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

Summary

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.

Abstract

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.

Introduction

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 neurotoxicity^1,2. 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 ....

Protocol

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. Films can be stored at -20 °C.
   4. Reconstitute monomeric Aβ42 film in 10 µL of fresh dimethyl sulfoxide (DMSO) by agitation in a ....

Discussion

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 interaction^13,14. Using these two a.......

Materials

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