Cleavage of S-2302 and PK by FXII species. (A) FXII-WT (WT), FXII-R353A, FXII-T (T), or FXII-S544A (200 nM) were incubated with S-2302 (200 μM) in standard buffer in the absence of a surface. Continuous formation of amidolytic activity was monitored at 405 nm. (B-C) Varying concentrations of S-2302 (1.6-1000 μM) were incubated with (B) 5 nM αFXIIa or (C) 200 nM FXII-T in the absence of a surface. Changes in OD 405 nm/minute were measured on a plate reader and converted to pNA generated per minute. Each point is a mean ± 1 SD for 3 separate experiments. (D) Same as reactions described in panel A except that incubations were conducted in the presence of a surface (PTT-A reagent, 10% final volume). Note that PTT-A reagent only increases the amidolytic activity of FXII-WT, compared with reactions in panel A because, of the 4 FXII species tested, it is the only 1 that is cleaved after Arg353 and has an active site serine at residue 544. (E) PK-WT (200 nM) was incubated with 200 nM FXII-WT, FXII-R353A, FXII-T, FXII-S544A, or control vehicle (C). Generation of kallikrein (or kallikrein and αFXIIa in the case of FXII-WT) was continuously monitored by cleavage of S-2302 (200 μM). (F-G) Varying concentration of PK were incubated with (F) 25 pM αFXIIa or (G) 15 nM FXII-T in the absence of a surface. Kallikrein generation was determined by measuring the rate of S-2302 (200 μM) cleavage. Each point is a mean ± 1 SD for 3 separate experiments. (H) Generation of kallikrein from PK-WT (200 nM) was followed by continuous monitoring of S-2302 (200 μM) cleavage in the presence or absence of a surface (70 μM Poly-P), and in the presence or absence of 200 nM FXII-T (T). (I) Kinetic parameters for cleavage of S-2302 and PK by αFXIIa or FXII-T determined from the curves in panels B, C, F, and G.