Figure 6.
Aβ42 binding to both HK and FXII, and PK and FXI binding to HK are involved in Aβ42-induced plasma contact system activation. (A) Aβ42 pulls down FXII, cHK, FXI, and PK from pooled NHP. NHP was incubated with biotinylated Aβ42 (B-Aβ42). The B-Aβ42/bound protein complexes were pulled down by Dynabeads M-280 Streptavidin, eluted with SDS sample buffer, and analyzed by western blot. Purified human FXII, FXI, PK, and HK were used to validate the specificity of the antibodies for each protein (lanes 1-4). Each protein was detected in the NHP input (lane 5) and the supernatant after B-Aβ42 pulldown (lane 6). B-Aβ42 pulled down FXII and FXIIa (activated by Aβ42-induced contact system activation during the pulldown process), cHK (HK was cleaved due to contact system activation, so most, if not all, cHK was pulled down by Aβ42), FXI, and some PK (lane 7). During the pulldown process, B-Aβ42 activated the contact system leading to FXII activation, decreased PK (since it was activated to PKa, which was not detectable by this antibody), FXI molecular size shift, and HK cleavage (compare lanes 5-7). TF was not pulled down by B-Aβ42. (B) 3E8 anti-HK antibody blocked PK and FXI, but not HK, pulldown by B-Aβ42. Since the contact system was activated during the pulldown process, we used FXII-DF human plasma to avoid B-Aβ42-induced contact system activation. All proteins except for FXII were detected in FXII-DF plasma (lane 1). B-Aβ42 pulled down HK, FXI, and PK, but not TF (lane 2). 3E8 blocked FXI and PK pulldown by B-Aβ42, but not HK (lane 3). (C) HK deficiency in KN-DF plasma prevented FXI and PK pulldown by B-Aβ42. All proteins except HK were detected in KN-DF plasma, and protein levels were similar between PNP and KN-DF plasma (lanes 1 vs 2). B-Aβ42 pulled down FXII, but neither FXI nor PK from KN-DF plasma (lane 3). (D,E) Plates were coated with PK (D) or FXI (E) and then incubated with HK in the presence or absence of 3E8 anti-HK antibody. In the absence of 3E8, HK binds to PK (D) or FXI (E). In the presence of 3E8, binding is blocked between HK and PK (D) or FXI (E). The control IgG did not have an effect on HK binding to PK or FXI. The experiments were performed in triplicate and repeated 3 times. Data are denoted as mean ± SEM. ***P ≤ .001. P > .05 was not significant (n.s.).

Aβ42 binding to both HK and FXII, and PK and FXI binding to HK are involved in Aβ42-induced plasma contact system activation. (A) Aβ42 pulls down FXII, cHK, FXI, and PK from pooled NHP. NHP was incubated with biotinylated Aβ42 (B-Aβ42). The B-Aβ42/bound protein complexes were pulled down by Dynabeads M-280 Streptavidin, eluted with SDS sample buffer, and analyzed by western blot. Purified human FXII, FXI, PK, and HK were used to validate the specificity of the antibodies for each protein (lanes 1-4). Each protein was detected in the NHP input (lane 5) and the supernatant after B-Aβ42 pulldown (lane 6). B-Aβ42 pulled down FXII and FXIIa (activated by Aβ42-induced contact system activation during the pulldown process), cHK (HK was cleaved due to contact system activation, so most, if not all, cHK was pulled down by Aβ42), FXI, and some PK (lane 7). During the pulldown process, B-Aβ42 activated the contact system leading to FXII activation, decreased PK (since it was activated to PKa, which was not detectable by this antibody), FXI molecular size shift, and HK cleavage (compare lanes 5-7). TF was not pulled down by B-Aβ42. (B) 3E8 anti-HK antibody blocked PK and FXI, but not HK, pulldown by B-Aβ42. Since the contact system was activated during the pulldown process, we used FXII-DF human plasma to avoid B-Aβ42-induced contact system activation. All proteins except for FXII were detected in FXII-DF plasma (lane 1). B-Aβ42 pulled down HK, FXI, and PK, but not TF (lane 2). 3E8 blocked FXI and PK pulldown by B-Aβ42, but not HK (lane 3). (C) HK deficiency in KN-DF plasma prevented FXI and PK pulldown by B-Aβ42. All proteins except HK were detected in KN-DF plasma, and protein levels were similar between PNP and KN-DF plasma (lanes 1 vs 2). B-Aβ42 pulled down FXII, but neither FXI nor PK from KN-DF plasma (lane 3). (D,E) Plates were coated with PK (D) or FXI (E) and then incubated with HK in the presence or absence of 3E8 anti-HK antibody. In the absence of 3E8, HK binds to PK (D) or FXI (E). In the presence of 3E8, binding is blocked between HK and PK (D) or FXI (E). The control IgG did not have an effect on HK binding to PK or FXI. The experiments were performed in triplicate and repeated 3 times. Data are denoted as mean ± SEM. ***P ≤ .001. P > .05 was not significant (n.s.).

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