Influence of HRG on FXII autoactivation and FXI activation. (A) FXII was incubated with 100μM polyphosphate for 15 minutes in HBS buffer containing 2mM CaCl₂ and 12.5μM ZnCl₂. After 10 minutes, aliquots were removed and FXIIa generation was determined by monitoring the hydrolysis of 500μM Biophen CS11–22. The data points represent the mean ± SE of 2 separate experiments. Data were analyzed by nonlinear regression (line) to determine the IC₅₀. (B) To examine the influence of HRG on FXI activation, 10nM FXIIa was incubated with 0 to 1μM HRG, 100nM HK, 10% APTT-SP, and 120nM FXI in HBS buffer containing 12.5μM ZnCl₂ and 2mM CaCl₂. Aliquots were removed, and FXIa generation was determined by monitoring the hydrolysis of 600μM S-2366. Rates of chromogenic substrate cleavage in the presence of HRG were normalized relative to that obtained in its absence. The data were fit to a rectangular hyperbola equation (line) to determine the IC₅₀. The data points represent the mean ± SE of 2 experiments.