OC 41.5 - Two positively charged patches in the EGF-1 domain of Factor XII synergistically contribute to surface binding and activation

Background: Factor XII (FXII) contributes to medical device-associated thrombosis by binding to foreign surfaces. We and others identified its EGF-1 domain as a surface binding module.

Aims: To explore the contribution of two positively charged patches in the FXII EGF-1 domain to surface binding and contact activation.

Methods: We studied surface binding and activation of recombinant FXII variants in buffer or FXII-deficient plasma in pull-down assays, chromogenic substrate conversion assays and clotting assays.

Results: FXII EGF-1 contains 11 positively charged residues. These can be divided into two distinct patches (Figure 1). The first patch (upstream; K73, K74, K76, H78, K81 and H82) is FXII-specific. The second patch (downstream) contains three histidines (H99, H105 and H110) which are conserved in HGFA and two FXII-specific lysines (K87 and K113).
We generated glutamine-substitution FXII variants, neutralizing FXII-specific positively charged residues in the upstream patch (Δupstream), in the downstream patch (Δdownstream) or in both (Δcombined). Compared to wild-type (WT) FXII, kaolin binding was decreased by 55%, 3% and 97% for Δupstream, Δdownstream and Δcombined. Kaolin-triggered prekallikrein activation in buffer was decreased by 71%, 43% and 86%, respectively. All variants were equally susceptible to activation by plasma kallikrein in solution.
Reconstitution of WT FXII in plasma results in an aPTT clotting time of 36 (±2) seconds. By comparison, reconstitution with Δupstream, Δdownstream or Δcombined leads to clotting times of 99 (±8), 48 (±2) and 223 (±13) seconds, respectively. Correspondingly, kaolin-triggered kallikrein generation decreased by 69%, 62% and 89%.
Finally, we raised VHHs against FXII EGF-1. These prolong aPTT clotting times (lead candidate 113 (±2) seconds vs control VHH 38 (±1) and inhibit kaolin-driven kallikrein generation in plasma (lead candidate: 74% vs control VHH 8%).

Conclusion(s): We here identify crucial residues for surface binding of FXII, which may serve as a druggable target.