This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (Aβ) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). Aβ, which forms neurotoxic amyloid aggregates in Alzheimer's disease (AD), has been shown to interact with fibrinogen. This Aβ-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. Aβ-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the Aβ-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of Aβ and Aβ-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of Aβ-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate Aβ-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified Aβ-fibrinogen interaction inhibitor.
All Science Journal Classification (ASJC) codes
- Chemical Engineering(all)
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)
- Alzheimer's disease
- Issue 141
- Scanning electron microscopy