(Circulation. 2000;101:1158.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa
Presented in part at the annual meeting of the American College of Cardiology, New Orleans, La, March 7–10, 1999, and published in abstract form (J Am Coll Cardiol. 1999;33:2A).
From the Department of Cardiology, University of Freiburg (K.P., C.B.), Freiburg, Germany; Department of Cardiology, University of Heidelberg (J.G., W.K.), Heidelberg, Germany; Sealy Center for Molecular Cardiology, University of Texas (M.S.R.), Galveston, Tex; and the German Cancer Research Center (S.K., M.Z.-W., M.L.), Heidelberg, Germany.
Correspondence to Dr Karlheinz Peter, Internal Medicine III, University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany. E-mail peter{at}mm31.ukl.uni-freiburg.de
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Abstract |
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Background—Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot.
Methods and Results—The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly4Ser)3 was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the Bß15-22 peptide of fibrin (ß-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv59D8 and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to ß-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin.
Conclusions—A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.
Key Words: anticoagulants • antibodies • thrombosis • molecular biology
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Introduction |
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Inhibition of thrombin by either the indirect thrombin inhibitor heparin or direct thrombin inhibitors such as hirudin reduces thrombus formation after arterial injury in animal models1 and in humans with unstable coronary syndromes.2 3 Furthermore, thrombin inhibitors potentiate fibrinolysis induced by plasminogen activators.4 Several animal experiments demonstrated that hirudin is more effective than heparin in preventing platelet-dependent arterial thrombosis, rethrombosis after reperfusion, and thrombus growth.4 5 6 7 However, clinical trials with direct thrombin inhibitors have only been partially successful.8 High concentrations of hirudin were very effective in inhibiting thrombin but are associated with frequent hemorrhagic complications.4 A strategy for circumventing this problem is the targeting of hirudin to fibrin.
Fibrin targeting can be achieved with the monoclonal antibody (mAb) 59D8, which selectively binds to the amino-terminus of the fibrin ß-chain that becomes exposed after cleavage of fibrinopeptide B by thrombin.9 Because exposure of this epitope is an early event in the conversion of fibrinogen to fibrin, it is likely that mAb 59D8 accumulates at sites of high thrombin activity, such as a developing arterial clot.9 Coupling of mAb 59D8 to plasminogen activators resulted in enhanced thrombolytic potency and specificity in vitro and in vivo.10 11 A chemical conjugate between hirudin and 59D8 effectively inhibited fibrin deposition on experimental clots12 and demonstrated potent antithrombotic activity in nonhuman primates.13 Nevertheless, chemical coupling of hirudin to mAbs has several limitations, the major ones being low yield and loss of hirudin activity.12 We tried to bypass these limitations by the use of recombinant technology.
Because hirudin needs a free amino- as well as a free carboxy-terminus for antithrombin activity,14 a direct fusion at the termini of hirudin was expected to result in a functional loss. Therefore, a factor Xa cleavage site was introduced between mAb 59D8 and hirudin. This cleavage site was chosen for 2 reasons. First, factor Xa cleaves at the C-terminus of its recognition sequence (Ile-Glu-Gly-Arg) and thus liberates the free amino-terminus of hirudin. Second, factor Xa is a major part of the activated coagulation system at the site of arterial clots15 and may therefore allow a preferential liberation of functional hirudin at the clot. Without an activated coagulation system, the fusion protein would be inert. However, as a clot develops, the combination of fibrin targeting and dependence on cleavage by factor Xa could result in an effective thrombin inhibition at the clot without systemic anticoagulation.
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Methods |
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Materials and Cells
Horseradish peroxidase (HRP)–conjugated sheep anti-M13 mAb and mouse anti-E-tag mAb were obtained from Pharmacia, mouse anti-c-myc mAb from Cambridge Research Biochemicals, and goat anti-mouse HRP-conjugated polyclonal antibody (Ab) from Dianova. Bß15-22, also termed ß-peptide, with the amino acid sequence Gly-His-Arg-Pro-Leu-Asp-Lys, was purchased from MWG Biotech. Hirudin was a gift of Knoll AG (Ludwigshafen, Germany). The hybridoma secreting the fibrin-specific mAb 59D8 was generated as described previously,9 and cells were grown on DMEM, 10% fetal calf serum, 2 mmol/L L-glutamine, penicillin (10 IU/mL), and streptomycin (10 µg/mL) (all from Gibco) with 5% CO2 at 37°C.
Construction of a Functional Single-Chain Antibody
cDNA of 59D8 hybridoma cells was prepared with mRNA purification
columns (oligo-dT) and M-MuLV (both from Pharmacia). Amplification of
the antibody variable regions and the insertion of the linker
sequence were achieved by polymerase chain reaction (PCR). Primer mixes
that contained sequences from conserved regions of the variable
regions of the heavy (VH) and light
(VL) chains were obtained from Pharmacia. The
linker sequence (Gly4Ser)3
was inserted by the addition of a linker fragment.
Clone Selection With the M13 Phage System
The PCR products encoding the functional single-chain
antibody fragments (scFv) were cloned into the vector pCANTAB5E
(Pharmacia). In this vector, an amber stop codon allows expression of
soluble scFv in the nonsuppressor Escherichia coli strain
HB2151 and display of scFv on the M13 phage surface by fusion to the
pIII adsorption protein in the suppressor strain TG1. The supernatant
of TG1 clones was used for the following panning procedure: A tissue
culture flask with a surface area of 25 cm2 was
coated with 50 µg of ß-peptide at 4°C overnight, washed 5 times
with PBS, and blocked for 2 hours at 37°C with 2% nonfat dry milk in
PBS. The phage-containing supernatant was added and incubated for 2
hours at 37°C. Nonadhering phages were removed by washing 20 times
with PBS. A TG1 culture was added to the flask for reinfection with
bound phages and incubated for 1 hour at 37°C at 250 rpm. This
panning procedure was repeated 9 times. Positive clones were tested for
phage binding on immobilized ß-peptide by use
of an HRP-conjugated anti-M13 sheep mAb. The best binding clones with
the expected fragment size (
750 bp) were used to transform HB2151.
Periplasmic extracts from the individual clones were analyzed
for binding to immobilized ß-peptide by an anti-E-tag
mAb.
Cloning of scFv59D8 Into the Expression Vector pHOG21,
Fusion With the Factor Xa Recognition and Hirudin Sequences, and
Transfer to pOPE51
DNA of scFv clone 33 was cloned into pHOG2116 ,
mutated at position 6 to glutamine,17 and cloned into
pOPE5118 (Figure 2
). DNA coding for hirudin
(Biermann) was used as a template for PCR with the sense primer
CAGCAAGATCTAAACTCAAGCGGC-ATCGAAGGTCGTGTTGT-TTACACCGACTGTACTG
and the antisense primer
AGATGATCTAGAGGATCCTTACTGCAGAT-ATTCTTCTGGG. The factor Xa
recognition sequence (bold) and the restriction site BglII
(underlined) are encoded by the sense primer and the restriction site
XbaI (underlined) by the antisense primer. The ligation
products were transformed into XL1-blue.
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Preparation of scFv From Inclusion Bodies
From overnight cultures of XL1-blue, 250 µL was
transferred to 5 mL of LB medium containing 100 µg/mL ampicillin and
100 mmol/L glucose and incubated at 37°C and 280 rpm until an
OD600 nm of 0.8 was reached. Protein expression
was induced by addition of
isopropyl-ß-D-thiogalactopyranoside (IPTG; 20
µmol/L) and cultured at room temperature for 4 hours. Cells were then
centrifuged (6000 rpm, 15 minutes) and resuspended in 165 µL
of ice-cold buffer (50 mmol/L Tris-HCl, 100 mmol/L NaCl,
1 mmol/L EDTA, pH 7.0). After freezing and thawing, the sample was
centrifuged (12 000g, 4°C, 30 minutes),
resuspended in 500 µL of ice-cold TE buffer (10 mmol/L Tris-HCl,
1 mmol/L EDTA, pH 7.4), and incubated for 1 hour at room
temperature. Lysozyme (Boehringer Mannheim) was then added to a
final concentration of 200 µg/mL, and the incubation was continued
for 1 hour, followed by the addition of NaCl (0.5 mol/L) and
Triton-X-100 (2.5%) and a final incubation for 1 hour. After
centrifugation (12 000g, 4°C, 1 hour),
the pellet was washed twice with 3 mol/L urea, and 50 mmol/L
Tris-HCl (pH 7.0) and finally solubilized by rotation overnight at
4°C in 250 µL of 6 mol/L GdHCl, 100 mmol/L Tris-HCl, pH 7.0.
After centrifugation (12 000g, 4°C, 1
hour), the supernatant was dialyzed against TA buffer (0.4 mol/L
arginine-HCl in 100 mmol/L Tris-HCl, pH 7.0).
ELISA With Immobilized ß-Peptide
Microtiter plates were coated with 1 µg of ß-peptide or the
control peptide GRGDSP in 100 µL of 0.05 mol/L
Na2CO3 (pH 9.6) overnight
at 4°C. The plate was then washed 4 times with PBS and blocked with
blocking buffer (2% nonfat dry milk in PBS) for 2 hours at room
temperature. Samples (100 µL) were incubated for 2 hours at room
temperature. After the plate was washed 5 times with PBS, 100 µL of
mAb solution (either anti-M13 mAb, anti-c-myc mAb [both 1 to 5000
diluted], or 1 µg/mL anti-E-tag mAb) in blocking buffer was added
and incubated for 2 hours at room temperature. Either ABTS (Sigma) or
TMB solution (Biorad) was used, and samples were measured at
wavelengths of 405 or 655 nm, respectively.
Purification by Immobilized Metal Affinity
Chromatography and Ionic Exchange
Chromatography of scFv59D8 Expressed in the
pHOG21 Vector
A 10-mL column of chelating sepharose (Pharmacia) was
equilibrated with 0.1 mol/L CuSO4. The samples
were loaded in 50 mmol/L Tris-HCl, 1 mol/L NaCl, pH 7.0. After the
column was washed with 200 mL of 50 mmol/L Tris-HCl, 1 mol/L NaCl,
50 mmol/L imidazole, pH 7.0, the bound scFv construct was eluted
with 40 mL of 50 mmol/L Tris-HCl, 1 mol/L NaCl, 250 mmol/L
imidazole, pH 7.0. The eluate was dialyzed against PBS buffer and
further purified by ionic exchange chromatography on a
MonoS column (Pharmacia) in 50 mmol/L MES buffer (pH 6.5) with a 0
to 1 mol/L NaCl gradient.
Affinity Chromatography of scFv and Factor Xa
Cleavage
Coupling of ß-peptide to sepharose was performed as described
previously.10 Columns containing ß-peptide–conjugated
sepharose were loaded and washed with TA buffer. Bound protein was
eluted by 0.1 mol/L glycine, pH 2.8, and 1-mL fractions were collected
and adjusted to pH 7.0 with 0.5 mol/L Tris buffer. Fractions containing
significant amounts of product were pooled and dialyzed against TA
buffer. For factor Xa cleavage, typically 150 µg of
scFv59D8-Xa-hirudin (2 µg/µL) was cleaved by
15 µg of factor Xa (1 µg/µL, Boehringer Mannheim) for
various times in 50 mmol/L Tris-HCl, 100 mmol/L NaCl, and
1 mmol/L CaCl2 (pH 8.0) at room
temperature.
Measurement of Thrombin Inhibition by
scFv59D8-Xa-Hirudin After Factor Xa Cleavage
Inhibition of thrombin was determined by cleavage of the
chromogenic substrate S-2238 (Chromogenix). After factor Xa
(0.1 µg/µL) cleavage (5 hours, room temperature), 20 µL of
thrombin solution (human thrombin, 2.5 U/mL; Sigma) was added to 100
µL of sample in assay buffer (20 mmol/L sodium dihydrogen
carbonate, 0.15 mol/L NaCl, and 0.1% bovine serum albumin, pH
7.4) and incubated at room temperature for 10 minutes. S-2238 (50 µL,
0.833 mg/mL) was then added, and after 10 minutes of incubation, the
reaction was stopped by the addition of 50 µL of 20% acetic acid.
Absorbance was measured at 405 nm.
Whole Blood Clot Assay
Except for minor modifications, clot assays were performed as
described previously.12 Clots were initiated by the
addition of CaCl2 (16.6 mmol/L) and 2.5
vol% of Actin7 FS-activated PTT reagent (Dade
International) to anticoagulated blood (citric acid, 11 mmol/L)
from healthy volunteers. Whole blood was immediately drawn into a
silicone tubing (4-mm inner diameter), and clots were allowed to form
at 37°C for 1 hour. Quantification of clot size was performed by
labeling of blood with 125I-fibrinogen (Amersham)
to a final activity of 37 500 cpm/mL. The silicon tubing was cut into
1.5-cm fragments, and the formed clots were extruded and washed 5 times
in TA buffer. In each assay, the starting size of clots chosen for
further experiments varied not more than ±5%. Appositional clot
growth was evaluated by the incubation of clots for 10 hours at 37°C
on a rotator (60 rpm). Clots were incubated in recalcified citrated
whole blood (trace labeled with 125I-fibrinogen
at a final activity of 112 500 cpm/clot) either with the addition of
native hirudin or scFv59D8-Xa-hirudin or without
addition. The clots were then washed 10 times with TA buffer, and
inhibition of appositional clot growth was evaluated on a
-counter.
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Results |
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To construct a single-chain antibody directed against fibrin (scFv), mRNA was prepared from 5x107 59D8 hybridoma cells and reverse transcribed with an oligo-dT primer. The variable regions of the heavy and light chains (VH and VL, respectively) were amplified by polymerase chain reaction (PCR) with primers that anneal to conserved regions at the 5'- and 3'-ends of the variable regions. PCR products of 348 and 339 bp were obtained for VH and VL, respectively (Figure 1
). After addition of a
(Gly4Ser)3 linker by fusion
PCR, the scFv product (Figure 1) was cloned into pCANTAB5E
(Figure 2
) for phage display of the scFv
clones. After 10 rounds of panning on immobilized
ß-peptide, 144 phage clones were tested for binding to the
ß-peptide by phage ELISA with a horseradish peroxidase
(HRP)–conjugated mAb against M13 and by DNA restriction
analysis. Twenty-four clones with strong binding to ß-peptide
and the expected size of the insert were used for transformation of
HB2151. The binding properties of soluble scFvs secreted into the
supernatant were compared by ELISA with a mouse anti-E-tag mAb and a
secondary HRP-conjugated goat anti-mouse antibody (Ab) (Figure 3). Clone 33 demonstrated the best
binding properties (Figure 3) and was therefore chosen for
sequencing and further characterization. Complementarity determining
regions and framework regions of both variable regions and the
linker region are highlighted in Figure 4.
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For enhanced expression and purification of soluble
scFv59D8, clone 33 was transferred to the
expression vector pHOG21 (Figure 2). This plasmid contains a tag
sequence coding for 6 histidine residues at the scFv C-terminus, thus
facilitating purification by immobilized metal affinity
chromatography. However, an additional purification
step with ion-exchange chromatography was necessary to
obtain a pure product (Figure 5A). An
analysis of several eluted fractions by SDS-PAGE is shown in
Figure 5B. The functional integrity of the highly purified
scFv59D8 was tested by binding on
immobilized ß-peptide (Figure 5C). The yield of
purified scFv59D8 was 0.2 mg from 1 L of
bacterial culture.
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To further increase the yield, glutamic acid at position 6 of the
heavy chain was mutated by PCR to glutamine, because this substitution
has been shown to give increased yields of scFvs.17
Indeed, the yield of functional soluble scFv59D8
was increased 4 times by this single amino acid substitution.
The factor Xa recognition sequence and the hirudin sequences were fused to the scFv59D8 by PCR. However, only a low yield of soluble fusion protein was obtained with the pHOG21 expression vector. This was probably due to the high cysteine content (10%) of hirudin that might interfere with the folding process of soluble scFv. To obtain higher levels of the scFv59D8-Xa-hirudin fusion protein, we chose the expression vector pOPE51,17 which facilitates the production of large amounts of fusion proteins as inclusion bodies in the periplasmic space. When this expression system was used, up to 10 mg of highly purified scFv59D8-Xa-hirudin could be obtained from a bacterial culture of 5 L.
The fusion protein scFv59D8-Xa-hirudin was
analyzed by SDS-PAGE and tested for its binding to
ß-peptide and its susceptibility to factor Xa cleavage.
The molecular weight of the intact fusion protein
scFv59D8-Xa-hirudin was 36 kDa, that of the
cleavage product scFv59D8-Xa was 29 kDa, and
that of hirudin was 7 kDa (Figure 6).
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We evaluated the functional characteristics of
scFv59D8-Xa-hirudin by measuring its binding to
ß-peptide and by determining its antithrombin activity
after binding to ß-peptide. Binding to
ß-peptide was comparable to the binding of equimolar
amounts of the Fab' fragment of the original mAb 59D8 as measured in
ELISA (Figure 7A). The antithrombin
activity of the scFv59D8-Xa-hirudin was
determined in the presence and absence of factor Xa.
ScFv59D8-Xa-hirudin was allowed to bind to
ß-peptide, and the nonbound fusion protein was washed
away. The binding function and antithrombin activity of bound
scFv59D8-Xa-hirudin could thus be evaluated
simultaneously. The uncleaved
scFv59D8-Xa-hirudin revealed no antithrombin
activity, whereas scFv59D8-Xa-hirudin in the
presence of factor Xa demonstrated marked antithrombin activity (Figure 7B).
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The ability of the fusion protein to inhibit clot growth was tested in
a whole blood clot assay. Native hirudin and
scFv59D8-Xa-hirudin were directly compared for
their ability to inhibit appositional clot growth.
ScFv59D8-Xa-hirudin was able to inhibit clot
growth significantly better than native hirudin (Figure 8).
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Discussion |
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Fibrin targeting allows for local enrichment of fibrinolytic agents at the site of the thrombus at low systemic concentrations and thus represents a strategy to increase fibrinolytic potency.10 11 Furthermore, a chemical conjugate of the fibrin-specific mAb 59D8 and the direct thrombin inhibitor hirudin inhibited fibrin deposition on experimental clots12 and demonstrated an increase in antithrombotic potency in baboons.13 To increase the yield and activity of antibody-targeted hirudin and to further improve the risk/benefit ratio of anticoagulation, we have developed a recombinant fusion molecule consisting of an antifibrin single-chain antibody and hirudin. In addition to fibrin targeting, the generation of a free N-terminus, which is essential for the antithrombin activity of hirudin, forms the basis of a unique pharmacological approach. By the addition of the factor Xa recognition sequence, the fusion protein inhibited thrombin only in the presence of factor Xa. Because this factor is part of the activated clotting system and is an important determinant of the procoagulant activity of whole blood clots and arterial thrombi,15 the designed fusion protein represents an anticoagulant that promises to be preferentially active at the site where it is needed.
The potential therapeutic use of single-chain antibody fusion proteins has several major advantages. The variable regions of antibodies comprise the smallest fragments containing a complete antibody binding site, and fusion molecules can be created without loss in binding function of the scFv. Therefore, scFvs are attractive tools for the targeting of drugs, toxins, and radionuclides. The fusion protein scFv59D8-Xa-hirudin with the small molecular size of 36 kDa is expected to be only minimally, if at all, immunogenic, and its small size may improve thrombus accessibility and penetration. It can be produced in bacteria in large amounts, in a short time, and at low cost, and it can be highly purified by affinity chromatography with ß-peptide columns, thus providing an ideal situation for drug preparation on a large scale.
Fibrin is an obvious target to concentrate antithrombotic or fibrinolytic agents at the clot. Sufficient amounts of fibrin are present even in platelet-rich thrombi.13 In addition to mAb 59D8, the mAb MA-15C5, directed against the fragment D-dimer of cross-linked human fibrin, has been used successfully to target plasminogen activators to clots.19
Several reports imply that direct thrombin inhibitors may be superior to heparin.3 4 5 6 7 8 This could be explained by a number of distinct mechanisms. In contrast to heparin, which only inhibits thrombin as a soluble molecule, hirudin can also inhibit thrombin that is bound to the clot or to soluble fibrin degradation products.20 21 Heparin binds to various other partners besides thrombin and is thereby inhibited.22 In contrast to heparin, hirudin has no natural inhibitors.7 Furthermore, hirudin can displace thrombin from platelet thrombin receptors.7 In an experimental study, hirudin but not heparin was even able to dissolve preexisting mural thrombi.7 Nevertheless, the experimental advantages of hirudin compared with heparin have not been reflected by superior clinical performance. Bleeding complications with higher doses of hirudin appear to be the major limitation.4 Fusion proteins, such as the one described, provide a promising new development based on the strategy of targeting to and activation at the existing or developing thrombus. This may result in highly efficient inhibition of thrombin and at the same time in fewer bleeding complications.
In summary, a fusion protein has been developed that combines fibrin targeting and antithrombin activity after activation by factor Xa. This recombinant anticoagulant promises to be active only when and where it is needed, thus providing a pharmacological approach that may facilitate an effective anticoagulation without systemic bleeding complications.
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (SFB 320) and by the Medical Faculty of the University of Heidelberg with grants to Dr Peter, Dr Little, and Dr Bode. The expert technical assistance of Simone Bauer and Armin Keller is gratefully acknowledged.
Received July 21, 1999; revision received September 21, 1999; accepted October 7, 1999.
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  C. von zur Muhlen, D. von Elverfeldt, J.A. Moeller, R.P. Choudhury, D. Paul, C.E. Hagemeyer, M. Olschewski, A. Becker, I. Neudorfer, N. Bassler, et al. Magnetic Resonance Imaging Contrast Agent Targeted Toward Activated Platelets Allows In Vivo Detection of Thrombosis and Monitoring of Thrombolysis Circulation, July 15, 2008; 118(3): 258 - 267. [Abstract] [Full Text] [PDF]
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 K. Danielyan, B.-S. Ding, C. Gottstein, D. B. Cines, and V. R. Muzykantov Delivery of Anti-Platelet-Endothelial Cell Adhesion Molecule Single-Chain Variable Fragment-Urokinase Fusion Protein to the Cerebral Vasculature Lyses Arterial Clots and Attenuates Postischemic Brain Edema J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 947 - 952. [Abstract] [Full Text] [PDF]
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 P. Stoll, N. Bassler, C. E. Hagemeyer, S. U. Eisenhardt, Y. C. Chen, R. Schmidt, M. Schwarz, I. Ahrens, Y. Katagiri, B. Pannen, et al. Targeting Ligand-Induced Binding Sites on GPIIb/IIIa via Single-Chain Antibody Allows Effective Anticoagulation Without Bleeding Time Prolongation Arterioscler. Thromb. Vasc. Biol., May 1, 2007; 27(5): 1206 - 1212. [Abstract] [Full Text] [PDF]
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 S. Zaitsev, K. Danielyan, J.-C. Murciano, K. Ganguly, T. Krasik, R. P. Taylor, S. Pincus, S. Jones, D. B. Cines, and V. R. Muzykantov Human complement receptor type 1-directed loading of tissue plasminogen activator on circulating erythrocytes for prophylactic fibrinolysis Blood, September 15, 2006; 108(6): 1895 - 1902. [Abstract] [Full Text] [PDF]
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 M. Schwarz, G. Meade, P. Stoll, J. Ylanne, N. Bassler, Y. C. Chen, C. E. Hagemeyer, I. Ahrens, N. Moran, D. Kenny, et al. Conformation-Specific Blockade of the Integrin GPIIb/IIIa: A Novel Antiplatelet Strategy That Selectively Targets Activated Platelets Circ. Res., July 7, 2006; 99(1): 25 - 33. [Abstract] [Full Text] [PDF]
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B.-S. Ding, C. Gottstein, A. Grunow, A. Kuo, K. Ganguly, S. M. Albelda, D. B. Cines, and V. R. Muzykantov Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature Blood, December 15, 2005; 106(13): 4191 - 4198. [Abstract] [Full Text] [PDF]
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