Diversity of Plasmatic Thrombin Pools Regulate Thrombin Generation and Blood Clotting: Interference By a Novel Nanobody

Background: Thrombin has a multitude of roles in the initiation and propagation of (anti)coagulation, in platelet activation and fibrin clot formation. The assessment of thrombin generation (TG) and thrombin-dynamics, combined with multi-omics data, has provided in-depth understanding of the plasma factors contributing to the (in)activation of thrombin in triggered plasma and whole-blood. By default, the generated thrombin is considered to act as a single pool. Here, we present evidence for at least two major proteolytically active thrombin pools with non-overlapping functions in blood clotting.

Methods: A novel Syn-Nb-AF106 anti-fibrin nanobody (Nb106) was characterized, targeting a species-conserved thrombin-binding site exposed by fibrinopeptide A cleavage from fibrinogen (Sun et al, this meeting). Thrombin generation was assessed by calibrated automated thrombography using various triggers with control (multi-donor) and (reconstituted) coagulation factor-deficient plasmas, platelet-rich plasma or whole blood. Fibrin clot formation was evaluated by scanning electron microscopy (SEM). Clot retraction was macroscopically assessed.

Results: In tissue factor-triggered thrombin generation, we discovered that Nb106 dose-dependently reduced the thrombin peak level and endogenous thrombin potential (ETP) to 54-62%, without affecting kinetic thrombin generation parameters. We observed a similar reduction in platelet-free plasma, platelet-rich plasma and whole blood. Biochemically, Nb106 displaced thrombin from a binding site on cleaved fibrinogen (Sun et al., abstract). Nb106 inhibited tissue factor-induced thrombin generation, using plasma deficient in factor IX, XI or XII, but not using plasmas deficient in fibrinogen or antithrombin. Reconstitution experiments showed that the inhibition depended on the fibrinogen concentration. A-specific control nanobodies were without effect. Together, the fibrin(ogen)-dependent and thrombin-inhibiting effect indicated that Nb106 operates by releasing proteolytically-active thrombin from fibrin to allow inactivation by antithrombin.

Time-dependent, late spiking experiments revealed that Nb106 reduced the generation of thrombin only when added at 0-15 minutes after trigger, but no longer after 30 minutes, suggesting that the inhibitory capacity was due to a shortened thrombin binding to fibrin rather than to a continued protection towards antithrombin. In plasmas from a cohort of 64 healthy subjects, application of Nb106 showed a consistent reducing effect on the parameters peak and ETP, with a moderate positive correlation with the fibrinogen level. Nb106 suppressed the thrombin generation amplitude, but not the kinetics in the presence of a panel of direct oral anticoagulants (DOAC). Strikingly, brightfield microscopy and SEM indicated that Nb106, but not control nanobodies, in triggered plasma, platelet-rich plasma and whole-blood, fully abrogated the formation of elongating fibrin fibers. In agreement with this, it strongly suppressed whole-blood clot retraction.

Conclusion: Collectivity, these data point to the existence of two major pools of proteolytically active thrombin, both of which contributing to thrombin generation, that are formed upon plasma and blood coagulation. One pool of soluble thrombin steers the proteolytic coagulation cascade, while another pool is bound to growing fibrin fibers. The latter pool still cleaves the conventional AMC thrombin substrate, is temporarily protected against antithrombin, and is required for fibrin fiber extension and blood clotting and clot retraction. The Nb106, replacing thrombin from its binding site on fibrin, selectively annuls the second pool, thereby blocking the clotting process. The replacement ensures thrombin inactivation by antithrombin.

Perspective: The novel anti-fibrin nanobody Syn-Nb-AF106 or a derivative has the potential to act as an alternative anticoagulant in thrombotic diseases with pathological fibrin formation.