Fig 2: The FXII-FVII proteolysis pathway responds to tissue injuryMice were treated i.p. with vehicle (filled circle: corn oil for CCl4; filled square: PBS for hPEPD), CCl4 (0.5 g/kg) or hPEPD (4 mg/kg); blood samples and various organs were collected 24 h later. A.-C. Plasma levels of mPEPD and/or hPEPD, mSRC and mAß1-42 were measured by ELISA. Error bars indicate SD (n = 3); data were log transformed before two-way ANOVA, followed by Tukey multiple comparisons test. ** p < 0.01; *** p < 0.001; **** p < 0.0001. D. Tissue levels of ERBB1 and ERBB2 and their phosphorylation status were measured by IB. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a loading control. Each lane represents a sample from a different mouse.

Fig 3: Aß1-42 binds to FN2D in FXII and activates FXII but is degraded by FVIIaA. hAß1-42 (0, 0.05, 0.5 and 11.1 µM) was incubated with FXII (0.97 nM) in ZnCl2-containing PBS at RT; FXII activation was measured by a chromogenic assay. Each value is mean ± SD (n = 3). B. hAß1-42 (200 nM) was incubated with FXII or a mutant (20 nM) in PBS at RT; Aß1-42 aggregated during the incubation. FXII or its mutant remaining in solution (S) or binding to Aß1-42 aggregates (A), the latter of which was re-dissolved in 2% SDS, were measured by IB. C. hAß1-42 (2.2 µM) was incubated alone, or with FVIIa (10 nM) and TF (10 nM) in CaCl2-containing PBS at RT for indicated times; the aggregates were re-dissolved in 2% SDS and mixed with the supernatant fraction, separated by SDS-PAGE and stained by silver. FVIIa and TF were incubated without Aß1-42, as a control.

Fig 4: The FXII-FVII proteolysis pathway detects and degrades Aß1-42WT mice, FXII-/- mice and FVIItTA/tTA mice were treated with vehicle or hAß1-42 i.p.; blood samples were collected from the mice at 6 h after treatment. A. Plasma concentrations of Aß1-42, measured by ELISA. B.-J. Changes in plasma coagulation factors, measured by IB (7.5 µl plasma per sample) or ELISA. Arrows indicate cleaved fragments. Error bars in A and J indicate SD (n = 3). Data were analyzed by two-way ANOVA in A or one-way ANOVA in J, followed by Tukey multiple comparisons test. Data in A were log transformed before ANOVA, **** p < 0.0001; ns, not significant.

Fig 5: SRC binds to PRD in FXII and activates FXII but is degraded by FVIIaA. mSRC (40 nM) was incubated with FXII or a mutant (0.5 µM) in PBS at 37°C for 2 h, followed by IP and IB. B. mSRC (0, 20 and 200 nM) was incubated with FXII (0.97 nM) in ZnCl2-containing PBS at RT; FXII activation was measured by a chromogenic assay. Each value is mean ± SD (n = 3). C. mSRC (0.17 µM) was incubated alone or with FVIIa (10 nM) and TF (10 nM) in CaCl2-containing PBS at RT for indicated times, separated by SDS-PAGE and stained by silver. FVIIa and TF were incubated without mSRC as a control.