Figure 2.
Figure 2. PGN activates the intrinsic coagulation pathway in challenged baboons. (A-C) Activation of contact coagulation cascade in baboons challenged with either high-dose (n = 5) or low-dose PGN (n = 3). Time-course changes in FXIIa-AT (A), kallikrein-C1inh (B), and FXIa-AT (C) are shown. Statistical analysis and data representation are consistent with Figure 1. (D) Binding of purified contact pathway initiators to immobilized PGN was evaluated in vitro using biotinylated purified human FXII or prekallikrein. Data shown are mean ± SEM from 1 experiment run in quadruplicate. Dissociation constants (KD) were estimated from the best-fit binding curves using GraphPad Prism and average KD from 2 to 3 independent binding experiments are shown in the inset. (E) The effect of PGN (0.1-100 µg/mL) on autoactivation of purified FXII was evaluated in a continuous assay using the S2302 chromogenic substrate. No significant changes in the rate of chromophore (pNA) generation were observed. (F) The effect of PGN (1-200 µg/mL) on the enzymatic function of active proteases α-FXIIa (50 nM) and α-kallikrein (α-Kal, 25 nM). Specific activities are represented after normalization of initial velocities (V0) to the amount of enzyme used (micromolar pNA per minute per picomole enzyme). For both proteases, we found a strong linear correlation between the enzymatic activity and PGN concentration (Pearson r = 0.9878 (P < .0001) for α-FXIIa and r = 0.9344 (P = .0007) for α-Kal). The best fit linear regression (solid line) and 95% confidence interval (CI; dotted lines) are graphically depicted for each. (G) The effect of PGN on AT-heparin inhibition of α-FXIIa (100 nM) proteolysis of the S2302 chromogenic substrate. Observed residual α-FXIIa activity (micromolar pNA per minute) at different AT concentration (0-2 µM, represented on a log scale) in the presence of constant saturating levels of heparin (200 µg/mL) are shown (blue circles). PGN effect on AT-heparin inhibition of α-FXIIa was assessed at 50 µg/mL (green triangles) and 200 µg/mL (red squares). PGN increases AT-heparin IC50 in a dose-dependent manner (inset). (F-G) Data are represented as mean ± SEM (n = 3).

PGN activates the intrinsic coagulation pathway in challenged baboons. (A-C) Activation of contact coagulation cascade in baboons challenged with either high-dose (n = 5) or low-dose PGN (n = 3). Time-course changes in FXIIa-AT (A), kallikrein-C1inh (B), and FXIa-AT (C) are shown. Statistical analysis and data representation are consistent with Figure 1. (D) Binding of purified contact pathway initiators to immobilized PGN was evaluated in vitro using biotinylated purified human FXII or prekallikrein. Data shown are mean ± SEM from 1 experiment run in quadruplicate. Dissociation constants (KD) were estimated from the best-fit binding curves using GraphPad Prism and average KD from 2 to 3 independent binding experiments are shown in the inset. (E) The effect of PGN (0.1-100 µg/mL) on autoactivation of purified FXII was evaluated in a continuous assay using the S2302 chromogenic substrate. No significant changes in the rate of chromophore (pNA) generation were observed. (F) The effect of PGN (1-200 µg/mL) on the enzymatic function of active proteases α-FXIIa (50 nM) and α-kallikrein (α-Kal, 25 nM). Specific activities are represented after normalization of initial velocities (V0) to the amount of enzyme used (micromolar pNA per minute per picomole enzyme). For both proteases, we found a strong linear correlation between the enzymatic activity and PGN concentration (Pearson r = 0.9878 (P < .0001) for α-FXIIa and r = 0.9344 (P = .0007) for α-Kal). The best fit linear regression (solid line) and 95% confidence interval (CI; dotted lines) are graphically depicted for each. (G) The effect of PGN on AT-heparin inhibition of α-FXIIa (100 nM) proteolysis of the S2302 chromogenic substrate. Observed residual α-FXIIa activity (micromolar pNA per minute) at different AT concentration (0-2 µM, represented on a log scale) in the presence of constant saturating levels of heparin (200 µg/mL) are shown (blue circles). PGN effect on AT-heparin inhibition of α-FXIIa was assessed at 50 µg/mL (green triangles) and 200 µg/mL (red squares). PGN increases AT-heparin IC50 in a dose-dependent manner (inset). (F-G) Data are represented as mean ± SEM (n = 3).

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