastoris. In
terms of plasminogen activation activity, SAKM3-L-K1 produced by
P. pastoris and B. subtilis showed comparable
activity (Fig. 2B). SAK-L-K1 and SAKM2-L-K1 from P. pastoris, on the other hand, demonstrated weaker activity as
judged by the smaller haloes. Because SAKM3-L-K1 was produced at a much
higher yield with P. pastoris, this mutein was purified from
P. pastoris for further characterization.
SAKM3-L-K1 Secreted by P. pastoris Folds Properly and Binds
Efficiently to Lysine-Agarose--
To show that Kringle-1 domain in
SAKM3-L-K1 is functional, the culture supernatant from
X33[pPICSAKM3LK1] was loaded to a lysine-agarose column.
Approximately 95% of SAKM3-L-K1 could be retained on the column and
recovered by EACA elution (Fig.
3A). This indicates that the
majority of SAKM3-L-K1 produced by P. pastoris retained
lysine binding capability. To further examine the formation of
disulfide bond in the Kringle-1 domain of SAKM3-L-K1, culture
supernatants from both X33 [pPICSAKM3LK1] (Fig. 3B,
lanes 1 and 3) and WB800[pSAKM3LK1]
(lanes 5 and 7) either in the presence or absence
of reducing agent were analyzed by Western blotting. In both hosts,
SAKM3-L-K1 migrated faster under the non-reducing condition
(lanes 3 and 7) presumably because of a more
compact conformation with disulfide bond formation. When run under the reducing conditions, protein from either host migrated as a sharp major
band with an apparent molecular size around 33 kDa (lanes 1 and 5). Electrophoresis of proteins under the non-reducing
conditions differentiated the nature of SAKM3-L-K1 produced by the two
hosts. Whereas in P. pastoris, majority of the protein
migrated at the fastest migration position (lane 3), the
opposite held true for B. subtilis (lane 7). This
observation suggests that the majority of SAKM3-L-K1 produced by
P. pastoris forms disulfide bonds and retains a compact
structure, whereas only a tiny portion of SAKM3-L-K1 produced by
B. subtilis achieves the proper folding configuration. This
offers a possible explanation for the observed difference in the
ability of SAKM3-L-K1 produced by either host to bind to lysine-agarose.
Each Domain in the Affinity-purified SAKM3-L-K1 from P. pastoris
Is Highly Functional--
To confirm that fusion of SAK and Kringle-1
in SAKM3-L-K1 does not interfere with the function of each domain,
SAKM3-L-K1 from P. pastoris was purified on a lysine-agarose
column and used for functional evaluations. As shown in Fig.
4A, with equimolar amounts of
both proteins the rate of plasminogen activation by SAKM3-L-K1 was
found to be around 96% that by SAK. To determine the functionality of
the Kringle-1 domain in SAKM3-L-K1, the affinity of this domain in
SAKM3-L-K1 to EACA, a lysine analog, was determined by isothermal
titration calorimetry. A typical example of heat changes accompanying
binding of EACA to the Kringle-1 domain of SAKM3-L-K1 was shown in Fig.
4B. The best-fit binding isotherm that resulted from
deconvolution of such data is shown in Fig. 4C. The
thermodynamic properties of the interaction in this particular experiment reveal a dissociation constant (Kd) of
14.3 µM (Ka of 69.8 mM Molecular Mass Determination of SAKM3-L-K1 from P. pastoris--
Because the apparent molecular mass of SAKM3-L-K1 on SDS
gel (33 kDa) differs from that calculated (26,168.2 Da), purified SAKM3-L-K1 was subjected to both N-terminal sequencing and MALDI-TOF mass spectrometry analyses. Sequence of the first six amino acid residues from SAKM3-L-K1 (SSSFDK) matched exactly with the predicted mature SAK sequence (Fig. 1A). Therefore, the Activated SAKM3-L-K1 Is Resistant to Plasmin
Digestion--
Because the SAKM3-L-K1 fusion is designed to introduce
a fibrin targeting capability to SAK via the Kringle-1 domain, it is essential that the Kringle-1 domain in SAKM3-L-K1 should not be cleaved
off in the presence of plasmin. Otherwise, the full impact of this
fusion in clot lysis will be significantly weakened. The last two
C-terminal residues in SAK are known to be lysine (Fig. 1A).
If plasmin, a trypsin-like protease, cleaves SAK or its fusions at this
site, any domain introduced C-terminally to SAK will be cleaved off. To
examine the stability of SAKM3-L-K1 in the presence of plasmin,
purified SAKM3-L-K1 was mixed with plasminogen at a 1:1 molar ratio,
and the stability of SAKM3-L-K1 was monitored at different time points
by SDS-PAGE under either reducing or non-reducing condition. As shown
in Fig. 5A (non-reducing
condition), 5 min after mixing SAKM3-L-K1 with plasminogen (lane
3), SAKM3-L-K1 was completely converted from the original intact
form with an apparent molecular mass of 29 kDa to a smaller form (28 kDa). This slight change in size reflects the removal of the first 10 amino acid residues (Fig. 1A) of SAKM3-L-K1 by plasmin
during the activation event. However, none of the processed SAKM3-L-K1 showed apparent molecular masses in the range around 16 kDa, which corresponds to that of SAK even after SAKM3-L-K1 had been mixed with
plasminogen for 1 h (Fig. 5A, lane 5). This
suggests that plasmin did not cleave the twin lysine site located
naturally at the C-terminal end of SAK.
SDS-PAGE of a duplicated set of samples run under the reducing
condition shows that plasminogen was completely converted to plasmin
with the appearance of a heavy chain (70 kDa) and a light chain (29 kDa) within 20 min after mixing with SAKM3-L-K1 (Fig. 5B,
lane 4). Western blotting of an equivalent gel probed with SAK-specific antibodies confirmed that the processed SAKM3-L-K1 remained stable for at least an hour in the presence of plasmin (Fig.
5C). The same result was observed on mixing SAKM3-L-K1 with plasminogen at a 1:3 molar ratio (data not shown). A faint band with an
apparent molecular mass of 22 kDa was observed after 20 min (Fig.
5C, lanes 4 and 5). The size of this
faint band suggests a low level of proteolytic cleavage of SAKM3-L-K1,
possibly close to the N-terminal region of the Kringle-1 domain.
SAKM3-L-K1 Binds to Partially Digested Fibrin--
Because exposed
lysine residues are needed for effective binding of the Kringle-1
domain, fibrin coated on the microtiter plate in the ELISA experiment
had to be partially digested for the binding study. Hence, plasmin was
used to digest the fibrin with varied time periods to generate the
optimal condition for Kringle-1 binding. Fig.
6 shows a typical result demonstrating the distinct difference between SAKM3-L-K1 and SAK in their ability to
bind to fibrin. Although SAK showed only a weak fibrin binding for the
entire time period, SAKM3-L-K1 bound readily to the fibrin. The fibrin
had to be at least slightly digested for any binding to occur.
Generation of newly exposed C-terminal lysine residues and increased
accessibility of the fibrin to SAKM3-L-K1 could both contribute to the
enhanced binding of SAKM3-L-K1 to the partially digested fibrin. In
this particular example, at the plateau (which last from 20 to 40 min
of fibrin digestion), the amount of SAKM3-L-K1 bound was 21 times that
of SAK.
Fibrin Clot Lysis Kinetics: Effects of SAKM3-L-K1 Versus
SAK--
Whether the enhanced fibrin binding ability by SAKM3-L-K1
could be translated to a faster clot lysis was investigated in a fibrin
clot lysis assay using an ELISA plate under two different conditions.
Under the first set of experimental condition, the thrombolytic agents
were not removed from the clots throughout the entire clot lysis
period. Fig. 7 shows the typical changes in clot turbidity that accompany clot lysis mediated by either agent.
SAK (closed triangles) mediated clot lysis began with a longer lag phase, and the time required for 50% clot lysis
(T50%) was obviously longer than that by
SAKM3-L-K1 (open triangles). Table
II summarizes the
T50% values with different amounts of
SAKM3-L-K1 or SAK. T50% mediated by SAK was
22-50% longer than that by SAKM3-L-K1. Because SAK in general is
introduced to patients by a short infusion and the in vivo
half-life of SAK in human is only 3 min (43), the infused SAK is
expected to be rapidly depleted from circulation once the infusion
process is terminated. To simulate this effect, clot lysis was examined under the second experimental condition in which any unbound agent (SAK
or SAKM3-L-K1) was removed by gently washing the clot surface 30 min
after incubation with the clots. The difference in
T50% values between SAK and SAKM3-L-K1 was even
more dramatic (Fig. 7, SAK (close circle) versus
SAKM3-L-K1 (open circle)). Table II shows that depending on
the concentration used in the study, SAK needed 40-80% longer time to
achieve 50% clot lysis. Approximately four times the amount for SAK
(200 nM) was required to attain the same rate of lysis
mediated by 50 nM SAKM3-L-K1. Whether the agents were
present throughout the lysis period or not, the enhancement of clot
lysis by SAKM3-L-K1 was increasingly obvious at lower dosages of either
thrombolytic agent used.
Plasma Clot Lysis Kinetics: Effects of SAKM3-L-K1 Versus
SAK--
To more closely simulate the physiological situation, clot
lysis experiment was repeated using human plasma under a constant flow
model. Constant plasma flows can supply plasminogen to the clot
surface. This can greatly influence clot lysis efficiency (44). Because
human plasma has a strong absorbance at 405 nm, measurement was taken
at 600 nm (at which human plasma has negligible absorbance) to ensure a
more accurate reflection of clot turbidity. Fig.
8 shows that when SAK was used as the
thrombolytic agent, a lag of ~30 min occurred before any obvious clot
lysis was detected. In contrary, clot lysis occurred almost
instantaneously after the introduction of SAKM3-L-K1. Based on the
average of triplicate assays, T50% value was 92 min (S.D. 1.5 min) for SAK and 17 min (S.D. 0.5 min) for SAKM3-L-K1.
Thus, SAKM3-L-K1 was at least five times more effective than SAK in
mediating plasma clot lysis.
An important genetic manipulation in this study involves the
production of properly folded, non-glycosylated SAK-Kringle-1 fusion
protein in P. pastoris. Glycosylation of SAK in P. pastoris has been found to result in a SAK with attenuated
plasminogen activator activity (41). The presence of the
oligosaccharide moiety is suggested to cause subtle changes in the
orientation of plasmin so that the complex is less optimal in
plasminogen activation. Two key residues in the glycosylation sites
were targeted in this study, Asn-28 and Thr-30. Our site-directed
mutagenesis studies suggest that Asn-28 is a critical residue for the
function of SAK. Replacement of Asn-28 with Asp or Ala reduced the
activity of SAK (Fig.
2B).2 A crystal
structure (16) of the ternary complex of microplasmin-SAK (active
plasminogen activator complex)-microplasmin (substrate) indicates that Asn-28 of SAK forms hydrogen bonds and other contacts with Gly-174 and Gln-177 of the microplasmin (the one in the active plasminogen activator complex). It also has intra-chain contacts with
Met-26, which has been shown to be important for SAK function (45).
Missing some or all of these contacts may explain the reduced activity
of SAK in SAKM1-L-K1 and SAKM2-L-K1. In contrast, replacement of Thr-30
with Ala is very well tolerated in SAK and results in the production of
large amounts of the non-glycosylated, functional SAKM3-L-K1 from
P. pastoris for biochemical and functional analyses.
Success of thrombolytic therapy heavily relies on the rapid and
complete restoration of blood flow in occluded blood vessels. tPA,
considered to be one of the best thrombolytic agents available, takes
on average 45 min to restore blood flow under an optimized treatment
regimen. Furthermore, only about 60% of the treated patients have
blood flow restored 90 min after the treatment. Development of more
potent and faster-acting thrombolytic agents that can speed up the clot
lysis process and have a higher reperfusion rate would be desirable. In
this study, by simply equipping staphylokinase with direct clot binding
capability, the efficacy of this already potent thrombolytic agent can
be improved further. In comparison with SAK, SAKM3-L-K1 resulted in a
5-fold reduction in T50% for plasma clot lysis
observed in the flow-cell model or a 4-fold reduction in concentration
needed for comparable clot lysis in a static fibrin clot lysis assay.
The observed enhancement of clot lysis by SAKM3-L-K1 appears to be the
effect of increasing the local concentration of this plasminogen
activator to the clot. Because SAK has no direct fibrin binding
ability, the amounts of clot-bound SAK would be strictly dependent on
the level of clot-bound plasmin(ogen). As shown in the fibrin binding
study (Fig. 6), SAK binds poorly to the plasmin-treated fibrin clot.
With 1 pmol of plasmin used in the clot treatment, the theoretical
maximum amount of clot-bound SAK is expected to be 1 pmol or less even
though 20 pmol of SAK were present in the binding reaction. In
contrast, the amount of SAKM3-L-K1 captured on plasmin-treated clots
can be 20 times more than that of SAK under the same conditions. This
was exactly what was observed in this study (Fig. 6). The high local
concentration of clot-bound SAKM3-L-K1 can allow direct interaction of
SAKM3-L-K1 with the adjacent clot-bound plasmin(ogen) to form
functional plasminogen activators or to capture more plasmin(ogen) from
plasma to the clot surface. Any plasminogen activated is likely to be
clot bound and is less susceptible to
Although conjugation of fibrin-targeting domains (e.g.
fibrin-specific antibody fragments) to either tPA or urokinase has been
shown to increase the potency of these agents (46-48), a recent report
suggests that introduction of Kringle-2 domain from tPA to SAK did not
lead to enhanced clot lysis under in vitro conditions (49).
These SAK fusions are K2/SAK/HIR and RGD/K2/SAK/HIR. The failure
to observe enhanced clot lysis by these recombinant fusions may
possibly be attributed to the positioning of the Kringle domain at the
N-terminal end of SAK and the use of Kringle 2 from tPA as the fibrin
targeting domain. Because plasmin has to activate SAK by cleaving the
N-terminal peptide at the twin lysine site (Fig. 1A), any
fibrin-targeting domain fused to the N-terminal end of the circulating
SAK fusion can potentially be cleaved off by any plasmin generated
during the plasminogen activation process. This may reduce the
clot-targeting potential of the fusion protein. This concern is
addressed in our design of SAKM3-L-K1 by placing the Kringle domain in
the C-terminal end of SAK and the confirmation of stability of the
fusion protein in the presence of plasmin (Fig. 5). Furthermore,
Kringle 2 from tPA has lower affinity to lysine and EACA relative to
the Kringle-1 domain from human plasminogen (29). The low affinity can
contribute to a higher off-rate of both the K2/SAK/HIR-fibrin complex
and the RGD/K2/SAK/HIR-fibrin complex. Before our findings, whether
introduction of a Kringle domain can improve the performance of SAK has
been questioned (50). Our study provides the first piece of evidence to
show that the performance of SAK can be further improved by introducing a fibrin targeting domain.
The use of the Kringle-1 domain from human plasminogen as a
fibrin-targeting domain in the design of the SAK fusion offers another
potential advantage. Because this domain originates from a human
protein, it should not induce any significant immune response. As a
foreign protein to human, SAK has been demonstrated to induce significant immune responses in human. This concern has been addressed by the recent development of the mutated versions of SAK with lower
immunogenicity (51) and the attachment of polyethylene glycol in a
site-specific manner to the cysteine substitution variants of
recombinant staphylokinase (43). The use of these SAK muteins in
combination with the Kringle-1 domain from human plasminogen may offer
the potential to develop potent and fast-acting thrombolytic agents
with low immunogenicity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-antiplasmin, present in plasma. In contrast, the
fibrin-bound plasminogen activator complex is much more resistant to
2-antiplasmin-mediated inhibition (15). The result is a
preferential plasminogen activation by SAK at the fibrin surface that
contributes to the fibrin specificity of SAK in a plasma milieu. This
fibrin specificity is made even stronger by the preferential binding of
SAK to plasmin(ogen) that is fibrin-bound (14). The fibrin-specific
property of SAK underlies an interesting observation in clinical
trials. The fibrinogen levels in patients treated with SAK remain close
to 100%, whereas patients treated with tPA have 32% of fibrinogen
cleaved and degraded in their plasma (9, 10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-factor signal
sequence in the P. pastoris expression vector pPICZ
A
(Invitrogen). pSAKLK1 was used as the template for PCR amplification
with AFACSAKF (5'-GACTCGAGAAGAGATCGAGCTCATTCGACAAAGG-3') as the forward
primer and SAKK1CPICB (5'-GAGCGGCCGCTTAACACTCAAGAATGTCGGAGTAG-3') as
the backward primer. A 730-bp PCR product was generated with XhoI at the 5' end and NotI at the 3' end. The
amplified sequence was digested with XhoI and
NotI and ligated to the similarly digested pPICZ
A to
generate pPICSAKLK1. pPICSAKLK1 was transformed to E. coli
TOP10F' and selected for resistance to zeocin (25 µg/ml) (Invitrogen). pPICSAKM2LK1 and pPICSAKM3LK1 were also generated using
the same approach with pSAKM2LK1 and pSAKM3LK1 as the PCR template, respectively.
A and pPICSAKLK1 integrated were similarly prepared
to serve as the negative and wild-type controls, respectively.
-amino-N-caproic acid (EACA) (Sigma). Eluants were
analyzed to determine yield and purity of SAKM3-L-K1 by SDS-PAGE and
Coomassie Blue staining. The fractions selected were pooled, dialyzed
against the binding buffer, and concentrated by ultrafiltration
(Millipore Corp.). Purified SAKM3-L-K1 was quantified
spectrophotometrically at 280 nm using a molar extinction coefficient
of 35,350 M
1 cm
1 (35).
20 °C in
aliquots. All plasma clot experiments were performed within 1 week of
blood collection. Clot perfusion using an optical flow cell was based on the model described by Hantgan et al. (37). Clotting was initiated by adding human thrombin (to 1.0 NIH units/ml) and
CaCl2 (to 30 mM) to freshly thawed PPP. 200 µl of the polymerizing plasma was transferred to the flow cell
(Hellma Cells, model 178.710-QS) to fill up the optical chamber (80 µl) and the inlet and outlet ports. Clotting was allowed to proceed
for 3 h at room temperature. The clot was then perfused with PPP
for 20 min at 20 µl/min, followed by perfusion with SAKM3-L-K1 or SAK
(200 nM in PPP, 20 µl/min) with the flow rate controlled
by a Bio-Rad peristaltic pump. The change in absorbance at 600 nm at
37 °C was continuously monitored using a Beckman DU-65
spectrophotometer (Beckman Instruments) operated with a Kinetics
Soft-Pac module. The experiment was performed three times for both
SAKM3-L-K1 and SAK.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Structure of SAK-L-K1. A,
design of SAK-L-K1 for its production in P. pastoris. Signal
peptide (SP) sequence from -factor was used to direct the
secretion of SAK-L-K1 and its derivatives. The two open
arrowheads mark the signal peptidase cleavage site and the plasmin
processing site, respectively. SAK is numbered according to its mature
sequence. The numbers below the individual domains represent
the amino acid residues in length for that particular region.
B, structural model of SAK-L-K1. A 20-amino acid linker
provides sufficient space to separate SAK from Kringle-1. The six
cysteine residues within the Kringle-1 domain are numbered from 1 to
6.
Glycosylation-site mutants of SAK-L-K1
View larger version (61K):
[in a new window]
Fig. 2.
Production of SAK-L-K1 and its derivatives in
P. pastoris. A, Coomassie Blue-stained
gel showing SAK-L-K1 and its muteins in the culture supernatants of
P. pastoris. Culture supernatants were collected at 19 h of culture. 24 µl of culture supernatant from each sample was
loaded. M, molecular weight marker. Lane 1,
P. pastoris X-33 [pPICZ A] (this serves as the negative
control); lane 2, X-33[pSAK-L-K1]; lane 3,
X-33[pSAKM2-L-K1]; lane 4, X-33 [pSAKM3-L-K1];
lane 5, B. subtilis WB800[pSAKM3-L-K1]. 24 µl
of culture supernatant was loaded. B, radial caseinolysis
assay comparing activities of equimolar amounts of the different
muteins. Numbers correspond to samples collected from the
culture supernatants as designated by the different lanes in
panel A.
View larger version (54K):
[in a new window]
Fig. 3.
SAKM3-L-K1 produced by P. pastoris can bind to lysine agarose and folds properly.
A, Coomassie Blue-stained gel showing affinity purification
of SAKM3-L-K1 using lysine-agarose column. M, molecular
weight maker. Lane 1, P. pastoris culture
supernatant; lane 2, column flow-through; lane 3,
eluate. B, Western blot analysis of SAKM3-L-K1 secreted by
P. pastoris and B. subtilis. Culture supernatants
were run on a 12% polyacrylamide gel containing SDS. The blot was
probed against polyclonal antibodies against SAK. Lanes 1 and 5 are samples from P. pastoris
X-33[pPICSAKM3LK1] and B. subtilis WB800[pSAKM3LK1],
respectively, prepared in buffer containing -mercaptoethanol.
Lanes 3 and 7 are the corresponding samples
prepared in buffer that did not contain
-mercaptoethanol.
Lanes 2, 4, and 6 are empty lanes to
minimize the effect of diffusion of the reducing agent across the
lanes. Western blot was done on a nitrocellulose membrane using
4-chloro-1-naphthol (Bio-Rad) as the color development reagent.
1), a
H of
3.1 kcal
mol
1, and a
S of 13.2 cal
mol
1K
1. Based on the average of duplicate
experiments, Kd for the interaction between
SAKM3-L-K1 and EACA was found to be 15 µM. As a
reference, the binding affinity of Kringle-1 domain and its derivative
carrying the N-terminal proactivation peptide of human plasminogen to
EACA, expressed in terms of the dissociation constant, has been
determined to be 17 µM by equilibrium dialysis (24), 12 µM by isothermal titration calorimetry (25), and 13.7 µM by fluorescence study (42). Thus, the
Kd obtained with SAKM3-L-K1 is within the range of
the reported values.
View larger version (13K):
[in a new window]
Fig. 4.
Experiments to show that each domain in
SAKM3-L-K1 from P. pastoris is functional. A,
plasminogen activation activities of SAKM3-L-K1 and SAK. Plasminogen (1 µM) and SAKM3-L-K1 or SAK (10 nM) were
incubated at 37 °C. The reaction was stopped at different time
points, and samples were assayed for plasmin activity using the
chromogenic substrate N-p-tosyl-Gly-Pro-Lys
p-nitroanilide (Sigma). The linear rate of color development
at 405 nm was calculated, and the activity was presented as WHO units
of plasmin (1 WHO unit = 2.84 optical density units/min,
established using a plasmin standard from Roche Applied Science).
Results shown represent the mean values and S.D. determined from
triplicate assays. , SAKM3-L-K1 (specific activity, 15.87 WHO
units/nmol of protein/min);
, SAK (specific activity, 16.47 WHO
units/nmol of protein/min). B, determination of
thermodynamic properties associated with binding of SAKM3-L-K1 with
EACA by isothermal titration calorimetry. The isotherm indicates heat
change accompanying titration of SAKM3-L-K1 with EACA. The experiment
consisted of an automated sequence of 30 injections, 5 µl each, from
a 13.5 mM stock solution of EACA in 150 mM
sodium phosphate, pH 7.4. EACA was titrated at 25 °C into the sample
cell containing 0.09 mM SAKM3-L-K1 in the same buffer. Each
injection occurred over a 10-s period with 4 min between injections.
C, deconvolution of the data in B. The
solid line represents the non-linear least squares best fit
deconvolution to the data points. The number of EACA binding sites per
SAKM3-L-K1, binding constant (Ka), and the values
for
H and
S are shown.
-factor
signal peptide was properly processed by the P. pastoris
signal peptidase. Results from mass spectrometry analysis determined
the molecular mass of SAKM3-L-K1 to be 26,320.99 which is in close
agreement with the calculated value. This value confirmed the absence
of N-linked glycosylation in SAKM3-L-K1 since the molecular
mass of the N-linked oligosaccharide unit would clearly be
larger than 153 Da.
View larger version (31K):
[in a new window]
Fig. 5.
Stability of SAKM3-L-K1 in the presence of
plasmin. SAKM3-L-K1 and plasminogen at a 1:1 molar ratio were
mixed, and samples were collected at different time points for SDS-PAGE
under non-reducing (A) or reducing (B)
conditions. C, Western blot probed against polyclonal
antibodies against SAK. Samples were prepared under reducing
conditions. Lane 1, plasminogen. Lane 2, purified
SAKM3-L-K-1. Lanes 3-5, plasmin processing of
purified SAKM3-L-K1 for 5 min, 20 min, and 1 h, respectively.
Lane 6, purified SAK. Lane 7, plasmin standard.
M, molecular weight marker; The asterisk marks
the position of the N-terminally processed SAKM3-L-K1. a and
b refer to the heavy (70 kDa) and light (29 kDa) chains of
plasmin, respectively.
View larger version (15K):
[in a new window]
Fig. 6.
A representative ELISA study showing fibrin
binding ability of SAK and SAKM3-L-K1. Fibrinogen (5 µg/ml) was
coated onto the wells of a Nunc-Immuno MaxiSorp module and left
overnight at 4 °C. After blocking the exposed sites with BSA, the
wells were incubated with a solution of phosphate-buffered saline
containing human thrombin (1 NIH unit/ml) and CaCl2
(20 mM) for 2 h at 37 °C. Cross-linked fibrin clots
on the wells were partially digested with plasmin for different time
periods. Binding of SAK and its derivative to the partially digested
fibrin was monitored as described under "Experimental Procedures."
The amount of SAK-specific antibody retained on the wells was estimated
by measuring the activity of bound horseradish peroxidase
(HRP). , SAKM3-L-K1;
, SAK.
View larger version (20K):
[in a new window]
Fig. 7.
A representative curve showing the time
course of fibrin clot lysis. Fibrin clots were formed on the wells
of an ELISA plate. SAK or SAKM3-L-K1 was used at 50 nM. The
decrease in absorbance at 405 nm with time was used to calculate the
relative clot turbidity at different time points. The clots were either
incubated with SAK ( ) or SAKM3-L-K1 (
) throughout the entire
period or incubated with SAK (
) or SAKM3-L-K1(
) for 30 min and
buffer-washed at the time indicated by an open arrowhead.
Control clots treated with buffer only (data not shown) showed that the
readings were stable throughout the incubation period. See
"Experimental Procedures" for experimental details.
Summary of the time required to lyse 50% of the fibrin clot
(T50%) with different amounts of SAK or SAKM3-L-K1
View larger version (18K):
[in a new window]
Fig. 8.
A typical time course of plasma clot
perfusion study. Plasma clot was formed from PPP in an optical
flow cell as described under "Experimental Procedures." The
decrease in absorbance at 600 nm with time was used to calculate the
relative clot turbidity at different time points. The clot was either
perfused with PPP throughout the experiment ( ) or at the time marked
by an open arrowhead, with PPP containing 200 nM
SAK (
) or SAKM3-L-K1 (
). In this example,
T50% was 93 min for SAK and 17.5 min for
SAKM3-L-K1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-antiplasmin-mediated inhibition (15).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. Fung and S. G. Sihota for the construction of pSAK-L-K1, Kenneth Ng for modeling the three-dimensional structure of SAK-L-K1, Ross T. A. MacGillivray at University of British Columbia for the cDNA clone of the human plasminogen gene, and R. G. Miele, L. Zhong, and S. Milosavljevic at University of Notre Dame for discussion of the Pichia cloning system.
![]() |
FOOTNOTES |
---|
* This work is supported by research grants from the Heart and Stroke Foundation of Canada (Alberta) (to S.-L. W.) and National Institutes of Heath Grant HL13423 (to F. J. C.).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: Div. of Cellular, Molecular and Microbial Biology, Dept. of Biological Sciences, University of Calgary, 2500 University Dr., N. W., Calgary, Alberta T2N 1N4, Canada. Tel.: 403-220-5721; Fax: 403-289-9311; E-mail: slwong@ucalgary.ca.
Published, JBC Papers in Press, March 19, 2003, DOI 10.1074/jbc.M210919200
2 S.-C. Wu, F. J. Castellino, and S.-L. Wong, unpublished data with B. subtilis.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
tPA, tissue
plasminogen activator;
EACA, -amino-N-caproic acid;
PPP, platelet poor plasma;
SAK, staphylokinase;
SAK-L-K1, staphylokinase-linker-Kringle-1 fusion;
T50%, time required for 50% clot lysis;
MALDI-TOF mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass
spectrometry;
ELISA, enzyme-linked immunosorbent assay;
HBS, HEPES-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Guzman, L. A., and Lincoff, A. M. (1997) J. Thromb. Thrombolysis 4, 337-343[CrossRef][Medline] [Order article via Infotrieve] |
2. | Hennekens, C. H., O'Donnell, C. J., Ridker, P. M., and Marder, V. J. (1995) J. Am. Coll. Cardiol. 25 Suppl. 7, S18-S22[CrossRef][Medline] [Order article via Infotrieve] |
3. | Tsikouris, J. P., and Tsikouris, A. P. (2001) Pharmacotherapy 21, 207-217[Medline] [Order article via Infotrieve] |
4. | Sinnaeve, P., and van de, W. F. (2001) Thromb. Res. 103 Suppl. 1, S71-S79[CrossRef][Medline] [Order article via Infotrieve] |
5. |
The GUSTO Angiographic Investigators.
(1993)
N. Engl. J. Med.
329,
1615-1622 |
6. |
The InTIME-II Investigators.
(2000)
Eur. Heart J.
21,
2005-2013 |
7. |
Zhu, Y.,
Carmeliet, P.,
and Fay, W. P.
(1999)
Circulation
99,
3050-3055 |
8. | Serizawa, K., Urano, T., Kozima, Y., Takada, Y., and Takada, A. (1993) Thromb. Res. 71, 289-300[Medline] [Order article via Infotrieve] |
9. |
Vanderschueren, S.,
Barrios, L.,
Kerdsinchai, P.,
Van den Heuvel, P.,
Hermans, L.,
Vrolix, M.,
De Man, F.,
Benit, E.,
Muyldermans, L.,
Collen, D.,
and Van de Werf, F.
(1995)
Circulation
92,
2044-2049 |
10. | Vanderschueren, S., Dens, J., Kerdsinchai, P., Desmet, W., Vrolix, M., De Man, F., Van den Heuvel, P., Hermans, L., Collen, D., and Van de Werf, F. (1997) Am. Heart J. 134, 213-219[Medline] [Order article via Infotrieve] |
11. | Collen, D. (1998) Nat. Med. 4, 279-284[Medline] [Order article via Infotrieve] |
12. | Suehiro, A., Tsujioka, H., Yoshimoto, H., Ueda, M., Higasa, S., and Kakishita, E. (1995) Thromb. Res. 80, 135-142[CrossRef][Medline] [Order article via Infotrieve] |
13. | Hauptmann, J., and Glusa, E. (1995) Blood Coagul. Fibrinolysis 6, 579-583[Medline] [Order article via Infotrieve] |
14. |
Sakharov, D. V.,
Lijnen, H. R.,
and Rijken, D. C.
(1996)
J. Biol. Chem.
271,
27912-27918 |
15. |
Silence, K.,
Collen, D.,
and Lijnen, H. R.
(1993)
J. Biol. Chem.
268,
9811-9816 |
16. | Parry, M. A., Fernandez-Catalan, C., Bergner, A., Huber, R., Hopfner, K. P., Schlott, B., Guhrs, K. H., and Bode, W. (1998) Nat. Struct. Biol. 5, 917-923[CrossRef][Medline] [Order article via Infotrieve] |
17. | Gase, A., Hartmann, M., Guhrs, K. H., Rocker, A., Collen, D., Behnke, D., and Schlott, B. (1996) Thromb. Haemostasis 76, 755-760[Medline] [Order article via Infotrieve] |
18. |
Schlott, B.,
Guhrs, K. H.,
Hartmann, M.,
Rocker, A.,
and Collen, D.
(1998)
J. Biol. Chem.
273,
22346-22350 |
19. |
Szarka, S.,
Sihota, E.,
Habibi, H. R.,
and Wong, S.-L.
(1999)
Appl. Environ. Microbiol.
65,
506-513 |
20. | Castellino, F. J., and McCance, S. G. (1997) CIBA Found. Symp. 212, 46-60[Medline] [Order article via Infotrieve] |
21. | Hoover, G. J., Menhart, N., Martin, A., Warder, S., and Castellino, F. J. (1993) Biochemistry 32, 10936-10943[Medline] [Order article via Infotrieve] |
22. | Wu, T. P., Padmanabhan, K. P., and Tulinsky, A. (1994) Blood Coagul. Fibrinolysis 5, 157-166[Medline] [Order article via Infotrieve] |
23. | Mathews, I. I., Vanderhoff-Hanaver, P., Castellino, F. J., and Tulinsky, A. (1996) Biochemistry 35, 2567-2576[CrossRef][Medline] [Order article via Infotrieve] |
24. | Lerch, P. G., Rickli, E. E., Lergier, W., and Gillessen, D. (1980) Eur. J. Biochem. 107, 7-13[Abstract] |
25. | Menhart, N., Sehl, L. C., Kelley, R. F., and Castellino, F. J. (1991) Biochemistry 30, 1948-1957[Medline] [Order article via Infotrieve] |
26. |
Sehl, L. C.,
and Castellino, F. J.
(1990)
J. Biol. Chem.
265,
5482-5486 |
27. | Chang, Y., Mochalkin, I., McCance, S. G., Cheng, B., Tulinsky, A., and Castellino, F. J. (1998) Biochemistry 37, 3258-3271[CrossRef][Medline] [Order article via Infotrieve] |
28. | Marti, D., Schaller, J., Ochensberger, B., and Rickli, E. E. (1994) Eur. J. Biochem. 219, 455-462[Abstract] |
29. | Byeon, I.-J. L., Kelley, R. F., Mulkerrin, M. G., An, S. S. A., and Llinás, M. (1995) Biochemistry 34, 2739-2750[Medline] [Order article via Infotrieve] |
30. | Wong, S.-L. (1989) Gene 83, 215-223[CrossRef][Medline] [Order article via Infotrieve] |
31. | Ye, R., Kim, J. H., Kim, B. G., Szarka, S., Sihota, S., and Wong, S.-L. (1999) Biotechnol. Bioeng. 62, 87-96[CrossRef][Medline] [Order article via Infotrieve] |
32. | Hemsley, A., Arnheim, N., Toney, M. D., Cortopassi, G., and Galas, D. J. (1989) Nucleic Acids Res. 17, 6545-6551[Abstract] |
33. | Wu, X.-C., Lee, W., Tran, L., and Wong, S.-L. (1991) J. Bacteriol. 173, 4952-4958[Medline] [Order article via Infotrieve] |
34. | Rosenfeld, S. A. (1999) Methods Enzymol. 306, 154-169[Medline] [Order article via Infotrieve] |
35. | Gill, S. C., and Von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve] |
36. |
Wu, S.-C.,
Yeung, J. C.,
Duan, Y.,
Ye, R.,
Szarka, S. J.,
Habibi, H. R.,
and Wong, S.-L.
(2002)
Appl. Environ. Microbiol.
68,
3261-3269 |
37. |
Hantgan, R. R.,
Jerome, W. G.,
and Hursting, M. J.
(1998)
Blood
92,
2064-2074 |
38. | Wong, S.-L., Ye, R., and Nathoo, S. (1994) Appl. Environ. Microbiol. 60, 517-523[Abstract] |
39. | Trexler, M., and Patthy, L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2457-2461[Abstract] |
40. | Zajicek, J., Chang, Y., and Castellino, F. J. (2000) J. Mol. Biol. 301, 333-347[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Miele, R. G.,
Prorok, M.,
Costa, V. A.,
and Castellino, F. J.
(1999)
J. Biol. Chem.
274,
7769-7776 |
42. | Douglas, J. T., von Haller, P. D., Gehrmann, M., Llinas, M., and Schaller, J. (2002) Biochemistry 41, 3302-3310[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Collen, D.,
Sinnaeve, P.,
Demarsin, E.,
Moreau, H.,
De Maeyer, M.,
Jespers, L.,
Laroche, Y.,
and Van de Werf, F.
(2000)
Circulation
102,
1766-1772 |
44. | Sakharov, D. V., and Rijken, D. C. (2000) Thromb. Haemostasis 83, 469-474[Medline] [Order article via Infotrieve] |
45. | Schlott, B., Hartmann, M., Gührs, K.-H., Birch-Hirschfeld, E., Gase, A., Vettermann, S., Collen, D., and Lijnen, H. R. (1994) Biochim. Biophys. Acta 1204, 235-242[Medline] [Order article via Infotrieve] |
46. | Haber, E., Quertermous, T., Matsueda, G. R., and Runge, M. S. (1989) Science 243, 51-56[Medline] [Order article via Infotrieve] |
47. | Runge, M. S., Bode, C., Matsueda, G. R., and Haber, E. (1988) Biochemistry 27, 1153-1157[Medline] [Order article via Infotrieve] |
48. | Dewerchin, M., Lijnen, H. R., Van Hoef, B., De Cock, F., and Collen, D. (1989) Eur. J. Biochem. 185, 141-149[Abstract] |
49. | Szemraj, J., Chabielska, E., Kawecka, I., Janiszewska, G., Buczko, W., and Pietrucha, T. (2001) Thromb. Haemostasis 86 Suppl. P, 438 |
50. |
Icke, C.,
Schlott, B.,
Ohlenschlager, O.,
Hartmann, M.,
Guhrs, K. H.,
and Glusa, E.
(2002)
Mol. Pharmacol.
62,
203-209 |
51. |
Laroche, Y.,
Heymans, S.,
Capaert, S.,
De Cock, F.,
Demarsin, E.,
and Collen, D.
(2000)
Blood
96,
1425-1432 |