Previously, A42 has been shown to form SDS-stable complexes with ApoE2 and 3

Previously, A42 has been shown to form SDS-stable complexes with ApoE2 and 3.25 Because SDS-stable complex formation between A and its binding partners may impair protein function, studies aimed at identifying the nature of the SDS-stable complexes involving A may shed light on new mechanisms by which A may contribute to pathological processes. We have previously shown that A42 binding to fibrin(ogen) delays fibrinolysis by interfering with the binding of plasminogen and plasmin to fibrin8 and that A42 binds to the -chain of fibrinogen fragment D.7 We also confirmed A42 binding to fibrin D-dimer (supplemental Figure 5). fibrinogen -chain (384-393). Furthermore, we identified an additional A-binding site within the C region of fibrinogen. A binding to this C region blocked plasmin-mediated fibrin cleavage at this site, resulting in the generation of increased levels of a plasmin-resistant fibrin degradation fragment. Overall, our study elucidates the A-fibrinogen interaction and clarifies the mechanism by which A-fibrinogen binding delays fibrinolysis by plasmin. These results may facilitate the development of effective therapeutics against the A-fibrinogen interaction to treat cerebrovascular abnormalities in AD. Introduction Accumulating Fluralaner evidence implicates fibrin(ogen), the main protein component of blood clots, in Alzheimer disease (AD) pathogenesis.1-3 Activation of the coagulation cascade results in the cleavage of soluble fibrinogen to fibrin, which polymerizes to form an insoluble network. Because fibrin is occlusive4 and proinflammatory,5 its clearance (fibrinolysis) by plasmin is a tightly regulated process. Disturbances to fibrinolysis may therefore have significant consequences for occlusive and inflammatory pathology in various diseases, including AD. Indeed, increased fibrin accumulation in the brains of AD patients and mouse models is correlated with areas of neuronal dysfunction.6 We have previously identified the AD-related peptide, -amyloid (A), as a factor capable of modulating fibrin clot structure and stability.7,8 A42 binds fibrinogen with a efficiently degrades fibrin-A coaggregates in vitro and in a rat model.13 Furthermore, long-term treatment with RU-505, a specific inhibitor of the A-fibrinogen interaction, results in reduced thrombosis, decreased AD pathology, and improved cognitive performance in a mouse model of AD.2 Although both novel therapeutics targeting the A-fibrinogen interaction in AD are effective in vitro and in vivo, low selectivity of the enzyme from and micromolar half-maximal inhibition (IC50) levels of RU-505 limit their capabilities Fluralaner for clinical development. To improve selectivity and potency of therapeutics against the A-fibrinogen interaction, a better understanding of the A-fibrinogen interaction is needed. Here, we analyzed the region within A responsible for A-fibrinogen binding using biochemical approaches and examined the structural aspects of binding between A and fragment D of fibrinogen using X-ray crystallography. In addition, we further investigated the mechanism by which A-fibrinogen binding delays fibrinolysis by plasmin. Methods Preparation of A42 and fibrinogen fragment D A42 (Anaspec) was reconstituted in a minimal volume of 0.1% NH4OH and then diluted to the desired concentration with IL-1a antibody 50 mM Tris or phosphate-buffered saline (pH 7.4). Solubilized A42 was spun at 12?000 for 15 minutes to remove aggregated material14 and the concentration was established by bicinchoninic acid assay (Thermo Scientific). Fibrinogen fragment D was prepared and purified as previously described. 15 Identification of fibrinogen-binding domains on A Fibrinogen- or fragment DCbinding assay using biotinylated A fragments. Synthetic N-terminally biotinylated A fragments 1 to 16, 15 to 25, 22 to 41, and 1 to 42 (50 nM; Anaspec) were incubated with fibrinogen (5 nM; Calbiochem) or fragment D (100 nM) for 1 hour at room temperature (RT) in 50 mM Tris pH 7.4 containing 500 mM NaCl, 0.01% bovine serum albumin (BSA), protease inhibitor cocktail (Roche), and 0.01% NP-40 or 0.05% Tween-20. Streptavidin-coated magnetic beads (Dynabeads M-280; Thermo-Fisher) were added for 30 minutes, washed, and eluted with nonreducing 1 lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific). Eluates were analyzed by sodium dodecyl sulfateCpolyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20% Tris-glycine gradient gel (Life Sciences) followed by western blot using a polyclonal antibody against fibrinogen (Dako). For the AlphaLISA assay, various concentrations (0.02-20 M) of N-terminally biotinylated A fragments 1 to 16, 15 to 25, 22 to 41, and 1 to 42 (50 nM, Fluralaner Anaspec) were incubated with 1 nM fibrinogen for 30 minutes at RT in a final volume of 10 L of assay buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, 0.1% BSA) in white 384-well plates (Greiner). The mixture was incubated with the anti-fibrinogen antibody, 20 g/mL streptavidin-conjugated donor, and protein ACconjugated acceptor beads (PerkinElmer) for 90 minutes at RT. Samples were read by a PerkinElmer EnVision plate reader. A-fibrinogen interaction inhibition assay using nonbiotinylated A fragments. Various concentrations (0.05-20 M) of 16 nonbiotinylated A fragments listed in supplemental Figure 1 (available on the Web site) (Anaspec or rPeptide) were.