Abstract
Factor XII (FXII) zymogen activation requires cleavage after arginine 353 located
in the activation loop. This cleavage can be executed by activated FXII (autoactivation),
plasma kallikrein (PKa), or plasmin. Previous studies proposed that the activation
loop of FXII is shielded to regulate FXII activation and subsequent contact activation.
In this study, we aimed to elucidate this mechanism by expressing and characterizing
seven consecutive N-terminally truncated FXII variants as well as full-length wild-type
(WT) FXII. As soon as the fibronectin type II domain is lacking (FXII Δ1–71), FXII
cleavage products appear on Western blot. These fragments display spontaneous amidolytic
activity, indicating that FXII without the fibronectin type II domain is susceptible
to autoactivation. Additionally, truncated FXII Δ1–71 is more easily activated by
PKa or plasmin than full-length WT FXII. To exclude a contribution of autoactivation,
we expressed active-site incapacitated FXII truncation variants (S544A). FXII S544A
Δ1–71 is highly susceptible to cleavage by PKa, indicating exposure of the activation
loop. In surface binding experiments, we found that the fibronectin type II domain
is non-essential for binding to kaolin or polyphosphate, whereas the following epidermal
growth factor-like domain is indispensable. Binding of full-length FXII S544A to kaolin
or polyphosphate increases its susceptibility to cleavage by PKa. Moreover, the activation
of full-length WT FXII by PKa increases approximately threefold in the presence of
kaolin. Deletion of the fibronectin type II domain eliminates this effect. Combined,
these findings suggest that the fibronectin type II domain shields the activation
loop of FXII, ensuring zymogen quiescence.
Keywords
factor XII - plasma kallikrein - bradykinin - contact system - fibronectin type II
domain - epidermal growth factor-like domain - polyphosphate - plasmin
