American Society of Hematology

Figure 5.

$Figure 5. Activation of FXII and FXI by single-chain FXII. (A) Western blots of a mixture of FXII-T (200 nM) and FXII-S544A (200 nM) in the absence of a surface (No Surface), or in the presence of PTT-A reagent (25% final volume) or Poly-P (70 μM). At indicated times, samples were removed into nonreducing sample buffer, size fractionated by SDS-PAGE, and analyzed by western blot using IgG D06 which recognizes formation of the FXIIa active site. The bottom row shows results for FXII-S544A and FXII-T incubated separately with 70 μM Poly-P. (B) FXI-S557A (30 nM) was incubated with 200 nM FXII-WT, FXII-T, or FXII-S544A in the absence (top row) or presence (bottom row) of 70 μM Poly-P. At indicated times, samples were removed into reducing sample buffer, size fractionated by SDS-PAGE, and analyzed by western blot using a goat-anti-human FXI polyclonal IgG.$

Activation of FXII and FXI by single-chain FXII. (A) Western blots of a mixture of FXII-T (200 nM) and FXII-S544A (200 nM) in the absence of a surface (No Surface), or in the presence of PTT-A reagent (25% final volume) or Poly-P (70 μM). At indicated times, samples were removed into nonreducing sample buffer, size fractionated by SDS-PAGE, and analyzed by western blot using IgG D06 which recognizes formation of the FXIIa active site. The bottom row shows results for FXII-S544A and FXII-T incubated separately with 70 μM Poly-P. (B) FXI-S557A (30 nM) was incubated with 200 nM FXII-WT, FXII-T, or FXII-S544A in the absence (top row) or presence (bottom row) of 70 μM Poly-P. At indicated times, samples were removed into reducing sample buffer, size fractionated by SDS-PAGE, and analyzed by western blot using a goat-anti-human FXI polyclonal IgG.

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