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Originally published In Press as doi:10.1074/jbc.M303241200 on May 7, 2003

J. Biol. Chem., Vol. 278, Issue 29, 26677-26686, July 18, 2003
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Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots*

Qun Lian, Steven J. Szarka {ddagger}, Kenneth K. S. Ng § and Sui-Lam Wong 

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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Acute myocardial infarction (also known as heart attack) is a leading cause of death in the Western world. It is commonly caused by the formation of a pathologic clot (thrombus) at a critical position that results in obstructing the blood flow to heart tissues. The use of blood clot dissolving agents is one of the well established methods in treating patients with acute myocardial infarction (1, 2). Results from several large scale clinical trials have firmly established the effectiveness of this approach in saving lives (1–3). Among the clinically approved blood clot dissolving agents including streptokinase, anisoylated plasminogen streptokinase activator complex (or anistreplase), tissue-specific plasminogen activator (tPA),1 and urokinase, tPA is the most commonly used blood clot dissolving agent in the Western world. Even though tPA is fibrin-specific, its short in vivo biological half-life and sensitivity to plasminogen activator inhibitors in circulation require the use of higher doses of tPA for effective clot lysis. At the pharmacological doses used, tPA exerts only partial fibrin specificity. This results in depletion of plasma proteins such as coagulation factors V and VIII, and to a certain degree, plasminogen and fibrinogen. Approximately 57% of the patients treated with tPA can restore their blood flow to an acceptable level within 90 min after receiving the treatment (2). Within this group, 10–30% of patients showed reocclusion shortly after clot dissolution. The reformed secondary clots are usually platelet-rich (4–6) and show strong resistance to lysis mediated by tPA (7). Furthermore, a low but significant percentage of the patients also suffer from stroke (3, 8). To address these shortcomings in the current thrombolytic therapy, we report the engineering of a "Y"-shaped staphylokinase-hirudin heterodimer to target freshly formed, thrombin-rich thrombi to initiate the clot lysis event. At the same time, the hirudin moiety can effectively minimize clot reformation during thrombolysis through the inhibition of thrombin. Staphylokinase (SAK) is a promising blood clot dissolving agent (10). Although SAK does not bind directly to fibrin, it can bind indirectly through the fibrin binding of plasmin(ogen) by forming a 1:1 stoichiometric SAK-plasmin(ogen) complex. The resulting SAK-plasmin complex can then function as the plasminogen activator to convert plasminogen to plasmin for clot lysis. Although SAK has comparable thrombolytic potency as tPA, several multicenter clinical trials demonstrate that staphylokinase shows a superior fibrin specificity in comparison to tPA (11–13). This high fibrin specificity (14) is accomplished by a mechanism whereby the plasminogen activation activity of any non-fibrin bound SAK-plasmin complex is inhibited by circulating {alpha}2-antiplasmin. In contrast, if the SAK-plasmin complex is fibrin bound, {alpha}2-antiplasmin is unable to bind to this complex. Consequently, the SAK-plasmin complex can activate plasminogen to generate plasmin locally on the surface of the clot. Furthermore, SAK is shown to bind preferentially to clot-bound plasmin(ogen) (15). SAK also has the ability to dissolve platelet-rich plasma clots and is much more efficient than tPA and streptokinase under both in vitro conditions and in animal models (16). Therefore, SAK is selected as the blood clot dissolving component in this engineered thrombolytic agent.

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 ~ 10–11–10–13 M, (17–19)), 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Assembly of K Coil and E Coil Sequences—The structural genes encoding both K coil and E coil sequences were assembled by using several long synthetic oligonucleotides (primers 1–4 for K coil and primers 5–8 for E coil) with a PCR-based strategy (22, 23). For the K coil sequence, portions of the primer 2 sequence are complementary to primers 1 and 3, respectively, and part of the primer 4 sequence is complementary to primer 3. The sequences of primers 1–4 are listed as follows: primer 1, 5'-GGGGATCCCCTGGACAGAAAGTTTCTGCTTGCAAACAG-3'; primer 2, 5'-CTTTTTGTTTTAAAGCGCTCACTTTCTGTTTAAGCGCGCTAACTTTCTGTTTGCAAGCAGAAAC-3'; primer 3, 5'-GTGAGCGCTTTAAAACAAAAAGTGTCAGCACTTAAGCAAAAAGTC TCTGCGCTGAAACAGTAATG-3'; primer 4, 5'-GGGCATGCGTTAACATTACTGTTTCAGCGCAG-3'. The assembled sequence encodes a portion of a linker sequence and the entire K coil sequence with a BamHI site at the 5' end and an HpaI site at the 3' end. This sequence was ligated to HincII-digested pUC19 as a blunt-end fragment to generate pUC19-K coil. The E coil sequence was assembled with a similar approach using four primers (primers 5–8) to generate a DNA fragment with a SpeI site at the 5' end and a SphI site at the 3' end. The fragment was ligated to HincII cut pUC19 to generate pUC19-E coil via a blunt-end ligation. Sequences of primers 5–8 are listed as follows: primer 5, 5'-GGACTAGTCCTGGACAGGAAGTTTCTGCTTGCGAACAG-3'; primer 6, 5'-CTTCTTGTTCAAGTGCTGAAACTTCTTGTTCTAATGCGCTCACTTCCTGTTCGCAAGCAGAAAC-3'; primer 7, 5'-CAGCACTTGAACAAGAAGTTAGCGCGCTTGAACAAGAAGTGAGCG CAT TAGAACAGTAATC-3'; primer 8, 5'-CCGCATGCCCGGGATTACTGTTCTAATGCG-3'. Nucleotide sequences of both the K coil and E coil sequences in pUC19-K coil and pUC19-E coil were determined and confirmed to be free of PCR errors.

Construction of B. subtilis Expression Vectors for SAKK and HE Production—Plasmid 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 [GenBank] and AF533146 [GenBank] , respectively.

Expression Studies in B. subtilis—Each 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-SAKK—For 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 (0–0.7 M NaCl) in the same buffer. The HE-containing fractions (0.25–0.35 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 200–300 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 0–0.5 M NaCl (in 150 ml) and the HE-SAKK containing fractions (0.1–0.15 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 (0–1.0 M NaCl in the same buffer). SAKK monomer-containing fractions (0.4–0.5 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 M–1 cm–1, 17,330 M–1 cm–1, and 20,730 M–1 cm–1, respectively.

Disulfide Bond Reshuffling—The 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 50–70 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 Spectrometry—Protein 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 Thrombin—Cross-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 35–3912, 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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  		FIG. 6.
Quantification and inhibition of clot-bound thrombin activity. Fibrin clots were formed by adding increasing amounts (expressed in terms of thrombin concentration in the clot formation process) of thrombin. A, activity of clot-bound thrombin. The clots were either washed extensively with HBS (open circle) or remained unwashed (closed circle). Thrombin activity was then determined. B, inhibition of clot-bound thrombin activities. Clot-bound thrombin activities from washed clots were determined in the presence of hirudin (closed triangle), HE-SAKK (closed square), or in the presence of HBS (control, open circle). C, correlation between the amounts of thrombin activities inhibited by either hirudin (closed triangle) or HE-SAKK (closed square) and the amounts of thrombin used in the clot formation process. The data presented in each panel represent the mean value (±S.D.) of three independent experiments.

 

Thrombin Inhibition Assays—These 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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  		FIG. 5.
Biological activities of purified HE-SAKK. A, staphylokinase activity determination. Plasminogen (1 µM) and SAK (5 nM) or HE-SAKK (5 nM) were incubated at 37 °C. At different time points, samples were collected and assayed for plasmin activity. Closed square and open circle represent the plasminogen activation activities of SAK and HE-SAKK, respectively. B, hirudin activity determination as reflected by the inhibition of thrombin activity. Thrombin activity was determined in the presence of increasing concentrations of hirudin (open circle with a solid line) or HE in HE-SAKK (before (closed triangle with a dash line) and after (closed square with a dotted line) the disulfide bond reshuffling). The data in both panels A and B represent the mean values (±S.D.) of three independent experiments.

 

The First Fibrin Clot Lysis Assay—In 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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  		FIG. 7.
Correlation between the clot lysis effect mediated by HE-SAKK and the thrombin concentrations used in the clot formation process. Clot lysis was mediated by either 75 nM SAK (closed square) or 75 nM HE-SAKK (closed circle).

 

The Second and Third Fibrin Clot Lysis Assays—In 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 Assays—Cross-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 Methods—Purification 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.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Design of the Y Shaped Heterodimer of SAK-K Coil and Hirudin-E Coil—To equip staphylokinase with the capability of targeting freshly formed thrombi and to minimize reocclusion, physical linkage of hirudin to staphylokinase is desirable. However, creation of simple linear fusions between these two molecules is not ideal because each component requires a free N terminus for its function. Removal of the positive charge of the {alpha}-amino group at the N terminus by either acetylation or addition of one extra amino acid severely reduces the inhibitory effect of hirudin to thrombin (19, 32, 33). The three-dimensional structure of the hirudin-thrombin complex and site-directed mutagenesis of hirudin illustrate that the N-terminal {alpha}-amino group of hirudin forms a hydrogen bond with Ser195 in the catalytic site of thrombin (34, 35). For staphylokinase, its processing by plasmin to remove the first 10 amino acids to expose the positively charged lysine residue at position 11 is essential for the activity of staphylokinase (24, 36). Although a processed version (deletion of the first 10 amino acids) of staphylokinase can be engineered using recombinant DNA technology, the presence of a positively charged residue at the N terminus of the processed staphylokinase is absolutely required (24). Therefore, a Y-shaped heterodimer of staphylokinase and hirudin was designed so that each molecule could have its N terminus free for its biological activity (Fig. 1A). We have previously demonstrated that fusion of proteins to the C-terminal end of staphylokinase does not affect the staphylokinase activity (24). The C-terminal region of hirudin is shown to bind to the thrombin cleft (also known as the anion-binding exosite) mainly through electrostatic interaction. With the presence of a flexible linker sequence, the addition of a dimerization domain to hirudin at the C-terminal end is expected to have a minimal effect on hirudin activity. In fact, several C-terminal hirudin fusions have been constructed and they all retain almost full activity (33, 37). To ensure the formation of heterodimers, a pair of engineered coiled coil sequences (also commonly known as a leucine zipper) designated K coil and E coil (20) were modified to serve as the heterodimeric domain (Fig. 1B). In our version of K coil and E coil, each sequence has approximately five heptad repeats (VSALKQK for K coil and VSALEQE for E coil). These residues occupy positions from "a" to "g" in a helical coiled coil sequence. It is well established that a minimum of three heptad repeats are required for successful dimerization and five heptad repeats are optimal for the formation of stable dimers (38). Position "f" of each coil is occupied by glutamine, which is polar and has a strong preference to form a {alpha}-helical structure. In the case of K coil, lysine residues are at positions "e" and "g" (Fig. 1B). In E coil, glutamate residues occupy the equivalent positions. With this arrangement, the E coil sequence is strictly negatively charged with a calculated pI of 3.29 and the K coil sequence is solely positively charged with a theoretical pI of 10.61. Because the pI for SAK and hirudin are 7.74 and 4.04, respectively, SAK in combination with K coil will be positively charged and hirudin with E coil will be negatively charged at physiological pH. These engineered molecules can be easily purified using ion exchange chromatography. Position "d" in the first helical turn of each coiled coil is occupied by cysteine. Once the proper heterodimer is formed, it can be locked in the heterodimeric state via a covalent disulfide bond. To ensure that each protein domain can fold independently, a hydrophilic, flexible linker of 20 amino acids ((GSTSG)3SGSPG) is inserted between SAK and K coil (Fig. 1A). Because hirudin is much smaller than staphylokinase, a shorter, 15-amino acid linker ((STSGG)2STSPG) is inserted between hirudin and E coil.



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  		FIG. 1.
Structure of HE-SAKK heterodimer. A, a molecular model of HE-SAKK. E coil and K coil serve as the heterodimerization domain. A cysteine residue located at position d of the first heptad repeat in each coiled coil allows the formation of an interchain disulfide bond as illustrated in the model. SAK represents staphylokinase. B, a helical wheel presentation of the coiled coil heterodimerization domain. Positions of the heptad repeat are marked from a to g.

 

Effects of Different Protease-deficient Strains on SAK-K Coil and Hirudin-E Coil Production—Secretion 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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  		FIG. 2.
Secretory production of hirudin-E coil and staphylokinase-K coil. A, Coomassie Blue-stained SDS-polyacrylamide gel. B, Western blot probed with hirudin-specific polyclonal antibodies. Lanes 1–4 in panels A and B are the culture supernatant from WB600(pUB18) (negative control), WB600(pHirudin-E coil), WB700(pHirudin-E coil), and WB800(pHirudin-E coil), respectively. Asterisk marks the position of the secreted hirudin-E coil. C, Coomassie Blue-stained SDS-polyacrylamide gel. D, Western blot probed with staphylokinase-specific polyclonal antibodies. Lanes 1–5 in panels C and D are the culture supernatant from WB600(pUB18) (negative control), WB600(pSAK), WB600(pSAK-K coil), WB700(pSAK-K coil), and WB800(pSAK-K coil), respectively. M represents the molecular weight markers.

 

Purification, Quantification, and Mass Spectrometry of Hirudin-E Coil and Staphylokinase-K Coil—Hirudin-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 (0.4–0.5 M NaCl) and the other was at a higher salt concentration (0.6–0.7 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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  		FIG. 3.
SDS-PAGE and Western blot analyses of purified hirudin-E coil, SAK-K coil, and HE-SAKK. A, Coomassie Blue-stained SDS-polyacrylamide gel for hirudin-E coil and SAK-K coil. Lanes 1 and 2 are the purified hirudin-E coil and SAK-K coil analyzed in the presence of reducing agent (mercaptoethanol). Lanes 4 and 5 are the same samples analyzed in the absence of reducing agent. Lane 3 is an empty lane without any sample loading. Its presence is to minimize diffusion of reducing agent from lanes 2 to 4. B, Western blot probed with hirudin-specific polyclonal antibodies. Lanes 1 and 2 are purified hirudin-E coil (before and after disulfide-bond reshuffling) analyzed in the absence of reducing agent. Lane 3 is an empty lane. Lane 4 is the purified hirudin-E coil analyzed in the presence of reducing agent. C, Coomassie Blue-stained SDS-polyacrylamide gel for purified HE-SAKK. Lanes 2 and 3 are purified HE-SAKK (before and after disulfide-bond reshuffling) analyzed in the absence of reducing agent. M represents the molecular weight markers.

 


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  		FIG. 4.
Characterization of purified HE-SAKK. A, Coomassie Blue-stained SDS-polyacrylamide gel for purified HE-SAKK. Only the staphylokinase-K coil band can be observed in lane 1. Failure to see the hirudin-E coil band in lane 1 is because of the poor staining of this protein with Coomassie Blue. B, Western blot probed with staphylokinase-specific polyclonal antibodies. C, Western blot probed with hirudin-specific polyclonal antibodies. Lanes 1 and 3 are purified HE-SAKK analyzed in the presence and absence of reducing agent, respectively. D, molecular mass determination of purified HE-SAKK by MALDI-TOF mass spectrometry. Peaks corresponding to various protonated HE-SAKK species are marked. The two peaks with the molecular weights of 12,092 and 21,043 represent the monoprotonated HE and SAKK species, respectively. This indicates the presence of low levels of non-cross-linked HE-SAKK in the purified HE-SAKK preparation.

 

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 HE—It 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-SAKK—B. subtilis has been shown to produce biologically active secretory proteins with disulfide bonds such as TEM-{beta}-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 {alpha}-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 Clots—There 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-SAKK—Washed 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 5–15 mg/patient (64–192 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 (1.3–2 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-SAKK—Because 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 (3–10 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 SAK—SAK 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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  		FIG. 8.
Fibrin-clot lysis mediated by various thrombolytic agents. SAK alone, SAK with hirudin (S+H), and HE-SAKK were used in this study in the absence (A) and presence (B) of fibrinogen (4 mg/ml). The concentration of both the thrombolytic agents and hirudin is 75 nM. A set of representative results of three independent experiments is presented here.

 

Effects of Clot Targeting and Antithrombotic Activities of HE-SAKK on Fibrin Clot Lysis—In 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 SAK—To 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, {alpha}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 1–2 µM and the {alpha}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 {alpha}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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  		FIG. 9.
Plasma-clot lysis mediated by various thrombolytic agents. Stability of the plasma clot (closed diamond) was monitored with the addition of plasma in the absence of any externally added thrombolytic agents. Other plasma clots are lysed in the presence of 7,200 nM SAK alone (open triangle), 600 nM HE-SAKK (open circle), or 1,200 nM SAK with 600 nM hirudin (closed square). A set of representative results of three independent experiments is presented here.

 

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.


   FOOTNOTES
 
* This work was supported by research grants from the Heart and Stroke Foundation of Canada (Alberta) and Natural Sciences and Engineering Research Council of Canada (NSERC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Current address: SemBioSys Genetics, Calgary, Alberta T1Y 7L3, Canada. Back

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. Back

2 S. Szarka and S.-L. Wong, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Larry Linton for the statistical analysis of the data, and Sau-Ching Wu and Chyi-Liang Chen for the supply of purified staphylokinase and staphylokinase-specific polyclonal antibodies, respectively.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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