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(Circulation. 2000;102:1766.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Polyethylene Glycol–Derivatized Cysteine-Substitution Variants of Recombinant Staphylokinase for Single-Bolus Treatment of Acute Myocardial Infarction
From the Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Belgium (D.C., E.D., H.M., M.D.M., L.J.,Y.L.); and the Department of Cardiology, UZ Gasthuisberg (P.S., F.V.d.W.).
Correspondence to D. Collen, MD, PhD, Center for Transgene Technology and Gene Therapy, University of Leuven, Campus Gasthuisberg O & N, Herestraat 49, B-3000 Leuven, Belgium. E-mail desire.collen{at}med.kuleuven.ac.be
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background—Thrombolytic therapy of acute myocardial infarction (AMI) is evolving toward bolus administration. Derivatization of proteins with polyethylene glycol (PEG) may reduce their clearance.
Methods and Results—A staphylokinase (SakSTAR) variant with 12
amino acid substitutions to reduce its antigenicity, SakSTAR (K35A,
E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R),
and with Ser in position 3 mutated into Cys (code SY161), was
derivatized with maleimide-PEG with Mr of
5000 (P5), 10 000 (P10), or 20 000 (P20). The PEGylated variants
recognized only one third of the antibodies elicited with wild-type
SakSTAR in AMI patients. In experimental animals, plasma clearances
were reduced 2.5- to 5-fold with P5, 5- to 20-fold with P10, and
20-fold with P20, and bolus injection induced pulmonary plasma
clot lysis at doses inversely related to their clearance.
Intravenous bolus injection of 5 mg of the P5, P10, or P20
variants in AMI patients was associated with plasma half-lives
(t1/2
) of 13, 30, and 120 minutes and clearances of 75,
43, and 8 mL/min, respectively, compared with 3 minutes and 360 mL/min
for SakSTAR. Injection of 5 mg P5 variant restored TIMI-3 flow within
60 minutes in 14 of 18 AMI patients (78%, 95% CI 55% to 91%) and of
2.5 mg in 7 of 11 patients (63%, 95% CI 35% to 85%), both in the
absence of fibrinogen degradation. The immunogenicity of the variants
was significantly (P<0.002) reduced.
Conclusions—The staphylokinase variant SY161-P5, derivatized with one linear polyethylene glycol molecule of Mr 5000, is a promising fibrin-selective agent for single-bolus coronary thrombolysis.
Key Words: myocardial infarction • thrombolysis • fibrinogen
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Thrombolysis has become routine therapy for acute myocardial infarction (AMI), given worldwide to >750 000 patients per year.1 Recent efforts to improve treatment have focussed on derivatives of tissue-type plasminogen activator (t-PA) that can be administered by bolus injection, including reteplase,2 a domain deletion variant given as a double bolus, and TNK-tPA,3 generated by site-directed mutagenesis, given as a single bolus.
Staphylokinase (SakSTAR), a 136–amino acid profibrinolytic bacterial protein, is at least equipotent to t-PA for coronary artery recanalization, significantly more fibrin-selective and obtainable in high yield by cytosolic expression in Escherichia coli.4 However, it has a relatively short plasma half-life,5 and its administration induces neutralizing antibody formation in a majority of patients.6 7
A comprehensive analysis of staphylokinase variants by sequential additive site directed mutagenesis identified several variants with intact specific activities and fibrin-selective thrombolytic potency, which recognized only one third of the antibodies elicited by treatment with wild-type staphylokinase.8 SakSTAR (K35A, E65Q, K74R, D82A, S84A, T90A, E99D, T101S, E108A, K109A, K130T, K135R) (code SY155) with a thrombolytic potency comparable to that of wild-type SakSTAR in patients with peripheral arterial occlusion, was selected for further clinical development. Variants of wild-type SakSTAR are identified by the substituted amino acids in single letter symbols followed by their position number in the mature staphylokinase sequence (136 amino acids) and by the substituting amino acids in single-letter symbols. However, this component was cleared from plasma at a rate similar to that of wild-type SakSTAR.
The plasma clearance and the immunogenicity of heterologous proteins may be reduced by derivatization with polyethylene glycol (PEG).9 10 Usually, PEG molecules are covalently attached to proteins by lysine residues. Staphylokinase contains as many as 20 lysine residues, some of which are essential for its activity, but it does not contain cysteine. Therefore, PEGylated SakSTAR was produced by site-directed substitution of selected amino acids with cysteine and derivatization with linear PEG molecules containing thiol-specific functional groups, yielding homogeneous end products with reduced clearances.11
In the present study, Ser in position 3 of staphylokinase was mutagenized to Cys, which was specifically PEGylated with maleimide-PEG with Mr 5000 to 20 000. In vitro and in vivo evaluation indicated that monoPEGylation prolongs the circulatory half-life of staphylokinase while maintaining its thrombolytic potency when bolus injected at a reduced dose in patients with AMI.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Reagents and Methods
The template vector for mutagenesis, pMEX602sakB (ie, pMEX.SakSTAR), and the recombinant DNA reagents and methods used have been described elsewhere.8 11 12
Construction of Expression Plasmids
The variant SakSTAR(S3C, K35A, E65Q, K74R, E80A, D82A, T90A,
E99D, T101S, E108A, K109A, K130T, K135R, (code SY161) was constructed
by PCR, using as template the previously described8 pMEX-derivative encoding SY155. To introduce the cysteine in
position 3, the fragments were amplified by cycling 30 times (5 seconds
at 94°C, 5 seconds at 50°C, 10 seconds at 72°C) with the
mutagenic primer LY459,
5'-CCTCATATGTCAAG-TTGTTTCGACAAAGGA (the modified bases
are underlined) and the external 3' end primer 819D,
5'-CAGGCTGAAAATCTTC-TCTCATCCGCC. The products were purified,
digested with HindIII, and cloned into the
StuI-HindIII vector fragment of pMEX-STAR.
Variant SY160 was constructed, which was identical to SY161 except for
a Gln instead of an Arg substitution in position 74, starting from the
pMEX-derivative encoding SY151.10
Expression and Preparative Purification of SakSTAR
Variants
Eighteen-liter cultures (in 1- to 2-L batches) of SY161 and
SY160 were grown for 20 hours in terrific broth medium13
supplemented with 100 µg/mL ampicillin and induced with 200
µmol/L IPTG during the last 3 hours. The cells were pelleted,
resuspended in 1/10 volume of 0.01 mol/L phosphate buffer, pH 6.0,
disrupted by sonication, and cleared by
centrifugation.
The compounds were purified on a 10x7 cm column of SP-Sepharose, equilibrated with 0.01 mol/L phosphate buffer, pH between 5.5 and 6.0, and eluted with a 1 mol/L NaCl gradient (3 column volumes). Fractions containing SakSTAR were pooled, solid NaCl was added to 2.5 mol/L, and the material was applied to a 10x20 cm column of phenyl-Sepharose, followed by stepwise elution with 0.01 mol/L phosphate buffer, pH between 5.5 and 6.0. After desalting on a 10x45 cm column of Sephadex G25, concentration on a 5x10 cm column of SP-Sepharose by elution with 1.0 mol/L NaCl, and gel filtration on a sterilized 6x60 cm column of Superdex 75 equilibrated with PBS buffer, pH 7.5, SakSTAR-containing fractions were pooled for derivatization with polyethylene glycol.
Chemical Cross-Linking of SakSTAR Cysteine Mutants With
Polyethylene Glycol
The cysteine side chain was targeted with activated
linear polyethylene glycol molecules with a
Mr of 5 kDa, 10 kDa, or 20 kDa. Mal-PEG
(Shearwater Polymers Europe) carries a maleimide group that reacts
specifically with thiol groups under mild conditions. Derivatization
was achieved by reduction with dithiothreitol, desalting on Sephadex
G25, and incubating the molecule (100 µmol/L) with 3x Mal-PEG
in 10 mmol/L phosphate buffer, pH 7.9, at room temperature. After
reaction for 60 minutes, the mixture was desalted on Sephadex G25,
concentrated on SP-Sephadex, and gel-filtered on Superdex G75. The
PEGylated SakSTAR variant containing fractions were pooled and adjusted
to 1 mg/mL, and the material was sterilized by filtration through a
0.22-µm Millipore filter.
Analytical Methods
The methodology for in vitro evaluation, pharmacokinetics, and
thrombolytic potency in hamsters has been described in
detail elsewhere.5 14 15 16
Thrombolytic and Immunogenic Properties of PEGylated
SakSTAR Variants in Patients
Patients were studied after giving informed consent, and the
protocol was approved by the Human Studies Committee of the University
of Leuven, Belgium. Thirty-five patients with AMI (age 
75 years),
examined within 6 hours and with ST-segment elevation (
1 mm in
at least 2 limb or 2 mm in at least 2 precordial leads) were
included. Exclusion criteria were as previously
described.5 6 7
SY161 carrying a single molecule of P5, P10, or P20 was given as a 5-mg IV bolus over a period of 3 minutes, in groups of 3 patients each (9 patients total). Plasma samples were drawn for pharmacokinetic analysis at 1, 10, 20, 30, 45, 60, 90, 120, 180, and 240 minutes, and Sak-related antigen was determined by ELISA.15 Thereafter, an additional 9 patients were treated with SY161-P5 and 6 patients with SY160-P5. Finally, 11 patients were treated with an intravenous bolus of 2.5 mg of the P5 variants.
After 60 minutes (and secondarily at 24 hours), a coronary angiogram was obtained to determine patency of the infarct-related artery, graded as described by the Thrombolysis in Myocardial Infarction (TIMI) study group, which constituted the primary end point. PTCA at the operator’s discretion was allowed after the angiogram for the primary end point. Aspirin (160 mg or 320 mg) was given on admission and daily thereafter. Heparin (Novo Nordisk) was administered from entry as a 5000-IU bolus followed by a 1000-IU/h infusion, adjusted at 6-hour intervals to maintain the aPTT within 55 to 75 seconds. Plasma samples were collected immediately before and at different time points after injection of PEG-Sak and analyzed as described elsewhere.15
Absorption of Antibodies Elicited With PEGylated SakSTAR
Variants
Plasma samples obtained 2 to 4 weeks after treatment with
staphylokinase-neutralizing activity >5 µg/mL were used and compared
with a plasma pool of 40 patients with AMI, obtained after treatment
with SakSTAR.
The plasma samples were serially diluted (50- to 400-fold) for the construction of individual calibration curves for antibody binding to each of the SakSTAR variants, absorbed for 10 minutes with 250 nmol/L of the SakSTAR variants, and residual binding to immobilized SakSTAR or SakSTAR variants was determined by biospecific interaction analysis as described elsewhere.15 Residual binding was expressed in percent of unabsorbed plasma, by use of the individual calibration curves.
Statistical Analysis
Data are expressed as mean values±SEM or as a median (15 to 85
percentile ranges). Significance levels were determined by paired or
unpaired Student’s t test or by Mann-Whitney rank sum test,
as appropriate. Fisher’s exact test was carried out when indicated in
the text. Two-tailed P<0.05 was considered to indicate
statistical significance. Confidence intervals were determined by the
Statexact software (Version 3.01, Cytel Software Corp).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Characterization of PEGylated SakSTAR Variants
The specific activities of the PEGylated variants purified to homogeneity from culture volumes of 18 L (Table 1
) were at least as high as that
of wild-type SakSTAR. The endotoxin content was <1 EU/mg. SDS gel
electrophoresis of a 10-µg sample revealed single main components
with apparent Mr of 30 000, 36 000, and
50 000 for P5, P10, and P20 derivatives, respectively (Figure 1). In addition, small amounts were
observed of unPEGylated monomers (Mr
16 000).
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The preparations were sterile on 3-day testing and did not provoke acute reaction, nor reduced weight, after intravenous bolus injection of 3 mg/kg body wt in mice (not shown).
Biological Properties of PEGylated SakSTAR Variants
Dose- and time-dependent lysis of
125I-fibrin–labeled human plasma clots submerged
in human plasma were obtained with equieffective concentrations
(causing 50% clot lysis in 2 hours; C50) ranging
from 0.19 to 0.39 µg/mL for the PEGylated SakSTAR variants, as
compared with a C50 value of 0.23 µg/mL for
wild-type SakSTAR (Table 1
). At these concentrations, no
significant fibrinogen degradation occurred (not shown).
PEGylated compounds remained fully active for up to 5 days at 37°C.
SDS-PAGE after 3 days at 37°C revealed that the covalent bonds
linking the maleimide-PEG molecule(s) to the cysteine in the
staphylokinase moiety remained intact (data not shown).
Pharmacokinetic Properties in Hamsters and Rabbits and in Patients
With AMI
The disposition of staphylokinase-related antigen from blood after
bolus injection of 100 µg/kg of the SakSTAR variants in hamsters or
rabbits occurred with initial plasma half-lives
(t1/2) and plasma clearances (Clp), summarized
in Table 2. The disposition rate of
staphylokinase-related antigen from blood after bolus injection of 5 mg
of the PEGylated SakSTAR variants in patients with AMI yielded initial
plasma half-lives (t1/2) and the plasma
clearances (Clp) summarized in Table 2.
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The clearances of PEGylated variants were inversely proportional to the molecular weight of the PEG molecules, with an average reduction of 2.5- to 5-fold with P5, 5- to 20-fold with P10, and 20-fold with P20, relative to wild-type SakSTAR.
Thrombolytic Properties of PEGylated SakSTAR Variants
in Hamster Pulmonary Embolism Model
SakSTAR and all PEGylated variants induced dose-related clot lysis
in a hamster pulmonary embolism model (Figure 2). SY161-P5 had a 2.5-fold lower
C50 value than wild-type SakSTAR, SY161-P10 had a
3-fold lower C50 value, and SY161-P20 had a
5-fold lower C50 value. Fibrinogen and
2-antiplasmin levels did not decrease after
bolus administration of any of the agents (not shown). The
thrombolytic potency of SY160-P5 was indistinguishable
from that of SY161-P5 (not shown).
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, SY155; 
, SY161-P5; •, SY161-P10; 
, SY161-P20
Thrombolytic and Immunogenic Properties in Patients
With AMI
Table 3 summarizes results of 12
patients treated with 5 mg SY161-P5 and of 6 patients treated with 5 mg
SY160-P5. Fourteen of the 18 patients had TIMI grade 3 flow at 60
minutes that persisted at 90 minutes (78%, 95% CI 55% to 91%).
After the qualifying 60-minute angiogram was obtained, PTCA was
performed in 4 patients with a TIMI flow 2. Of the 14 patients who
had TIMI grade 3 flow at 60 minutes, 13 had a corrected TIMI frame
count (CTFC) of 30 (20±6, mean±SEM), corresponding to a normal
flow, whereas the other patient had a CTFC of 46, which corresponds to
TIMI grade 2 flow. The mean percent stenosis in this group was
65±5 (mean±SEM) at 60 minutes and 58±5 at 24 hours
(P=0.09). Eight of these patients underwent PTCA or stent
placement after 24 hours for severe stenosis. Ejection
fraction, obtained echocardiographically during
hospital stay, was 64±12 for all patients (68±10 versus 57±13 for
patients with TIMI 3 or TIMI 2 flow, respectively,
P=0.10), indicating a preserved left ventricular
function. Treatment-related complications consisted of Mallory-Weiss
bleeding in one patient who also received abciximab during PTCA at 60
minutes and a large puncture site hematoma in another patient requiring
blood transfusion. No hemodynamic problems or allergic
reactions were observed.
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Table 3 also summarizes results of an additional 11 patients
treated with an intravenous bolus of 2.5 mg of the P5
variants, resulting in TIMI grade 3 flow at 60 minutes in 7 patients
(63%, 95% CI 35% to 85%). PTCA was performed at 60 minutes in the
patients with TIMI flow 0 and in 3 patients at 24 hours because of
severe stenosis.
Table 4 summarizes results of hemostasis
analyses in the patients given a 5-mg or a 2.5-mg bolus of
SY161-P5 or SY160-P5, as compared with a group of 12 patients given 30
mg SakSTAR over 30 minutes. Fibrinogen, plasminogen, and
2-antiplasmin at 240 minutes remained
essentially unchanged, reflecting the high fibrin-selectivity of these
agents. Median D-dimer levels increased from 190 ng/mL at baseline to
4300 and 3200 ng/mL respectively, indicating significant in vivo fibrin
digestion. Antibody-related SY160-P5– or SY161-P5–neutralizing
activity were low at baseline but thereafter increased to reach median
values at 3 to 4 weeks of 11 µg SakSTAR variant neutralized per
milliliter of plasma in patients treated with 5 mg (Table 4), as
compared with 21 µg SakSTAR neutralized per milliliter in 10 patients
treated with a 30-minute infusion of 30 mg wild-type SakSTAR
(P=0.002). With 2.5 mg of PEGylated variant, neutralizing
activity increased to a median value of 3 µg SakSTAR variant
neutralized per milliliter of plasma (P=0.0013 versus
wild-type SakSTAR) (Table 4).
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Absorption With SakSTAR Variants of Antibodies elicited in patients
by treatment with SakSTAR variants
Overt immunization (neutralizing activity at 3 to 4 weeks of 5
µg compound per mL plasma) was observed in 10 of 15 patients treated
with 5 mg and in 2 of 7 patients treated with 2.5 mg PEGylated SakSTAR
variants (Table 4). Wild-type SakSTAR absorbed >95% of the
antibodies binding to insolubilized SakSTAR, SY151, or SY155,
respectively, irrespective of the compound used for treatment,
indicating that no neoantigens were introduced by the substitution
mutagenesis. SakSTAR, SY151, and SY155, respectively, absorbed in
excess of 95% of the antibodies binding to insolubilized SakSTAR,
SY151, or SY155, respectively, from plasma of patients treated with
SakSTAR, SY160-P5, or SY161-P5, respectively (data not shown), which
supports the validity of the absorption method used. From pooled plasma
of 40 patients treated with SakSTAR, SY155 absorbed 36%, and SY151
bound 32%, which confirms the progressive elimination of B-lymphocyte
epitopes by the sequential additive mutagenesis.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study reports on the preclinical and initial clinical evaluation of polyethylene glycol-derivatized (PEGylated) variants of recombinant staphylokinase for single-bolus treatment of AMI. Introduction of cysteine in the SakSTAR molecule and derivatization with thiol-directed PEG derivatives allowed specific derivatization yielding homogeneous derivatives, whereas substitution in the NH2-terminal region, which is removed during processing of SakSTAR, yielded derivatives with high thrombolytic potency.
The selected cysteine-substitution variant SY161 that was derivatized with maleimide-PEG (Mal-PEG) with molecular weights of 5000 (P5), 10 000 (P10), or 20 000 (P20) was produced in sufficiently large amounts and conditioned for the evaluation of their pharmacokinetics and thrombolytic potencies in hamsters and of their thrombolytic potency/immunogenicity ratios in patients with AMI. PEGylated SY161 had an intact apparent specific activity, maintained fibrinolytic potency and fibrin-selectivity in a human plasma milieu, and had a markedly reduced reactivity with anti-SakSTAR antibodies in pooled immunized patient plasma. The clearances of the PEGylated SY161 derivatives in hamsters, rabbits, and in patients with AMI were reduced inversely proportionally to the molecular weight of the PEG molecules.
Intravenous administration of SY161-P5 as a 5-mg bolus in 12 patients and of SY160-P5 in 6 patients with AMI resulted in complete recanalization (TIMI grade 3 flow) in 14 patients (78%), and partial recanalization (TIMI grade 2 flow) in 2, without hemodynamic or allergic reactions and without measurable systemic plasminogen activation. A bolus dose of 2.5 mg still produced TIMI grade 3 flow at 60 minutes in 7 of 11 patients with AMI (63%).
After administration of variant SakSTAR, neutralizing antibody titers increased after 3 to 4 weeks to a median level that was significantly lower than after administration of wild-type SakSTAR. The antibodies induced by treatment with the SakSTAR variants were completely absorbed by SakSTAR, indicating that immunization was not due to neo-epitopes generated by the amino acid substitutions but to residual epitopes in the variants. However, although there was a significant reduction in immunogenicity, the residual immunization remained too high to warrant repeated use of the drug.
In summary, on the basis of the present study, SakSTAR (S3C-P5, K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K09A, K130T, K135R) (code SY161-P5) has been selected for clinical development toward fibrin-selective thrombolytic therapy by single intravenous bolus injection in patients with AMI.
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Acknowledgments |
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This study was supported in part by a sponsored research agreement between the Flanders Interuniversity Institute for Biotechnology (VIB) and Thromb-X NV, a spin-off company of the University of Leuven, in which Dr Collen has an equity interest and for which Dr Collen serves as a consultant. Dr De Maeyer is an employee of Thromb-X NV. Dr Sinnaeve is the recipient of a fellowship of the Fund for Scientific Research of Flanders. The authors thank Frans De Cock and Berthe Van Hoef for skillful technical assistance and Laura De Velder, research nurse at the CCU of the UZ Gasthuisberg Hospital.
Received April 11, 2000; revision received May 11, 2000; accepted May 11, 2000.
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