Figure 5.
Factor XII, Pro-HGFA, and chimera activation. (A) Reciprocal activation. PK (60 nM) was mixed with 12.5 nM of FXII, Pro-HGFA, HGFAHC/FXIILC, FXIIHC/HGFALC, or vehicle (PK) and S-2302 (200 μM) and incubated at 37°C. Changes in optical density (OD) of 405 nm were continuously monitored on a spectrophotometer. (B) Activation of proteins with FXII catalytic domains. FXII, ΔFXII, or HGFAHC/FXIILC (100 nM) was incubated with PKa (12.5 nM) at 37°C. At indicated times, aliquots were removed and tested for FXIIa generation by chromogenic assay. (C) Reciprocal activation. PK (60 nM) was mixed with 12.5 nM of FXII, HGFAHC/FXIILC, or ΔFXII and S-2302 (200 μM) and incubated at 37°C. Changes in OD of 405 nm were continuously monitored on a spectrophotometer. (D-E) Activation of proteins with Pro-HGFA catalytic domains. (D) Pro-HGFA (260 nM) was incubated with thrombin (26 nM) with (•) or without (○) dextran sulfate (10 μg/mL) at 37°C. (E) Pro-HGFA (red), ΔPro-HGFA (lavender), or FXIIHC/HGFALC (light blue), at 260 nM each, were incubated with thrombin (26 nM) in phosphate-buffered saline at 37°C. For panels D and E, at the indicated times, samples of reactions were stopped and HGFA generation determined by chromogenic assay. (F-G) Reciprocal FXII activation with PK. PK (60 nM) was incubated at 37°C with 12.5 nM of FXII, FXII-FN2, FXII-EGF1, FXII-FN1, FXII-EGF2, FXII-KNG, or FXII-PRR and 200 μM of S-2302 at 37°C. Changes in OD of 405 nm were continuously recorded on a spectrophotometer. (H-I) FXII activation by PKa. FXII, HGFAHC/FXIILC, and the FXII-HCD chimeras (100 nM) were incubated with 12.5 nM of PKa at 37°C. At the indicated times, aliquots were removed and tested for FXIIa generation by chromogenic assay.