Factor XII contact activation can be prevented by targeting 2 unique patches in its epidermal growth factor-like 1 domain with a nanobody

Background: Factor (F)XII triggers contact activation by binding to foreign surfaces, with the epidermal growth factor-like 1 (EGF-1) domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device–induced thrombosis. Objectives: To unravel and prevent EGF-1–mediated FXII surface-binding with a variable domain of heavy chain–only antibody (V_HH). Methods: FXII variants with glutamine substitutions of 2 positively charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain–specific V_HHs were raised to inhibit EGF-1–mediated FXII contact activation while preserving quiescence. Results: Two unique, positively charged patches in the EGF-1 domain were identified (upstream, 73K74K76K78H81K82H; downstream, 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in autoactivation of 94%, and prolongation of clot formation in activated partial thromboplastin time assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1–specific V_HHs were developed that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective V_HH “F2” binds the positively charged patches and thereby dose-dependently extends activated partial thromboplastin time clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence. Conclusion: The 2 unique, positively charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding, making both patches crucial for contact activation. Targeting these with FXII EGF-1–specific V_HHs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable targets in preventing medical device–induced thrombosis.