3E8 dose-dependently inhibits Aβ42-induced PK and FXII activation in human plasma. (A,B) The 3E8 anti-HK antibody dose-dependently inhibited Aβ42-induced PK activation in NHP. NHP was incubated with buffer, 3E8 anti-HK antibody (0.02 μM, 0.1 μM, 0.5 μM), or control IgG (0.5 μM) at 37°C for 20 minutes. Aβ42 (5 μM) was added and incubated at 37°C for 2 hours (A). Western blots were performed with antibodies against HK, PK, FXII, and TF. 3E8 dose-dependently protected HK from Aβ42-induced cleavage, as shown previously.³⁶  It also dose-dependently inhibited PK activation. Decreases in the PK band indicate activation of PK to PKa; PKa was not detectable by this antibody (lanes 1-5 in [A-B]). Control IgG did not show any effect on Aβ42-induced PK activation (lane 6 in [A-B]). (C) 3E8 dose-dependently inhibited Aβ42-induced kallikrein activity in human plasma. NHP was incubated with buffer, control IgG (50 nM), or various concentrations of 3E8 HK antibody (5 nM, 25 nM, and 50 nM) at 37°C for 20 minutes. Aβ42 (5 μM) and chromogenic substrate S-2302 (0.67 mM final concentration) were added, and absorbance at 405 nm was read for 60 minutes at 37°C. In the absence of 3E8, Aβ42 induced a dramatic increase in kallikrein activity. The 3E8 antibody dose-dependently inhibited Aβ42-induced kallikrein activation, while control IgG had no effect. (D-E) 3E8 anti-HK antibody dose-dependently inhibited FXII activation. 3E8 also dose-dependently inhibited FXII activation (FXIIa) (lanes 1-5 in [D-E]), while control IgG did not influence Aβ42-induced FXII activation (lane 6 in [D-E]). FXIIa and PK western blots were normalized against TF. n = 6. Data are denoted as mean ± SEM. *P < .05, **P ≤ .01, ***P ≤ .001. P > .05 was not significant (n.s.).