A Bispecific Antifibrin-antiplatelet Urokinase Conjugate (BAAUC) Induces Enhanced Clot Lysis and Inhibits Platelet Aggregation

Ruef, Johannes; Nordt, Thomas K.; Peter, Karlheinz; Runge, Marschall S.; Kübler, W.; Bode, Christoph

doi:10.1055/s-0037-1614638

Cited by 18 publications

(2 citation statements)

References 31 publications

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“…Thrombolytics that targeted fibrin and platelets simultaneously may have enhanced potency and clot specificity. A bispecific antifibrin-antiplatelet urokinase conjugate (BAAUC) was created by coupling urokinase to the monovalent Fab’ from the antifibrin monoclonal antibody 59D8 and the monovalent Fab’ from the anti-glycoprotein GPIIb/IIIa monoclonal antibody 7E3 (16). In vitro , this bispecific drug has the potency to lyse fibrin-rich and platelet-rich thrombi with high efficacy and to effectively inhibit platelet aggregation.…”

Section: Discussionmentioning

confidence: 99%

Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

Dan

Tan

Xia

et al. 2013

Experimental and Therapeutic Medicine

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The aim of this study was to construct a plasmid expressing glycoprotein IIb-IIIa (GPIIb/IIIa) and D-dimer single-chain bispecific antibody for the targeted therapy of thrombosis. The phosphorylated gene encoding the anti-GPIIb/IIIa single-chain variable fragment (scFv) and the gene encoding the anti-D-dimer scFv were amplified by PCR and linked in tandem by blunt-end ligation. The recombinant plasmid was transfected into the competent cell line HB2151 and identified by PCR and DNA sequencing. Then, the soluble recombinant antibody in bacterial lysates was purified by an NTA column and molecular sieve chromatography in turn. Finally, the binding specificity of the purified antibody was tested by enzyme-linked immunosorbent assay (ELISA). Results demonstrated that the construction of the expression plasmid was successful and the purified recombinant protein, which had a molecular weight of ∼56 kDa, was specific to GPIIb/IIIa and D-dimer. In conclusion, a plasmid expressing a bispecific antibody was constructed by a new method of blunt-end ligation. The soluble recombinant protein is a promising platform for target-oriented thrombolytic therapy.

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Section: Discussionmentioning

confidence: 99%

Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

Dan

Tan

Xia

et al. 2013

Experimental and Therapeutic Medicine

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show abstract

“…The in vitro platelet aggregation and clot lysis activity of different urokinase constructs were also compared alone, fused to a full-length IgG, fused only to either one of the monospecific Fab, and finally as a bispecific construct. The bispecific construct displayed the highest activity, inhibited platelet aggregation and was proposed as an antithrombotic therapy (Ruef et al 1999 ).…”

Section: Biomolecular Strategies For Targeting Fibrinmentioning

confidence: 99%

Engineered Molecular Therapeutics Targeting Fibrin and the Coagulation System: a Biophysical Perspective

et al. 2022

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The coagulation cascade represents a sophisticated and highly choreographed series of molecular events taking place in the blood with important clinical implications. One key player in coagulation is fibrinogen, a highly abundant soluble blood protein that is processed by thrombin proteases at wound sites, triggering self-assembly of an insoluble protein hydrogel known as a fibrin clot. By forming the key protein component of blood clots, fibrin acts as a structural biomaterial with biophysical properties well suited to its role inhibiting fluid flow and maintaining hemostasis. Based on its clinical importance, fibrin is being investigated as a potentially valuable molecular target in the development of coagulation therapies. In this topical review, we summarize our current understanding of the coagulation cascade from a molecular, structural and biophysical perspective. We highlight single-molecule studies on proteins involved in blood coagulation and report on the current state of the art in directed evolution and molecular engineering of fibrin-targeted proteins and polymers for modulating coagulation. This biophysical overview will help acclimatize newcomers to the field and catalyze interdisciplinary work in biomolecular engineering toward the development of new therapies targeting fibrin and the coagulation system.

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Plasma thiols redox status by laser‐induced fluorescence capillary electrophoresis

et al. 2004

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High concentrations of total plasma thiols such as cysteine and homocysteine are important risk factors for atherosclerosis and cardiovascular diseases. We have recently described a new laser-induced fluorescence capillary electrophoresis (CE-LIF) method to measure total plasma thiols, in which the baseline separation of cysteinylglycine, homocysteine, cysteine, and glutathione was achieved by adding the organic base N-methyl-D-glucamine to the run buffer. However, because the active fractions of homocysteine and cysteine responsible for vascular injuries are still unknown, research calls for a set up of methods able to analyze different forms of plasma thiols. In this paper, we present an improvement of our previous method that allows the measurement of different thiol forms. Total, reduced, and free thiols were measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulfosalicylic acid. After derivatization with 5-iodoacetamidofluorescein, samples were separated and measured by CE-LIF using a phosphate/borate buffer in the presence of 75 mmol/L N-methyl-D-glucamine. Oxidized thiols and protein bound thiols were calculated by difference, free minus reduced and total minus free form, respectively. Linearity, reproducibility, analytical recovery, and sensitivity were evaluated. The assay was used to measure the thiols redox status in 15 plasma samples from healthy volunteers.

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A Bispecific Antifibrin-antiplatelet Urokinase Conjugate (BAAUC) Induces Enhanced Clot Lysis and Inhibits Platelet Aggregation

Cited by 18 publications

References 31 publications

Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

Engineered Molecular Therapeutics Targeting Fibrin and the Coagulation System: a Biophysical Perspective

Plasma thiols redox status by laser‐induced fluorescence capillary electrophoresis

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