American Society of Hematology

Figure 1.

$Recombinant proteins. (A) Schematic diagrams of contact activation proteases. Catalytic domains are indicated by gray boxes, and noncatalytic domains by white boxes. Positions of active site serine residues are indicated by black bars. Sites of proteolysis during activation are indicated by arrows, with black arrows indicating sites of cleavage required for full protease activity. PK is an 86-kDa polypeptide that is cleaved after Arg371 to form α-kallikrein (PKa). A second cleavage after Arg140 produces β-kallikrein in some experiments. FXI is a 160-kDa dimer of two 80-kDa polypeptides (1 shown). It is converted to FXIa by cleavage after Arg369. The noncatalytic portions of PK and FXI contain 4 apple domains, designated A1 to A4 from the N-terminus. FXII is an 80-kDa polypeptide. Cleavage after Arg353 is responsible for formation of the protease αFXIIa. The noncatalytic portion of FXII contains fibronectin type 2 (F2), epidermal growth factor (EGF), fibronectin type 1 (F1), and kringle (K) domains, and a proline–rich region (PRR). (B) Nonreducing SDS-PAGE of ∼2-μg samples of purified PK or FXI. Left panel, Plasma PK (pPK), recombinant wild-type PK (WT), and PK variants with alanine replacing Arg371 (PK-R371A or R371A) or Ser559 (PK-S559A or S559A). Right panel, Plasma FXI (pFXI), recombinant wild-type FXI (WT), and FXI variants FXI-R369A (R369A) or FXI-S557A (S557A). (C) Plasma PK, PK-WT, PK-R371A, or PK-S559A, 200 nM, were incubated with (+) or without (−) 40 nM FXIIa at 37°C. After 10 minutes, reactions were stopped with reducing sample buffer, size fractionated by SDS-PAGE, and analyzed by western blot using polyclonal anti-PK IgG. For panel B, positions of molecular mass standards in kilodaltons are shown to the left of the images. For panel C, positions of standards for PK, the heavy chain (HC) and light chain (LC) of PKa, and a fragment representing part of β-kallikrein are shown at the right of the figure. (D) Cleavages of S-2302 by PK-WT, PK-R371A, or PK-S559A after digestion with FXIIa as described in panel C (FXIIa was blocked with 1 μM CTI). (E) Clotting times in an aPTT assay for pooled normal plasma (PNP), PK-deficient plasma (C) or PK-deficient plasma supplemented with plasma-derived PK or with recombinant PK-WT (WT), PK-R371A (371), or PK-S559A (559). Each symbol indicates 1 clotting time and the horizontal bars indicate means for each group ± 1 standard deviation. *P ≤ .05 compare with pooled normal plasma. HC, heavy chain; LC, light chain.$

Recombinant proteins. (A) Schematic diagrams of contact activation proteases. Catalytic domains are indicated by gray boxes, and noncatalytic domains by white boxes. Positions of active site serine residues are indicated by black bars. Sites of proteolysis during activation are indicated by arrows, with black arrows indicating sites of cleavage required for full protease activity. PK is an 86-kDa polypeptide that is cleaved after Arg371 to form α-kallikrein (PKa). A second cleavage after Arg140 produces β-kallikrein in some experiments. FXI is a 160-kDa dimer of two 80-kDa polypeptides (1 shown). It is converted to FXIa by cleavage after Arg369. The noncatalytic portions of PK and FXI contain 4 apple domains, designated A1 to A4 from the N-terminus. FXII is an 80-kDa polypeptide. Cleavage after Arg353 is responsible for formation of the protease αFXIIa. The noncatalytic portion of FXII contains fibronectin type 2 (F2), epidermal growth factor (EGF), fibronectin type 1 (F1), and kringle (K) domains, and a proline–rich region (PRR). (B) Nonreducing SDS-PAGE of ∼2-μg samples of purified PK or FXI. Left panel, Plasma PK (pPK), recombinant wild-type PK (WT), and PK variants with alanine replacing Arg371 (PK-R371A or R371A) or Ser559 (PK-S559A or S559A). Right panel, Plasma FXI (pFXI), recombinant wild-type FXI (WT), and FXI variants FXI-R369A (R369A) or FXI-S557A (S557A). (C) Plasma PK, PK-WT, PK-R371A, or PK-S559A, 200 nM, were incubated with (+) or without (−) 40 nM FXIIa at 37°C. After 10 minutes, reactions were stopped with reducing sample buffer, size fractionated by SDS-PAGE, and analyzed by western blot using polyclonal anti-PK IgG. For panel B, positions of molecular mass standards in kilodaltons are shown to the left of the images. For panel C, positions of standards for PK, the heavy chain (HC) and light chain (LC) of PKa, and a fragment representing part of β-kallikrein are shown at the right of the figure. (D) Cleavages of S-2302 by PK-WT, PK-R371A, or PK-S559A after digestion with FXIIa as described in panel C (FXIIa was blocked with 1 μM CTI). (E) Clotting times in an aPTT assay for pooled normal plasma (PNP), PK-deficient plasma (C) or PK-deficient plasma supplemented with plasma-derived PK or with recombinant PK-WT (WT), PK-R371A (371), or PK-S559A (559). Each symbol indicates 1 clotting time and the horizontal bars indicate means for each group ± 1 standard deviation. *P ≤ .05 compare with pooled normal plasma. HC, heavy chain; LC, light chain.

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