Concurrent interference of thrombin and fibrin fiber generation by a novel nanobody

Introduction Thrombin generation (TG) is required for fibrin formation, but the generated thrombin retains activity also after initial clot formation. Here, we present evidence that a substantial proportion of the generated thrombin serves to ensure the continued fibrin growth in plasma systems. This thrombin pool is fibrin-bound and temporarily protected against antithrombin inactivation.

Method A novel nanobody Syn-Nb-AF106 (Nb106) was isolated from llamas injected with human clotting factor concentrate. Selection and characterization proved that Nb106 recognizes thrombin bound to fibrin but not fibrinogen. Calibrated thrombography was used for assessment of thrombin generation (TG) with tissue factor and collagen-related peptide (CRP, platelet glycoprotein VI agonist) using platelet-free plasma, platelet-rich plasma (PRP) and whole blood. Conventional Z-Gly-Gly-Arg aminomethyl coumarin (AMC) was used as thrombin substrate. Fibrin clot formation was evaluated from mechanical clotting kinetics, turbidity assays, and scanning electron microscopy. Interference was tested of the fibrin modulators, GPRP, ancrod and protease III .

Results In tissue factor-triggered TG,we discovered that Nb106 dose-dependently reduced thrombin peak levels and endogenous thrombin potential (ETP) with>50%. We observed a similar reduction in human platelet-free plasma, PRP and whole blood, independent of the trigger. Atypical control nanobodies did not alter TG parameters. In mouse PRP, Nb106 was most effective upon co-stimulation with tissue factor and CRP. Mechanistically, Nb106 still suppressed TG in the presence of either GPRP (fibrin cross-linking inhibitor) or ancrod (serine protease, specifically cleaving fibrinopeptide A from fibrinogen). However, it did no longer affect TG parameters in the presence of protease III (serine protease cleaving the Bβ chain of fibrinogen, including fibrinopeptide B). This indicated that Nb106 specifically targets a novel site thrombin-binding site on fibrin, which becomes exposed upon fibrinogen cleavage of fibrinopeptide A.

In fibrin clotting experiments, ancrod shortened the Ca²⁺-dependent clotting time, whereas protease III prolonged it. Markedly, Nb106 failed to affect plasmatic clotting times, but it significantly and strongly blocked plasma turbidity changes induced by tissue factor. Furthermore, brightfield and scanning electron microscopy revealed that Nb106 dose-dependently suppressed the lateral and longitudinal fibrin fiber extension until complete inhibition, although non-fibrillar fibrin structures were still visible.

Conclusion Collectivity, these data point to the existence of a pool of fibrin-bound thrombin , which is protected against antithrombin inactivation. The Nb106, by binding to the thrombin binding site on fibrin exposed by fibrinopeptide A cleavage, can replace this thrombin, and hence prevents the extension of fibrin fibers, to ultimately abrogate the clotting process.

Conflict of Interest

BdL JK, DH, FS, RdLK and MR are employees of Synapse Research Institute Maastricht, part of the Diagnostica Stago group, where PGdG and JWMH are advisors. SS and JZ are founded by the China Scholarship Council.

Publication History

Article published online:
26 February 2024