SummaryThrombolysis is well established in the treatment of acute myocardial infarction. However, clinical application of thrombolytic agents has limitations with respect to efficacy and specificity. To achieve highly effective and at the same time clot-selective plasminogen activation urokinase was coupled to a bispecific antibody consisting of the mono-valent Fab’ from the antifibrin monoclonal antibody 59D8 and the monovalent Fab’ from the anti-glycoprotein GPIIb/IIIa monoclonal antibody 7E3. The bispecific antifibrin-antiplatelet urokinase conjugate (BAAUC) was synthesized and characterized. Assays with either immobilized platelets, GPIIb/IIIa or fibrin showed an increase in plasminogen activation compared to uncoupled urokinase by 10-fold, 58-fold and 13-fold, respectivley (p < 0.0001 each). In vitro clot lysis was performed on platelet-rich and fibrin-rich clots and revealed an up to 5-fold higher potency of BAAUC compared to uncoupled urokinase (p < 0.0001). In vitro platelet aggregation was effectively inhibited by the hybrid molecule, whereas urokinase had no effect. BAAUC and two monospecific urokinase-conjugates, UK-59D8-IgG and UK-7E3-(Fab’)2 were compared with each other with regard to similar tests. In vitro clot assays with platelet-rich and platelet-poor clots were performed. BAAUC achieved a significantly higher plasminogen activation compared to each of the monospecific conjugates (p < 0.05, respectively). We conclude that BAAUC, a bispecific plasminogen activator with antifibrin and antiplatelet properties has the potency to lyse both fibrin-rich and platelet-rich thrombi with high efficacy and to effectively inhibit platelet aggregation.
We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv(59D8)). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clot-bound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications.TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for anti-fXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule. Two step affinity chromatography with Ni(2+) and beta(15-22)-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv(59D8)-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv(59D8)-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.
In der Bevölkerung aber auch unter Ärzten besteht eine gewisse Skepsis gegenüber mathematischen Modellen zur Berechnung der Prognose von Intensivpatienten. Die Gründe dafür sind vielschichtig: seitens der Patienten gehört dazu vor allem die tiefe Angst davor, daß letztlich seelenlose Computerprogramme -Todesprogramme -nach rein wirtschaftlichen Gesichtspunkten über das individuelle Schicksal entscheiden könnten. Bei Ärzten besteht die Befürchtung, daß die eigene klinische Urteilsfähigkeit begrenzt und die therapeutische Kompetenz durch Scoring Systeme abgewertet werden könnte. Trotz der Vorbehalte nahm die Verbreitung von prognostischen Scores in den letzten Jahren rasch zu. Für den auf der Intensivstation tätigen Arzt heißt dies: er muß, um die Systeme sinnvoll einsetzen zu können, Grundkenntnisse über deren Entwicklung erwerben, die korrekte Anwendung beherrschen, insbesondere aber ihre Limitationen kennen. Diesem Zweck dient Systeme zeichnen sich durch eine hohe Spezifität aus, d.h. sie können das Über-leben mit circa 90% Wahrscheinlichkeit richtig voraussagen; sie weisen aber nur eine mittelmäßige Sensitivität auf, d.h. sie können die Wahrscheinlichkeit zu versterben nur mit 50-70% Sicherheit richtig voraussagen.
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