Abstract
In cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), the amyloid β (Aβ) peptide deposits along the vascular lumen, leading to degeneration and dysfunction of surrounding tissues. Activated coagulation factor XIIIa (FXIIIa) covalently cross-links proteins in blood and vasculature, such as in blood clots and on the extracellular matrix. Although FXIIIa co-localizes with Aβ in CAA, the ability of FXIIIa to cross-link Aβ has not been demonstrated. Using Western blotting, kinetic assays, and microfluidic analyses, we show that FXIIIa covalently cross-links Aβ40 into dimers and oligomers (kcat/Km = 1.5 × 105 m-1s-1), as well as to fibrin, platelet proteins, and blood clots under flow in vitro Aβ40 also increased the stiffness of platelet-rich plasma clots in the presence of FXIIIa. These results suggest that FXIIIa-mediated cross-linking may contribute to the formation of Aβ deposits in CAA and Alzheimer's disease.
Highlights
In cerebral amyloid angiopathy (CAA) and Alzheimer’s disease (AD), the amyloid  (A) peptide deposits along the vascular lumen, leading to degeneration and dysfunction of surrounding tissues
To test if A could be covalently cross-linked by factor XIIIa (FXIIIa), A was incubated with FXIIIa and changes in molecular mass were detected using Western blotting
With T101, there was a distinct band near 85 kDa, corresponding to the molecular mass of FXIIIa attached to A40, which has been reported previously [6]
Summary
To test if A could be covalently cross-linked by FXIIIa, A was incubated with FXIIIa and changes in molecular mass were detected using Western blotting. Because platelets contain the FXIII-A subunit, which can be activated by high concentrations of Ca2ϩ, A40 was incubated with platelets to test if platelet-derived FXIIIa could cross-link A40 to itself or to other proteins. The A bands formed with platelets had higher molecular mass than A dimers and trimers, suggesting A was cross-linked to platelet proteins. Both EDTA, which chelates the Ca2ϩ required for FXIIIa activity and platelet activation, and T101 prevented A oligomers from forming. To test whether FXIIIa cross-links A40 to blood clots formed under flow, plasma containing platelets and fluorescently tagged A40 were flowed through a microfluidic device. The Flemish mutation was cross-linked to fibrin to a greater extent than WT (Fig. 5, C and D)
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