Summary
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 (scFv59D8 ). 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 clotbound 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 antifXa activity,
it was a challenging question, whether TAP will be active as a N-terminally coupled
fusion molecule. Two step affinity chromatography with Ni2+ and β15-22 -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, scFv59D8 -TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted
TAP did not reveal anticoagulative activity at all. In summary, scFv59D8 -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.
Keywords Coagulation inhibitors - drug design - fibrinogen / fibrin
