From the
Division of Cellular, Molecular and Microbial Biology,
Division of Biochemistry, Department of
Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4,
Canada
Received for publication, March 28, 2003 , and in revised form, May 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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To increase the potency of SAK, to equip SAK with the capability to
differentiate freshly formed pathologic thrombi from physiological hemostatic
plugs formed earlier and to minimize reocclusion, hirudin, a potent thrombin
inhibitor (Kd
10111013
M,
(1719)),
serves as both the anti-thrombotic agent and the targeting component to
freshly formed clots in this engineered thrombolytic agent. SAK and hirudin
are joined together via a pair of engineered coiled coil sequences that act as
the heterodimerization domain
(20). A lysine-rich coiled
coil sequence (K-coil) is added to the C-terminal tail of SAK to generate
SAK-K coil (SAKK) and a glutamate-rich coiled coil sequence (E coil) is added
to the C-terminal end of hirudin to generate hirudin-E coil (HE). Each of
these proteins is produced from Bacillus subtilis via secretion to
form heterodimeric molecules (designated HE-SAKK) via an optimized
co-cultivation approach. With an in vitro treatment to reshuffle the
disulfide bonds in hirudin
(21), both SAK and hirudin in
the heterodimer are shown to retain biological activities comparable with
their parent molecules. In vitro fibrin and plasma clot lysis studies
demonstrate that HE-SAKK is a superior thrombolytic agent in comparison to
staphylokinase.
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EXPERIMENTAL PROCEDURES |
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Construction of B. subtilis Expression Vectors for SAKK and HE ProductionPlasmid pSAK-K coil is the B. subtilis vector for secretory production of SAKK. It is a derivative of the pUB18-SAK-K1 vector (24), which encodes a staphylokinase fusion carrying the kringle-1 domain from human plasminogen. pSAK-K coil was generated by replacing the BamHI/HpaI fragment encoding a portion of the linker sequence and the kringle-1 domain sequence in pUB18-SAK-K1 with the synthetic BamHI/HpaI K coil sequence from pUC19-K coil. Plasmid pHirudin-E coil is the B. subtilis vector for secretory production of hirudin-E coil and is a derivative of pUB18-hirudin-SAK (24), which produces a hirudin fusion with SAK at the C-terminal end. To construct pHirudin-E coil, the sak sequence (as a SpeI/SphI fragment) in pUB18-hirudin-SAK was replaced by a SpeI/SphI fragment encoding a portion of the linker sequence and the entire E coil sequence. The sequences of SAK-K coil and hirudin-E coil are available from GenBankTM with the accession numbers AF533145 and AF533146, respectively.
Expression Studies in B. subtilisEach of the pSAK-K coil and pHirudin-E coil plasmids was transformed into three engineered B. subtilis strains for expression studies. WB600 (25), WB700 (26), and WB800 (27) are strains deficient in 6, 7, and 8 extracellular proteases, respectively. Transformed cells were cultivated in super-rich medium (without glucose) (28) containing 10 µg/ml kanamycin at 37 °C and samples were collected at different time points. After normalization for cell density, culture supernatants were analyzed by SDS-PAGE under reducing or non-reducing conditions. Because all the expression vectors in this study are derivatives of pUB18 (29), WB600(pUB18) was used as the negative control.
Purification of HE, SAKK, and HE-SAKKFor HE purification, 50 ml of WB800(pHirudin-E coil) culture supernatant was collected by centrifugation and dialyzed against 4 liters of 17 mM potassium phosphate buffer (pH 6.0) at 4 °C overnight. The dialyzed sample was applied to a DE52 (Whatman, England) column (15 x 1.5 cm) equilibrated with the same buffer. After washing the column until the absorbance at 280 nm was less than 0.02, HE was eluted using a 150-ml linear salt gradient (00.7 M NaCl) in the same buffer. The HE-containing fractions (0.250.35 M NaCl) were combined, dialyzed, and concentrated in 0.1 M NaHCO3 buffer (pH 8.3) containing 0.05 M NaCl to a final volume of 200300 µl using a Centricon unit. The concentrated sample was then loaded onto a Bio-Prep SE 100/17 column and eluted with 12 ml of 0.1 M NaHCO3 buffer (pH 8.3) containing 0.05 M NaCl buffer at a flow rate of 0.4 ml/min. Fractions containing HE were pooled and concentrated by ultrafiltration.
To purify HE-SAKK, 50 ml of culture supernatant was collected, dialyzed, and applied to a DE52 column. HE-SAKK was separated with a linear salt gradient of 00.5 M NaCl (in 150 ml) and the HE-SAKK containing fractions (0.10.15 M NaCl) were further purified by gel filtration.
For SAKK purification, 50 ml of culture supernatant was collected, dialyzed against 4 liters of 17 mM potassium phosphate buffer (pH 7.0) at 4 °C overnight, and applied to a Macro-Prep High S column (Bio-Rad, 5 x 1.5 cm) previously equilibrated with the same buffer. After washing the column until A280 was less than 0.1, SAKK was eluted from the column with a 100-ml linear salt gradient (01.0 M NaCl in the same buffer). SAKK monomer-containing fractions (0.40.5 M of NaCl) were further purified by gel filtration using 0.1 M sodium phosphate buffer (pH 5.8) containing 0.1 M NaCl. Purified HE, SAKK, and HE-SAKK were quantified spectrophotometrically at 280 nm using molar extinction coefficients (30) of 3,400 M1 cm1, 17,330 M1 cm1, and 20,730 M1 cm1, respectively.
Disulfide Bond ReshufflingThe disulfide bond reshuffling of HE or HE-SAKK was performed based on the method of Chang (21) with modification. Briefly, each protein sample was dialyzed and concentrated to a final concentration of 5070 µM in 0.1 M NaHCO3 buffer (pH 8.3) in the absence of any extra salt to minimize dimer formation. The reshuffling process proceeded at 4 °C overnight in a microcentrifuge tube by adding cysteine (Cys) and cystine (Cys-Cys) to final concentrations of 4 and 2 mM, respectively. The efficiency of the reshuffling process was detected by non-reducing SDS-PAGE and Western blotting.
Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass SpectrometryProtein mass spectrometry analyses were performed at the Southern Alberta Mass Spectrometry Proteomics Research Centre, University of Calgary. Purified and concentrated samples were applied to a Voyager-DE STR MALDI-TOF biospectrometry work station (Applied Biosystems) using sinapic acid as the matrix. Whole protein spectra were recorded in a linear mode and bovine serum albumin was used as the calibration marker.
Quantification of Clot-bound ThrombinCross-linked fibrin clots were prepared by adding human thrombin (Sigma) at final concentrations from 0.1 to 2 NIH units/ml to 4 mg/ml human fibrinogen (Sigma) in HEPES-buffered saline (HBS, 0.02 M HEPES, 0.13 M NaCl, pH 7.4) containing 20 mM CaCl2 at room temperature. Immediately after mixing, 100 µl of the polymerizing solution was transferred to a microtiter plate (Falcon 353912, BD Biosciences). The clots were formed at room temperature for 2 h. Two sets of fibrin clots were formed. The first set of fibrin clots was washed with 100 µl of HBS followed by careful removal of the washing buffer. This step was repeated 5 times until no thrombin activity in the wash buffer was detected. The second set of clots was unwashed and were used as the control to determine the ratio of thrombin incorporated into the fibrin clot. 100 µlof560 µM thrombin-specific chromogenic substrate (N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, Sigma) was then added to each well and the color development was monitored over a 10-min period at 37 °C in the kinetic mode at 405 nm using a Ceres model UV900 plate reader (Bio-Tek Instruments Inc., Winooski, VT). Thrombin activity (Fig. 6A) was expressed as the initial rate of color development (mOD/min).
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Thrombin Inhibition AssaysThese assays were performed under two different conditions with hirudin and its derivatives (HE and HE-SAKK) as the inhibitors. The first series was performed with thrombin freely in solution although the second series was determined with clot-bound thrombin. For assays under the first condition (Fig. 5B), increasing amounts of hirudin and its derivatives (final concentrations from 0 to 30 nM) were incubated with thrombin (final concentration: 1 NIH unit/ml) at room temperature in HBS containing 0.2 mg/ml bovine serum albumin. The reaction was started by the addition of 50 µl of 560 µM thrombin-specific chromogenic substrate to the thrombin/hirudin mixture (50 µl). Thrombin activity was determined as described above. IC50 values of hirudin and its derivatives were then determined (Fig. 5B). To monitor the inhibition of the clot-bound thrombin activity by hirudin and its derivatives, each washed fibrin clot was incubated with 100 µl of either HBS or inhibitors (75 nM). After incubation at room temperature for 1 h, liquid was carefully removed without disturbing the clot, and the clot was then washed with HBS to remove any unbound inhibitors. Finally, chromogenic substrate was added to each clot and the rate of color development (at 405 nm) was measured. The activity of the non-inhibited clot-bound thrombin was plotted over the thrombin concentrations used to form the clot (Fig. 6B). The amount of clot-bound thrombin inhibited by the inhibitor was determined as the differences of thrombin activities between the clots treated with the inhibitor and the clots treated with HBS (Fig. 6C).
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The First Fibrin Clot Lysis AssayIn this assay, washed fibrin clots with different amounts of clot-bound thrombin were prepared according to the above mentioned conditions. Each clot was treated with 100 µl of 75 nM SAK or HE-SAKK for 1 h. The solution was then removed by pipetting. Each clot was washed once with HBS to remove any unbound thrombolytic agent. Clot lysis was initiated by the addition of 100 µl of 1 µM plasminogen in HBS. The lysis process was monitored at room temperature using the microtiter plate reader until the turbidity of the clot reached the minimal value. The extent of clot lysis expressed as the percentage of the original clot turbidity was plotted over the lysis time (minutes). T50% for clot lysis, which represented the time required to achieve a 50% lysis of the fibrin clot, was obtained from the graphs. T50% values for clots with different amounts of thrombin incorporated inside were then plotted over the thrombin concentration used in forming the clot to determine the thrombin effects on clot lysis mediated by HE-SAKK (Fig. 7).
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The Second and Third Fibrin Clot Lysis AssaysIn these two
assays, fibrin clots (formed in the presence of 0.8 NIH unit/ml of thrombin)
were generated as described above and the assays were all performed at room
temperature. In the second clot lysis assay, clot lysis was initiated by the
addition of 100 µl of a freshly prepared thrombolytic solution (75
nM SAK or HE-SAKK + 1 µM plasminogen). The clot lysis
process was monitored for 1 h. After this 1-h incubation, liquid (100
µl) was carefully removed to eliminate any unbound thrombolytic agent. In
this process, plasminogen was removed as well. Therefore, 100 µl of a 1
µM plasminogen solution was layered on each clot. Upon returning
the microtiter plate to the reader, the subsequent clot lysis process was
monitored. The third fibrin clot lysis assay was identical to the second one
except that all the plasminogen containing solution used in this set of assays
also contained 4 mg/ml fibrinogen.
Plasma Clot Lysis AssaysCross-linked plasma clots were prepared using freshly prepared, citrated platelet-poor human plasma pooled from healthy donors. Once prepared, plasma was aliquoted and stored at 20 °C until use. Clotting was initiated by adding human thrombin to 0.8 NIH unit/ml (final concentration) and CaCl2 to 20 mM (final concentration) at room temperature. Immediately after mixing, 50 µl of the polymerizing plasma was transferred to microtiter plate. The clots were formed at room temperature for 2 h and washed once with plasma. Clot lysis was performed by adding 50 µl of plasma containing freshly added thrombolytic agent (600 nM HE-SAKK, 7,200 nM SAK, or 1,200 nM SAK plus 600 nM hirudin) on each clot. The clot lysis process was monitored. After 1-h incubation, liquid (plasma with unbound thrombolytic agent and thrombin) at the surface of the clot was removed and the same amount of plasma was added to the clot. One plasma clot was incubated with 50 µl of plasma as the control to monitor the stability of the plasma clot during the assay period. T50% values were used to compare the clot lysis potencies of each agent.
Other MethodsPurification of plasminogen and specific activity determination of SAK, SAKK, and HE-SAKK were performed as described by Szarka et al. (24). Properties of both staphylokinase and hirudin-specific polyclonal antibodies used in this study were also described by Szarka et al. (24). Recombinant protein production yield was measured using the quantitative Western blot method as described previously (31). DNA sequencing was performed at the DNA Sequencing Laboratory, University Core DNA and Protein Services, University of Calgary. In the fibrin-clot lysis studies with different thrombolytic agents (i.e. SAK versus HE-SAKK), the T50% values were subject to the Student's t test for statistical significance analysis. A probability value of 5% (p < 0.05) was regarded as being significant.
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RESULTS AND DISCUSSION |
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Effects of Different Protease-deficient Strains on SAK-K Coil and Hirudin-E Coil ProductionSecretion of both SAK-K coil and hirudin-E coil is directed by the B. subtilis levansucrase signal peptide sequence. A strong and constitutively expressed promoter P43 is used to drive transcription. Analyses of culture supernatants from six (WB600), seven (WB700), and eight (WB800) extracellular protease-deficient strains harboring either pSAK-K coil or pHirudin-E coil indicated that SAK-K coil and hirudin-E coil could be produced via secretion (Fig. 2). The identity of each fusion was confirmed by Western blot studies with SAK- and hirudin-specific polyclonal antibodies, respectively. Production of both SAK-K coil and hirudin-E coil reached peak levels in a time window of 6 to 8 h after inoculation (data not shown). The calculated molecular weight of hirudin-E coil is 12,080 and a diffused protein band, which was not present in the negative control WB600(pUB18), was observed in the culture supernatant of WB600, WB700, and WB800 carrying the pHirudin-E coil plasmid (Fig. 2, panels A and B). This band showed an apparent molecular weight of 21,500. Because engineered proteins carrying flexible linkers (such as single chain antibody fragments) always show larger apparent molecular weights from SDS-PAGE, the apparent molecular weight of hirudin-E coil in the range of 21,500 is not unexpected. All three protease-deficient strains could produce hirudin-E coil with the same apparent molecular weight and with comparable production yields (Fig. 2, panels A and B). In contrast, SAK-K coil produced from WB600(pSAK-K coil) and WB700(pSAK-K coil) showed an apparent molecular weight (20,000) that was different from the one (28,000) produced from WB800(pSAK-K coil) (Fig. 2, panels C and D). Although the calculated molecular weight for SAK-K coil is 21,029, our data suggest that SAK-K coil with an apparent molecular weight of 28,000 from WB800(pSAK-K coil) is the intact form of SAK-K coil. Western blot studies support this suggestion because the presence of multiple bands of SAK-K coil in the range from 20 to 28 kDa was observed only from the culture supernatants of WB600(pSAK-K coil) and WB700(pSAK-K coil) (Fig. 2D). Our data illustrate the importance of using WB800 for the production of intact SAK-K coil. Because WB800 differs from WB700 by inactivation of a wall bound protease WprA (27, 39), this protease, either directly or indirectly, accounts for the observed degradation of SAK-K coil. Judging from the observed ladder of degraded SAK-K coil and their molecular masses, the major cleavage sites are mainly located within the K coil sequence because staphylokinase by itself is resistant to the residual proteases from WB600 and WB700 (40). The K coil sequence is almost identical to the E coil sequence with the exception that lysine rather than glutamate is located at positions e and g of the coiled coil sequence. If WprA in WB700 cleaves the K coil sequence directly, this protease is suggested to cut lysine-rich sequences.
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Purification, Quantification, and Mass Spectrometry of Hirudin-E Coil and Staphylokinase-K CoilHirudin-E coil was purified to homogeneity from the culture supernatant of WB800(pHirudin-E coil) using a two-step purification scheme: a DE-52 cellulose column and a Bio-Rad Bio-Prep SE100/17 gel filtration column (Fig. 3A, lane 1). Staphylokinase-K coil was also purified to homogeneity (Fig. 3A, lane 2) using a similar approach (a MacroS column and a Bio-Rad Bio-Prep SE100/17 column). Interestingly, staphylokinase-K coil was found to be well resolved into two peaks in the cation exchange column. One was eluted at a lower salt concentration (0.40.5 M NaCl) and the other was at a higher salt concentration (0.60.7 M NaCl). Analysis of these samples by SDS-PAGE under reducing and non-reducing conditions demonstrated that the form that eluted under the lower salt condition was the monomeric staphylokinase-K coil although the other form was the homodimer of staphylokinase-K coil (data not shown). Although the purified hirudin-E coil sample seemed to be relatively pure in a Coomassie Blue-stained SDS-polyacrylamide gels, the presence of homodimeric hirudin-E coil in this sample could only be observed via the Western blot study (Fig. 3B). In fact, hirudin-E coil stained very poorly in SDS-polyacrylamide gels. Because Coomassie Blue is reported to bind preferentially to arginine and aromatic residues in proteins (41), the low content of these residues in hirudin-E coil provides an explanation for this observation. By using known amounts of purified hirudin-E coil and SAK-K coil to generate standard curves for these proteins in quantitative Western blot studies, the production yields of hirudin-E coil and SAK-K coil in the culture supernatant before purification were estimated to be 20 (1.66 µM) and 99 mg/liter (4.7 µM), respectively. Because there is a significant discrepancy between the calculated and the apparent molecular masses of hirudin-E coil and SAK-K coil, purified samples were analyzed by MALDI-TOF mass spectrometry. By using bovine serum albumin as the reference, monomeric hirudin-E coil and SAK-K coil showed molecular weights of 12,083 and 21,046, respectively (data not shown, also see Fig. 4D). These values matched closely with the predicted values (12,080 for hirudin-E coil and 21,029 for SAK-K coil).
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Production, Purification, and Mass Spectrometry of Heterodimeric Hirudin-E Coil and SAK-K Coil (HE-SAKK)Complicated by the presence of homodimers, a co-cultivation method was developed to produce heterodimeric HE-SAKK by adjusting the initial inoculation density of WB800(pHirudin-E coil) and WB800(pSAK-K coil) at different ratios (1:1, 2:1, and 5:1). The best production yield of HE-SAKK heterodimer could be accomplished using the 2:1 ratio (data not shown). This observation was consistent with the measurement that HE and SAKK were individually produced at a level of 1.66 and 4.7 µM, respectively. Because HE-SAKK has a calculated pI of 5.15, this protein was purified to homogeneity using a two-step purification scheme (a DE52-cellulose column and a gel filtration column). The secretory production yield of HE-SAKK under the co-cultivation condition was estimated to be 50 mg/liter (1.51 µM). Although the apparent molecular mass of HE-SAKK in SDS-PAGE was 43 kDa (Fig. 4A), mass spectrometric analysis (MALDI-TOF MS) showed that the mono-protonated HE-SAKK molecule had a molecular weight of 33,122 (Fig. 4D), which agreed very well with the theoretical prediction (33,091). The result also supported the idea that a disulfide bridge was formed between the heterodimeric coiled coil sequences to make HE-SAKK a single entity. Low levels of heterodimeric HE-SAKK that did not form the disulfide bond in the coiled-coil region were also observed from the MALDI-TOF mass spectrogram.
Biological Activities of SAKK and HEIt is vital to confirm that both SAK and hirudin in the heterodimer retain the full biological activities as their parent molecules. Using purified monomeric SAKK for plasminogen activation assays, the data confirmed that indeed staphylokinase-K coil showed identical specific activity as the free staphylokinase (Fig. 5A). In contrast, the IC50 of hirudin-E coil (13.5 nM) for thrombin inhibition is 2.5 times higher than that (5.2 nM) of hirudin. Therefore, the thrombin inhibitory effect of hirudin in hirudin-E coil is significantly lower than that of non-fused hirudin (Fig. 5B). This is because of the improper pairing of the cysteine residues in the hirudin moiety in hirudin-E coil as illustrated in the disulfide-bond reshuffling experiment described below.
Reshuffling of Disulfide Bonds in Hirudin-E Coil and HE-SAKKB.
subtilis has been shown to produce biologically active secretory proteins
with disulfide bonds such as TEM--lactamase and single chain antibody
fragments (27,
31,
42). These proteins have
either one or two pairs of disulfide bonds and the cysteine residues involved
in disulfide bond formation are arranged in a sequential manner (i.e.
the first cysteine in the sequence pairs with the second cysteine and the
third cysteine pairs with the fourth one). In the case of hirudin, there are
three pairs of disulfide bonds and the cysteine residues that form disulfide
bonds are not arranged in a sequential manner (i.e.
Cys6-Cys14, Cys16-Cys28, and
Cys22-Cys39). When the purified hirudin-E coil sample
was analyzed by Western blotting (Fig.
3B), three bands were observed under the non-reducing
condition for SDS-PAGE. The 40-kDa band corresponded to the homodimer of
hirudin-E coil whereas the two bands with the apparent molecular masses of 21
and 23 kDa were the monomeric hirudin-E coil
(Fig. 3B, lane
1). In contrast, after reduction, all forms of hirudin-E coil migrated as
a single band with the apparent molecular weight of 24,000
(Fig. 3B, lane
4) using molecular weight markers for Western blotting as the reference.
These data indicated that not all the hirudin-E coil molecules produced from
B. subtilis had the correct pairing of cysteine residues. The fast
migrating 21-kDa band was likely to be the properly folded form as it had the
most compact structure reflected by its fast migration. The slower migrating
band corresponded to hirudin-E coil with either partial disulfide bond
formation or incorrectly paired disulfide bonds. Hirudin-E coil without any
disulfide bond formation (under the reducing condition) migrated slowest
because it was in a fully extended configuration. Because staphylokinase-K
coil does not contain any intramolecular disulfide bonds, it showed the same
mobility in SDS-PAGE under both reducing and non-reducing conditions
(Fig. 3A, lane 2
versus lane 5). To overcome the problem associated with mispairing of
cysteine residues in hirudin-E coil, purified hirudin-E coil was allowed to
reshuffle its disulfide bonds in the presence of 4 mM cysteine and
2 mM cystine. After this treatment, both monomeric and dimeric
hirudin-E coil molecules were in a more compact structure and migrated faster.
As shown in Fig. 3B
(lane 2), the majority of monomeric hirduin-E coil showed an apparent
molecular mass of 21 kDa. Thrombin inhibition assays
(Fig. 5B) indicated
that reshuffled hirudin-E coil showed an activity comparable with hirudin. The
IC50 of hirudin and the reshuffled hirudin-E coil were 5.2 and 5.5
nM, respectively. Purified HE-SAKK also showed a faster migration
after the reshuffling treatment (Fig.
3C). Before reshuffling, IC50 of hirudin in
HE-SAKK was 22 nM. After reshuffling, the IC50 value
decreased to 5.6 nM (data not shown). SAK in the HE-SAKK
heterodimer also showed activity comparable with staphylokinase (data not
shown). In this study, inefficiency of the B. subtilis expression
system in producing secretory proteins with complicated disulfide bond
profiles is clearly illustrated. A similar observation for the production of
misfolded human pancreatic
-amylase that also has a complicated
disulfide bond profile has also been reported
(43). However, as illustrated
in the present study, the use of an in vitro disulfide bond
reshuffling protocol provides a simple and effective solution to overcome this
problem. In this protocol, no disulfide isomerase or disulfide oxidoreductase
is needed.
Generation of Thrombin-rich Fibrin ClotsThere is plenty of
evidence supporting the idea that freshly formed blood clots are rich in
thrombin (44,
45). Thrombin binds to fibrin
directly through an interaction at its anion-binding exosite
(46). With time, thrombin
bound to or trapped within blood clots will leak out and the thrombin content
in an aged clot will decrease gradually. Therefore, the clot-bound thrombin
would potentially serve as an interesting marker to differentiate a freshly
formed clot from an aged clot
(47). For acute myocardial
infarction patients, the pathologic thrombi are freshly formed and therefore
should be thrombin-rich. In contrast, a physiological hemostatic plug that has
been formed for a while should be thrombin poor. Consequently, HE-SAKK should
be able to differentiate these two different types of clots. To determine
whether the hirudin domain in HE-SAKK can serve as a targeting agent to direct
HE-SAKK to fibrin clots in proportion to the thrombin content, one has to
first determine whether fibrin clots formed in the presence of higher levels
of thrombin can trap more thrombin or not. Fibrin clots were formed in a final
volume of 100 µl with different levels of thrombin (the final concentration
of thrombin ranged from 0.05 to 2 NIH units/ml). Two sets of fibrin clots were
generated. One set was washed with HBS to remove any unbound thrombin while
the other set was not washed. Thrombin activity was then determined using a
thrombin-specific chromogenic substrate. Within the range tested, the amounts
of thrombin retained in the washed fibrin clots were proportional to the
amounts of thrombin added during the clot formation process
(Fig. 6A). Because
thrombin activity in the washed clots was 79.5 ± 4.2% of that in
the unwashed clots, this indicated that considerable amounts of thrombin could
remain tightly bound to the fibrin clots. It is important to note that the
assayable clot-bound thrombin activity may not truly reflect the actual amount
of all the clot-bound thrombin molecules because the thrombin-specific
chromogenic substrate may not be able to freely diffuse into the center of the
highly cross-linked fibrin clots.
Inhibition of Clot-bound Thrombin Activity by Hirudin and HE-SAKKWashed fibrin clots formed in the presence of different amounts of thrombin were used in this study. 100 µl of hirudin (75 nM) or HE-SAKK (75 nM) was layered on top of each clot that occupied a volume of 100 µl. The mixtures were incubated for 60 min. The concentration of hirudin or HE-SAKK at a final concentration of 75 nM was selected in this study because the clinical doses of SAK used in thrombolysis (48) are usually 515 mg/patient (64192 nM in circulation). After several extensive washes to remove any unbound hirudin or HE-SAKK, thrombin activity within these fibrin clots was determined. When fibrin clots were formed with low doses of thrombin (up to 1.2 NIH units/ml), both hirudin and HE-SAKK were equally effective and 92% of the clot-bound thrombin activity was inhibited (Fig. 6B). For fibrin clots formed in the presence of higher levels (1.32 NIH units/ml) of thrombin, hirudin was slightly more effective than HE-SAKK in inhibition of clot-bound thrombin. This is probably because of the smaller size of hirudin (i.e. 6.97 versus 33 kDa for HE-SAKK) so that it can diffuse more easily into the interior of the clot. With clots formed in the presence of thrombin at a final concentration of 2 NIH units/ml, HE-SAKK could still inhibit 84% of the clot-bound thrombin activity. When the differences of the thrombin activities between the clots treated with the thrombin inhibitors (hirudin or HE-SAKK) and the clots treated with HBS (control) were plotted over the thrombin concentrations used in forming the clot, it clearly showed a thrombin dose-dependent inhibition mediated by both hirudin and HE-SAKK (Fig. 6C). Therefore, a fibrin clot with higher levels of clot-bound thrombin should bind higher levels of HE-SAKK.
Lysis of Thrombin-rich Fibrin Clots with HE-SAKKBecause
HE-SAKK can be targeted to fibrin clots depending on the level of the
clot-bound thrombin, it would be of interest to determine whether a fibrin
clot with higher levels of clot-bound thrombin can be lysed faster. To address
this question, an in vitro clot lysis assay was established. Two
series of fibrin clots were formed in the presence of different amounts of
thrombin. After washing to remove any unbound thrombin, one set of clots was
incubated with HE-SAKK (100 µl at a concentration of 75 nM). The
second set of clots was incubated with staphylokinase (same concentration as
HE-SAKK). After a 1-h incubation, unbound thrombolytic agents (i.e.
HE-SAKK and SAK) were removed by washing the clots with HBS. A 100-µl
plasminogen solution (1 µM, physiological concentration) was
then added to initiate the clot lysis event. The clot lysis process was
monitored by the reduction of the clot turbidity with time. This assay was
designed to examine the clot lysis effect of the clot bound HE-SAKK. Removal
of the unbound thrombolytic agents was designed with the objective to account
for the short in vivo half-life (310 min) of SAK in human
(49). As shown in
Fig. 7, it took SAK 250 min to
achieve a 50% clot lysis (T50%) and the
T50% values remained fairly constant for clots formed with
different thrombin concentrations. Interestingly, the T50%
values for clot lysis mediated by HE-SAKK indeed decreased as the amounts of
thrombin used in clot formation (i.e. the amounts of clot-bound
thrombin) increased. T50% values reached the lowest value
of 100 min and remained constant at that level when the thrombin
concentrations used in clot formation was 0.8 NIH unit/ml or higher. The
inability to observe a faster clot lysis when the thrombin concentration used
in clot formation was higher than 0.8 NIH unit/ml was likely because of the
inability of plasminogen (92 kDa) to penetrate into fibrin clots that have
small pores. Various studies including confocal and electron microscopies
(50,
51) have demonstrated that
fibrin clots formed in the presence of low concentrations of thrombin consist
of thick fibers with large pores. With high concentrations of thrombin, the
fibrin clots have much thinner fibers and these fibers form a network with
small pores. Therefore, even though more HE-SAKK (33 kDa) can be trapped
inside the clots in proportion to the amounts of fibrin-bound thrombin, those
HE-SAKK molecules located in the interior of the clot would not be able to
activate plasminogen. This is simply because plasminogen is too big to diffuse
into the interior of the clot as illustrated by the superficial accumulation
of plasminogen only on the clot surface in the confocal microscopic study
(52). In comparison with
fibrin clots formed under in vitro conditions, it is logical to
predict that HE-SAKK would be able to lyse the freshly formed pathologic
thrombi more efficiently. Pathologic thrombi are formed in the presence of
plasminogen so that some plasminogen molecules will be trapped in or bound to
the thrombi. In contrast, the interior of the fibrin clots formed under the
in vitro conditions with purified fibrinogen and thrombin is
essentially plasminogen free. Based on this in vitro study, HE-SAKK
indeed can promote clot lysis in proportion to the amounts of the clot-bound
thrombin up to a certain degree.
HE-SAKK Lyses Thrombin-rich Fibrin Clots Faster Than SAKSAK by itself has no ability to bind to the fibrin clot directly unless it forms a SAK-plasmin(ogen) complex. Both plasmin and plasminogen have fibrin binding capability via the kringle domains within these molecules (53). Under physiological conditions, plasminogen molecules are present in the circulation when staphylokinase is infused into the patient. Therefore, the above mentioned clot lysis assay may underestimate the potency of staphylokinase in clot lysis because only SAK molecules that have been physically trapped inside the clot via diffusion would be able to mediate the clot lysis event under the assay condition. To overcome this problem, a second in vitro fibrin clot lysis assay was established. Fibrin clots were formed in the presence of thrombin at a final concentration of 0.8 NIH units/ml in an enzyme-linked immunosorbent assay plate. These clots were then washed and incubated with either SAK or HE-SAKK in the presence of plasminogen for 1 h. Solution containing unbound thrombolytic agents (100 µl) was removed from each well. Because these clots were quite fragile, they were not washed. Plasminogen (1 µM, 100 µl) was then added to each well to continue the clot lysis event. The change in clot turbidity including the first hour incubation with thrombolytic agents in the presence of plasminogen was then monitored. Under this assay condition, the T50% values for SAK and HE-SAKK were 192 ± 3 and 152 ± 2.3 min, respectively (Fig. 8A). This represents a 21% reduction of T50% for HE-SAKK. The difference observed for these values was statistically significant (t = 18.543, degree of freedom = 4, p < 0.05). The larger T50% value for HE-SAKK under this assay condition reflected removal of some of the clot-bound HE-SAKK molecules during the step to remove the unbound HE-SAKK agent because of the partial clot lysis in the presence of both HE-SAKK and plasminogen during the first hour of preincubation.
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Effects of Clot Targeting and Antithrombotic Activities of HE-SAKK on
Fibrin Clot LysisIn addition to the clot targeting ability,
HE-SAKK should have an antithrombotic effect. To examine the antithrombotic
effect mediated by HE-SAKK, a third in vitro fibrin clot lysis assay
was developed. In this assay, fibrin clots formed in the presence of 0.8 NIH
units/ml of thrombin were washed to remove the unbound thrombin. Thrombolytic
agents (SAK or HE-SAKK) in the presence of both plasminogen and fibrinogen
were added to the washed fibrin clots and incubated for 1 h. Any solution
(100 µl) containing unbound thrombolytic agents, plasminogen, and
fibrinogen was then removed with the objective to remove any unbound
thrombolytic agent. However, in the process, plasminogen and fibrinogen were
also removed as well. Therefore, 100 µl of HBS containing both plasminogen
(1 µM) and fibrinogen (4 mg/ml) was added to the assay to
restore the plasminogen and fibrinogen contents. The change in clot turbidity
including the first hour of incubation with thrombolytic agents was monitored.
Interestingly, the preformed fibrin clot increased in size as reflected by an
increase in turbidity during the initial phase of incubation with SAK,
plasminogen, and fibrinogen (Fig.
8B). This observation indicated that the clot formation
process was even faster than the clot lysis process. In a later stage, clot
lysis became dominant. In contrast, the growth of the preformed clot was not
as dramatic when HE-SAKK was used as the thrombolytic agent. In this assay,
the T50% values for SAK and HE-SAKK were 242 ± 5
and 168 ± 5 min, respectively (Fig.
8B). The difference in these values was statistically
significant (t = 18.088, degree of freedom = 4, p <
0.05). The longer time required for 50% clot lysis in this assay reflected the
simultaneous occurrence of both clot formation and clot lysis events. In
comparison with the T50% value of SAK,
T50% for HE-SAKK showed a reduction by 30%. To illustrate
the importance of the hirudin-mediated clot targeting effect in HE-SAKK for
improved clot lysis, a control experiment was performed by adding SAK and
hirudin together with plasminogen and fibrinogen to the preformed fibrin clot
during the first hour of incubation. In the presence of hirudin, the growth of
the preformed fibrin clot under this condition could also be suppressed.
However, without the covalent linkage of hirudin to SAK, it took a longer time
(T50% = 200 min) for free SAK to lyse the fibrin clot
(Fig. 8B).
HE-SAKK Lysed Plasma Clot Much Better Than SAKTo examine
the potency of HE-SAKK in clot lysis under conditions that are more relevant
to physiological conditions, a plasma clot assay was applied to monitor the
effectiveness of HE-SAKK in clot lysis. This is important because plasma
contains various factors such as plasminogen, fibrinogen, prothrombin,
2-antiplamin, and other factors that may influence both the
clot formation and clot lysis events. Under this condition, thrombin (0.8 NIH
unit/ml) was added to plasma to induce clot formation. The clots were washed
with plasma and then resuspended in plasma containing either SAK or HE-SAKK
for 1 h. Plasma containing unbound thrombolytic agents was then removed and
replaced with 50 µl of plasma. Changes in clot turbidity were monitored
including the first hour of incubation with thrombolytic agents. In this
assay, the concentration of each thrombolytic agent was adjusted to give a
T50% of
120 min. The difference in the concentration
of these thrombolytic agents required to reach such a clot lysis rate was
compared. This method was used for comparison because SAK at a final
concentration of 600 nM (even up to 1,200 nM) failed to
show any measurable clot lysis whereas HE-SAKK worked effectively at this
concentration. As shown in Fig.
9, when the washed plasma clot was placed in plasma without any
added thrombolytic agent, the turbidity of the washed plasma clot increased
gradually within the first 120 min. The growth of the clot was expected to be
mediated by thrombin bound to the surface of the clot. No clot lysis was
observed as expected. The addition of SAK to plasma greatly stimulated the
growth of the plasma clot within the first 60-min incubation. To achieve a
T50% value of 160 min, SAK at a final concentration of
7,200 nM was required. The drastic growth of the plasma clot at the
initial stage can be contributed by several factors. 1) Not only thrombin
bound to the surface of the clot can promote clot growth, but thrombin buried
inside the clot can be re-exposed during the fibrinolytic event mediated by
SAK (54). 2) It has been well
established that with excess plasmin generation in plasma, plasmin can
generate more thrombin from prothrombin via three different mechanisms
(55). Plasmin can activate
factor XII to factor XIIa that can then activate kallikrein from prokallikrein
(56). Both factor XIIa and
kallikrein are involved in the initial phase of the intrinsic coagulation
pathway that leads to thrombin generation. Furthermore, plasmin can also
activate factor V, which is one of the key components in the prothrombinase
complex that mediates the conversion of prothrombin to thrombin
(57). Last, plasmin has been
shown to increase the activity of the factor VIIIa/IXa complex
(58). All these mechanisms can
lead to more thrombin generation in plasma. With a low dose of SAK, no
systemic activation of plasmin resulted. However, under the experimental
conditions used in this study, 7,200 nM staphylokinase can induce
systemic plasminogen activation because the physiological concentration of
human plasminogen is 12 µM and the
2-antiplasmin concentration is only about half of the
concentration of plasminogen
(59). With more thrombin
generation in plasma, thrombin can activate factor XIII, which mediates the
cross-linking between fibrin chains and the covalent immobilization of
2-antiplasmin to fibrin
(60). A thrombin-activable
fibrinolysis inhibitor, which is a plasma carboxypeptidase that selectively
removes C-terminal lysine residues from fibrin
(61), can also be activated.
This will reduce the binding of both plasmin(ogen) and SAK-plasmin(ogen)
complex to fibrin because these molecules bind to fibrin via the interactions
between the kringle domains of plasmin(ogen) and the C-terminal lysine
residues in fibrin generated during fibrinolysis. All these factors will make
the plasma clots more resistant to fibrinolysis and can account for the
requirement of SAK at high concentrations for effective plasma clot lysis
within 160 min.
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In contrast to the SAK-mediated plasma clot lysis, HE-SAKK-mediated clot lysis did not show any significant growth of the plasma clot. Furthermore, the concentration (600 nM) of HE-SAKK required to achieve a T50% of 120 min was 12 times lower than that for SAK. These data illustrated the dramatic effect of HE-SAKK in preventing clot growth and promoting clot lysis. This is the result of the combination of both the clot targeting effect and thrombin inhibition effect of hirudin. For the clot lysis event mediated by SAK in the presence of 600 nM hirudin, although no rapid growth of plasma clot could be observed, the SAK concentration required to generate a T50% of 122 min was 1,200 nM, which is two times higher than that of HE-SAKK.
Various attempts to introduce antithrombotic activity to staphylokinase have been reported (24, 62, 63) including our systematic generation of both staphylokinase-hirudin and hirudin-staphylokinase. However, all these reported structures are in the linear fusion format. Hirudin activity in staphylokinase-hirudin is significantly weaker.2 On the other hand, although both staphylokinase and hirudin can potentially be biologically active in hirudin-staphylokinase, the targeting effect to thrombin-rich clots mediated by hirudin would be either partially or completely lost because of the proteolytic processing of staphylokinase. Therefore, HE-SAKK reported here represents a successfully engineered bifunctional thrombolytic agent that truly possesses both fibrinolytic and antithrombotic activities that are comparable with their parent molecules. At the same time, HE-SAKK can target to thrombin-rich clots. The clot targeting capability, the potential to minimize reocclusion, and the efficient clot lysis make HE-SAKK a very promising agent for the treatment of acute myocardial infarction. Although immunogenicity of HE-SAKK can be a concern, the use of an engineered version of staphylokinase with lower immunogenicity (9) or the polyethylene glycol derivatives of HE-SAKK can potentially minimize the immunogenicity problem.
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FOOTNOTES |
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Current address: SemBioSys Genetics, Calgary, Alberta T1Y 7L3, Canada.
¶ To whom correspondence should be addressed: Division of Cellular, Molecular and Microbial Biology, Dept. of Biological Sciences, University of Calgary, 2500 University Drive, N.W., Alberta T2N 1N4, Canada. Tel.: 403-220-5721; Fax: 403-289-9311; E-mail: slwong{at}ucalgary.ca.
1 The abbreviations used are: tPA, tissue-specific plasminogen activator;
APSAC, anisoylated plasminogen streptokinase activator complex; HBS,
HEPES-buffered saline; HE, hirudin-E coil; HE-SAKK, hirudin-E
coil-staphylokinase-K coil heterodimer; MALDI-TOF MS, matrix-assisted laser
desorption/ionization-time of flight mass spectrometry; SAK, staphylokinase;
SAKK, staphylokinase-K coil; T50%, time required for 50%
clot lysis.
2 S. Szarka and S.-L. Wong, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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