Figure 4.
Studies of the FXII PRR. (A) FXII-ΔPRR. Nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of purified wild-type FXII (left), and the FXII-HCD chimera FXII-PRR and FXII lacking the amino acids Thr278 and Leu338 comprising the FXII PRR (FXII-ΔPRR; right). Each lane was loaded with 2 μg of protein. Positions of molecular mass standards in kilodaltons are shown to the left of the image. (B) Autoactivation. Two hundred nanomolar FXII (blue), FXII-ΔPRR (green), or FXII-Ser340 467 (mustard) were incubated with S-2302 (200 μM) and poly-P (70 μM). Reactions without surface are indicated by the dashed lines. Changes in optical density (OD) of 405 nm were continuously monitored on a spectrophotometer. (C) aPTT assays. Shown are average clotting times (±1 standard deviation) for normal plasma (NP) or FXII-deficient plasma supplemented with the recombinant proteins (400 nM) listed in panel B. (D) Autoactivation of FXII and FXII-Ser340 467. FXII or FXII-Ser340 467 (200 nM) were incubated with 70 μM of poly-P (PP), 20 μM of ellagic acid (EA), or 10 μg/mL of dextran sulfate (DS). After 3 hours, samples were removed and size fractionated by reducing SDS-PAGE (12% polyacrylamide). (E) Activation of FXII and FXII-Ser340 467 by PKa. FXII or FXII-Ser340 467 (200 nM) were incubated with 25 nM of PKa at 37°C. After 1.5 hours, samples were removed into nonreducing (left) or reducing (right) buffer. For panels D and E, controls (C) are unincubated proteins. Note that the FXII-Ser340 467 preparation had traces of light chain, but it does not increase in the presence of a surface. For panels D and E, positions of standards for zymogen FXII and the heavy and light chains of FXIIa are indicated at the right of the image. Positions of molecular mass standards in kilodaltons are shown to the left of the images. (F) Activation of FXII and FXII-Ser340 467 by PKa chromogenic assay. Aliquots of the reactions described in panels D and E were tested for FXIIa generation using a chromogenic substrate assay. Results were compared with a standard curve constructed with purified FXIIa.

Studies of the FXII PRR. (A) FXII-ΔPRR. Nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of purified wild-type FXII (left), and the FXII-HCD chimera FXII-PRR and FXII lacking the amino acids Thr278 and Leu338 comprising the FXII PRR (FXII-ΔPRR; right). Each lane was loaded with 2 μg of protein. Positions of molecular mass standards in kilodaltons are shown to the left of the image. (B) Autoactivation. Two hundred nanomolar FXII (blue), FXII-ΔPRR (green), or FXII-Ser340 467 (mustard) were incubated with S-2302 (200 μM) and poly-P (70 μM). Reactions without surface are indicated by the dashed lines. Changes in optical density (OD) of 405 nm were continuously monitored on a spectrophotometer. (C) aPTT assays. Shown are average clotting times (±1 standard deviation) for normal plasma (NP) or FXII-deficient plasma supplemented with the recombinant proteins (400 nM) listed in panel B. (D) Autoactivation of FXII and FXII-Ser340 467. FXII or FXII-Ser340 467 (200 nM) were incubated with 70 μM of poly-P (PP), 20 μM of ellagic acid (EA), or 10 μg/mL of dextran sulfate (DS). After 3 hours, samples were removed and size fractionated by reducing SDS-PAGE (12% polyacrylamide). (E) Activation of FXII and FXII-Ser340 467 by PKa. FXII or FXII-Ser340 467 (200 nM) were incubated with 25 nM of PKa at 37°C. After 1.5 hours, samples were removed into nonreducing (left) or reducing (right) buffer. For panels D and E, controls (C) are unincubated proteins. Note that the FXII-Ser340 467 preparation had traces of light chain, but it does not increase in the presence of a surface. For panels D and E, positions of standards for zymogen FXII and the heavy and light chains of FXIIa are indicated at the right of the image. Positions of molecular mass standards in kilodaltons are shown to the left of the images. (F) Activation of FXII and FXII-Ser340 467 by PKa chromogenic assay. Aliquots of the reactions described in panels D and E were tested for FXIIa generation using a chromogenic substrate assay. Results were compared with a standard curve constructed with purified FXIIa.

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