Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The Alzheimer’s disease peptide Aβ promotes thrombin generation through activation of coagulation factor XII

doi: 10.1111/jth.13209

Figure Lengend Snippet: Thrombin generation was measured by CAT. (A) Aβ42-induced thrombin generation was blocked by a monoclonal antibody against FXIIa [69] (4 µM), but not by IgG. (B) Aβ42’s enhancement of thrombin generation was not inhibited by the extrinsic coagulation pathway inhibitor ASIS (60 nM). (C) Thrombin generation was not enhanced in human plasma from a FXII-deficient individual in the presence of Aβ42. Deficiency of FXII in this plasma was confirmed by Western blot (WB; inset). Aβ42 had no effect when thrombin generation was triggered by 3 pM FXIa. (D) Thrombin generation was enhanced in WT mouse plasma but not FXII−/− mouse plasma in the presence of Aβ42. Mouse plasma contained 240 nM ASIS to block TF-mediated thrombin generation stemming from TF contamination during blood draw. ASIS does not affect Aβ42-mediated enhancement of thrombin generation (Fig. 2B). All experiments were performed in duplicate, and averaged curves are presented.

Article Snippet: For FXII-dependent FXI activation, 0.8 mM Pefachrome FXIa (Centerchem) was added to 5 nM FXII, 15 nM FXI (Haematologic Technologies), and 3 μM Aβ or vehicle.

Techniques: Coagulation, Clinical Proteomics, Western Blot, Blocking Assay

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The Alzheimer’s disease peptide Aβ promotes thrombin generation through activation of coagulation factor XII

doi: 10.1111/jth.13209

Figure Lengend Snippet: (A) Aβ42 (3 µM) triggered autoactivation of FXII as determined by chromogenic substrate assay. (B) In the presence of both FXII and prekallikrein (PPK), Aβ42 dose-dependently promoted activation of FXII as seen through the reduction of FXII zymogen levels at 80 kDa and the appearance of the FXIIa heavy chain at 52 kDa. Dextran sulfate 500 kDa (DS500) and kaolin were used as positive controls. (C) Aβ42 (3 µM) triggered FXII-dependent FXIa generation by chromogenic substrate assay. The signal was not due to non-specific cleavage of chromogenic substrate by FXIIa, or by autoactivation of FXI, as seen in controls where FXII or FXI were omitted. (D) FXI activation can be seen through the appearance of the 50 kDa FXIa heavy chain band following incubation of FXII, FXI, PPK, and HK with Aβ42 or kaolin. Levels of FXIa heavy chain were increased in Aβ42- (p <0.05) and kaolin- (p <0.01) treated samples compared to vehicle. All lanes presented are from the same blot. (E) Aβ42 oligomers were more potent in promoting FXII-dependent FXI activation than freshly dissolved Aβ42. Aβ42 fibrils had no effect. All Aβ42 preparations were 3 µM. (F) Aβ42 oligomers were more potent than Aβ40 oligomers in promoting FXII-dependent FXI activation. (G) Aβ42 oligomers (TEM image, top inset) promoted FXII-dependent FXI activation much more strongly than Aβ42 Dutch oligomers (TEM image, bottom inset). Chromogenic substrate assays were performed multiple times with representative results shown. Representative immunoblots are from 3 experiments.

Article Snippet: For FXII-dependent FXI activation, 0.8 mM Pefachrome FXIa (Centerchem) was added to 5 nM FXII, 15 nM FXI (Haematologic Technologies), and 3 μM Aβ or vehicle.

Techniques: Activation Assay, Incubation, Western Blot

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The Alzheimer’s disease peptide Aβ promotes thrombin generation through activation of coagulation factor XII

doi: 10.1111/jth.13209

Figure Lengend Snippet: (A) Western blot demonstrating that biotinylated Aβ42 oligomers pull down FXII from human plasma. (B) Incubation of Aβ42 oligomers with human plasma leads to FXII cleavage. (C) ELISA measuring FXIa-C1inh complex formation in normal and FXII-deficient human plasma. Oligomers of Aβ42 but not Aβ42 Dutch promoted the formation FXIa-C1inh (p <0.001; Aβ42 vs. vehicle). (D) ELISA measuring FXIa-α1AT complex formation in normal human plasma. Aβ42 oligomers promoted increased FXIa-α1AT complex formation (p <0.0001; Aβ42 vs. vehicle). Results are expressed as % of kaolin-activated normal plasma and presented as mean ± SD of experiments performed in triplicate.

Article Snippet: For FXII-dependent FXI activation, 0.8 mM Pefachrome FXIa (Centerchem) was added to 5 nM FXII, 15 nM FXI (Haematologic Technologies), and 3 μM Aβ or vehicle.

Techniques: Western Blot, Clinical Proteomics, Incubation, Enzyme-linked Immunosorbent Assay