Factor XII (FXII) Locarno is a natural variant with proline replacing Arg353 at the activation cleavage site, preventing conversion to the fully active protease factor XIIa (FXIIa). Recently, we showed that FXII restricted to a single chain form (sc-FXII) by replacing Arg353 with alanine expresses proteolytic activity that is enhanced by cofactors such as polyphosphate.To determine if the Pro353 substitution affects the activity of sc-FXII.Wild type FXII (FXII-WT), FXII-R353A, and FXII Locarno (FXII-R353P) were tested for their abilities to activate prekallikrein, and to induce thrombin generation and coagulation in plasma in a factor XI-dependent manner.FXII-WT is converted to FXIIa by autoactivation in the presence of polyphosphate, and by incubation with kallikrein. FXII-R353P and FXII-R353A were not converted to FXIIa by these methods. Despite this, FXII-R353A converts prekallikrein to kallikrein, and the reaction is enhanced by polyphosphate. FXII-R353P also converts prekallikrein to kallikrein, but at a slower rate than FXII-R353A. In FXII-deficient plasma induced to clot with silica, FXII-R353A is a better promoter of factor XI-dependent thrombin generation and coagulation than FXII-R353P.The activity of sc-FXII is sensitive to perturbations in the activation loop, which contains residue 353. Homology modeling based on the crystal structure of the FXII homolog tissue plasminogen activator suggests that Pro353 introduces changes in the shape and flexibility of the activation loop that disrupt key interactions that support an active conformation in sc-FXII.