The effect of APC on [Ca²⁺]_i in human brain endothelial cells. (A) [Ca²⁺]_i was monitored in single cells when challenged with (i) APC (50 nM); (ii) APC (10 nM) and hirudin (40 nM); (iii) mutant Ser360Ala-APC (50 nM); (iv) zymogen protein C (50 nM); (v) APC (50 nM) pretreated for 5 minutes with monoclonal anti-APC IgG (C3) (1:1 molar ratio); and (vi) boiled APC (50 nM). Ten to 20 cells were measured in representative experiments. Positions of bars indicate the stimulation with the noted substances. (B) [Ca²⁺]_i in single cells was measured in the presence of 0.02, 0.1, 0.5, 10, 50, and 100 nM APC administered for 60 seconds. Each set of curves is representative of 3 identical experiments. (C) Bars represent the mean [Ca²⁺]_i ± SEs after exposure to APC (50 nM), APC (10 nM) and hirudin (40 nM), mutant Ser360Ala-APC (50 nM) (mut-APC), zymogen protein C (50 nM) (zym-PC), C3 antibody pretreated APC (50 nM) (C3 APC), and heat-inactivated APC (50 nM). (D) Dose-response relationship for the effect of APC (20 pM to 100 nM) on [Ca²⁺]_i. [Ca²⁺]_i changes from basal levels ± SEs were taken from 3 identical experiments. Data for log [APC] versus the change in [Ca²⁺]_i were fitted to a sigmoidal curve (dashed line).