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Materials

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Biochemistry

Analysis of β-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Published: November 30th, 2018

DOI:

10.3791/58475

1Patricia and John Rosenwald Laboratory of Neurobiology and Genetics, Rockefeller University, 2Electron Microscopy Resource Center, Rockefeller University
* These authors contributed equally

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

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.

Alzheimer's disease (AD), a neurodegenerative disease leading to cognitive decline in elderly patients, predominately arises from abnormal beta-amyloid (Aβ) expression, aggregation and impaired clearance resulting in neurotoxicity1,2. Despite the well-characterized association between Aβ aggregates and AD3, the precise mechanisms underlying the disease pathology are not well understood4. Increasing evidence suggests that neurovascular deficits play a role in the progression and severity of AD5, as Aβ directly interacts with ....

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1. Preparation of Aβ42 and Fibrinogen for Analysis

  1. Prepare monomeric Aβ42 from lyophilized powder
    1. Warm Aβ42 powder to room temperature and spin down at 1,500 x g for 30 s.
    2. Add 100 µL of ice-cold hexafluoroisopropanol (HFIP) per 0.5 mg of Aβ42 powder and incubate for 30 min on ice.
      CAUTION: Use care when handling HFIP and perform all steps in a chemical hood.
    3. Prepare 20 µL aliquots and let the films air dry in a chemical hood for 2-3 h. Fi.......

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In the in vitro clotting (turbidity) assay, the enzyme thrombin cleaves fibrinogen, resulting in the formation of the fibrin network24. This fibrin clot formation causes scattering of the light passing through the solution, resulting in increased turbidity (Figure 1), plateauing before the end of the reading period (Figure 2, green). When the fibrinogen was incubated in the presence of Aβ42, .......

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The methods described here provide a reproducible and rapid means of assessing fibrin clot formation in vitro. Furthermore, the simplicity of the system makes the interpretation of how Aβ affects the fibrin clot formation and structure relatively straight-forward. In this lab's previous publication, it was shown that these assays can be used to test compounds for their ability to inhibit the Aβ-fibrinogen interaction13,14. Using these two a.......

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Authors thank Masanori Kawasaki, Kazuyoshi Aso, and Michael Foley from Tri-Institutional Therapeutics Discovery Institute (TDI), New York for synthesis of Aβ-fibrinogen interaction inhibitors and their valuable suggestions. Authors also thank members of the Strickland lab for helpful discussion. This work was supported by NIH grant NS104386, the Alzheimer's Drug Discovery Foundation, and Robertson Therapeutic Development Fund for H.A., NIH grant NS50537, the Tri-Institutional Therapeutics Discovery Institute, Alzheimer's Drug Discovery Foundation, Rudin Family Foundation, and John A. Herrmann for S.S.

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Name Company Catalog Number Comments
Fibriogen, Plasminogen-Depleted, Plasma EMD Millipore 341578 keep lid parafilm wrapped to avoid exposure to moisture
Beta-Amyloid (1-42), Human Anaspec AS-20276
Thrombin from human plasma Sigma-Aldrich T7009
1,1,1,3,3,3-Hexafluoro-2-propanol, Greater Than or Equal to 99% Sigma-Aldrich 105228
DIMETHYL SULFOXIDE (DMSO), STERILE-FILTERED Sigma-Aldrich D2438
Pierce BCA Protein Assay Kit Thermo Scientific 23225
Tris Base Fischer Scientific BP152
HEPES Fischer Scientific BP310
NaCl Fischer Scientific S271
CaCl2 Fischer Scientific C70
Filter Syringe, 0.2µM, 25mm Pall 4612
Millex Sterile Syringe Filters, 0.1 um, PVDF, 33 mm dia. Millipore SLVV033RS
Solid 96 Well Plates High Binding Certified Flat Bottom Fischer Scientific 21377203
Spectramax Plus384 Molecular Devices 89212-396
Centrifuge, 5417R Eppendorf 5417R
Branson 200 Ultrasonic Cleaner Fischer Scientific 15-337-22
Lab Rotator Thermo Scientific 2314-1CEQ
Spare washers for cover slip holder Tousimis 8766-01
Narrow Stem Pipets - Sedi-Pet Electron Microscopy Sciences (EMS) 70967-13
Sample holder for CPD (Cover slip holder) Tousimis 8766
Mount Holder Box, Pin Type Electron Microscopy Sciences (EMS) 76610
Round glass cover slides (12 mm) Hampton Research HR3-277
10% Glutaraldehyde Electron Microscopy Sciences (EMS) 16120
Ethanol Decon Labs 11652
24 well plate Falcon 3047
Na Cacodylate Electron Microscopy Sciences (EMS) 11652
SEM Stubs, Tapered end pin. Electron Microscopy Sciences (EMS) 75192
PELCO Tabs, Carbon Conductive Tabs, 12mm OD Ted Pella 16084-1
Autosamdri-815 Critical Point Dryer with Gold/Palladium target Tousimis
Denton Desk IV Coater Denton Vacuum
Leo 1550 FE-SEM Carl Zeiss
Smart SEM Software Carl Zeiss

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