Identification and characterization of profibrinolytic single variable domain (VHH) antibodies

Introduction Camelid single-domain antibodies (VHHs, or nanobodies) combine high stability, small molecule size, and strong antigen-binding affinity. Their unique properties make them valuable tools for both, therapeutic and diagnostic approaches. We describe the generation and identification of nanobodies that bind plasminogen/plasmin with high affinity. Functional evaluation demonstrated that these nanobodies enhance the activation of plasminogen by tissue- and urokinase-type plasminogen activators (tPA/uPA). Method A VHH library derived from an immunized alpaca was screened in order to identify high-affinity binders to human plasminogen/plasmin. Lead candidates, designated A11 and C5, were expressed, purified, and further characterized using ELISA, biolayer interferometry (BLI), as well as functional assays in the purified matrix, plasma and whole blood. Results As determined by BLI, A11 and C5 showed high-affinity binding to human plasminogen and plasmin (KD≤50 nM) but no binding to orciplamin, indicating binding to kringle domain structures. Binding of VHHs to plasmin increased fluorogenic substrate conversion, demonstrating allosteric modulation of the enzyme. Competitive analysis by ELISA revealed identical or overlapping epitopes of both VHHs on plasmin(ogen). In plasminogen-activation assays, both nanobodies significantly enhanced plasmin formation, reducing the amount of tPA or uPA required for effective conversion. Accelerated fibrinolysis in the presence of the nanobodies was also confirmed in whole blood by thrombelastometry and in plasma by an aPTT-based mechanical lysis assay. Conclusion Currently applied thrombolytic agents, such as tPA or uPA, act by converting plasminogen into plasmin. These enzymes require high systemic concentrations, which often cause an unfavorable side-effect profile including a substantial bleeding risk. The plasmin(ogen)-binding nanobodies A11 and C5 described here constitute a new class of fibrinolytic enhancers. By facilitating the activation of plasminogen through uPA/tPA, these molecules may enable more efficient clot dissolution with potentially lower systemic exposure of enzymes and an associated lower risk of bleeding. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany