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 the components of the circulatory system⁶. Aβ has a high-affinity interaction with fibrinogen^7,8, which also localizes to Aβ deposits in both AD patients and mouse models^9,10,11. Furthermore, the Aβ-fibrinogen interaction induces abnormal fibrin-clot formation and structure, as well as resistance to fibrinolysis^9,12. One therapeutic possibility in treating AD, is alleviating circulatory deficits by inhibiting the interaction between Aβ and fibrinogen^13,14. We, therefore, identified several small compounds inhibiting the Aβ-fibrinogen interaction using high throughput screening and medicinal chemistry approaches^13,14. To test the efficacy of Aβ-fibrinogen interaction inhibitors, we optimized two methods for the analysis of in vitro fibrin clot formation: clot turbidity assay and scanning electron microscopy (SEM)¹⁴.

Clot turbidity assay is a straight-forward and rapid method for monitoring fibrin clot formation using UV-visible spectroscopy. As the fibrin clot forms, light is increasingly scattered and the turbidity of the solution increases. Conversely, when Aβ is present, the structure of the fibrin clot is altered, and the turbidity of the mixture is reduced (Figure 1). The effect of inhibitory compounds can be assessed for the potential to restore clot turbidity from Aβ-induced abnormalities. While the turbidity assay allows for rapid analysis of multiple conditions, it provides limited information on the clot shape and structure. SEM, in which the topography of solid objects is revealed by electron probe, allows for the analysis of the 3D architecture of the clot^15,16,17,18 and the assessment of how the presence of Aβ and/or inhibitory compounds alters that structure^9,14. Both spectrometry and SEM are classical laboratory techniques that have been used for various purposes, for example, spectrophotometry is used for monitoring amyloid aggregation^19,20. Similarly, SEM is also used to analyze fibrin clot formed from the plasma of Alzheimer's, Parkinson's and thromboembolic stroke patients^21,22,23. The protocols presented here are optimized for assessing fibrin-clot formation in a reproducible and rapid manner.

The following protocol provides the instructions for the preparation of an in vitro fibrin-clot both with and without Aβ. It also details the methods to analyze the effect of Aβ on fibrin clot formation and structure. The effectiveness of these two methods for measuring the inhibition of the Aβ-fibrinogen interaction is demonstrated using TDI-2760, a small inhibitory compound¹⁴. These methods, both individually and together, allow for rapid and straightforward analysis of in vitro fibrin clot formation.

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 bath sonicator for 10 min at room temperature.
   5. Add 190 µL of 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.4) and pipette up and down gently.
   6. Remove the protein aggregates by centrifugation at 20,817 x g at 4 °C for 20 min.
   7. Incubate the supernatant at 4 °C for overnight and next day centrifuge the solution at 20,817 x g at 4 °C for 20 min to discard any further protein aggregates.
   8. Measure Aβ42 concentration by bicinchoninic acid (BCA) assay. Serial dilute purified bovine serum albumin (BSA) from 1 mg/mL to 0.0625 mg/mL to produce a protein standard, add 10 µL of each standard to the wells of a multi-well plate in triplicate. Dilute Aβ samples 1:4 and add 10 µL to the wells in triplicate. Mix BCA solutions A and B and add 200 µL to each well. Incubate for 30 min at 37 °C and read on a plate reader at 562 nm. Keep the remaining Aβ solution on ice and use this preparation for turbidity assay and SEM.
2. Prepare Fibrinogen solution
   1. Measure 20 mg of lyophilized fibrinogen powder into a 15 mL tube and re-suspend with pre-warmed 2 mL of 20 mM hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer (pH 7.4).
   2. Incubate in a 37 °C water bath for 10 min.
   3. Filter solution through a 0.2 µm syringe filter and store at 4 °C for 30 min. Take out the solution from 4 °C and filter again through 0.1 µm syringe filter to remove fibrinogen aggregates or pre-existing fibrin.
   4. Measure the fibrinogen concentration by BCA assay. Follow the instructions from Step 1.1.8. Keep the remaining fibrinogen solution at 4 °C and use this preparation for turbidity assay and SEM.

2. Clot Turbidity Assay

1. To each experimental well of the 96-well plate, add 20 µL of 30 µM Aβ42 solution from Step 1.1.8 so that its final concentration is 3.0 µM in 200 µL of the buffer. Add the same volume of 5% DMSO in 50 mM Tris buffer (pH 7.4) to buffer control wells in the 96-well plate.
   NOTE: The exact volume of Aβ42 at this step is not important. For low concentration preparations, a higher volume can be used, and the volume of the clot formation buffer can be adjusted. The final volume should remain 200 µL.
2. If testing inhibitory compounds, dilute the compound to the working concentration as DMSO. Add compound or DMSO alone for a control to the wells with Aβ42 and mix well.
   NOTE: Aβ42 solution should form a discrete droplet at the bottom of the well, the compound or DMSO should be pipetted directly into the center of the droplet.
3. Dilute the fibrinogen stock solution from Step 1.2.4 in clot formation buffer (20 mM HEPES buffer (pH 7.4), 5 mM CaCl₂, and 137 mM NaCl) and add it to Aβ42 containing and control wells of the 96-well plate. Adjust the volume of the fibrinogen solution so that its final concentration becomes 1.5 µM in total 200 µL reaction volume. Pipette the solution slowly and avoid forming bubbles. Incubate the plate at room temperature for 30 min shaking on a rotating platform.
   NOTE: The volume of fibrinogen solution may vary depending on its stock concentration, but the total volume in each well should be 170 µL while incubation.
4. Prepare thrombin solution by dissolving commercially purified thrombin powder in ddH₂O to make a stock solution of 50 U/mL. Dilute to the 5 U/mL working solution in 20 mM HEPES buffer (pH 7.4) directly before use.
5. After 30 min of incubation, simultaneously add 30 µL of thrombin solution (5 U/mL) into the fibrinogen solutions in the 96-well plate from Step 2.3 using multichannel pipette. Addition of thrombin will immediately initiate the clot formation. Therefore, add thrombin directly into the center of the fibrinogen solution with care to avoid forming bubbles.
   NOTE: The activity of thrombin can vary between experimental conditions, as well as thrombin lots. The correct concentration required to robustly produce clots may have to be determined empirically.
6. Read the absorbance of in vitro clot on a plate reader immediately following the thrombin addition. Measure the absorbance at 350 nm over the course of 10 min, every 30-60 s. Perform the entire assay at room temperature.
   NOTE: Some inhibitor compounds may alter the solution absorbance at 350 nm, in which case the turbidity can be measured at 405 nm.

3. Scanning Electron Microscopy

1. Preparation of clot, fixation, and washing
   1. Place clean siliconized glass circle cover slides (12 mm) into a 12-well or 24-well plate using forceps.
   2. Prepare fibrinogen in clot formation buffer. See protocol step 1.2 for details.
   3. To evaluate the effect of Aβ-fibrinogen interaction inhibitors on fibrin clot structure, follow the instructions for incubation as described for the turbidity assay (Step 2). Always include control wells which contain fibrinogen in the absence of both Aβ and inhibitors.
   4. Pipette 80 µL of the fibrinogen mixture from Step 3.1.3 on the cover slides. Gently spread the solution so that it is evenly distributed on the cover slide.
   5. Dilute thrombin stock (50 U/mL) into 20 mM HEPES buffered saline to a final concentration of 2.5 U/mL and add 20 µL directly to the center of to the fibrinogen solution without mixing.
      NOTE: If required, a higher thrombin working concentration (5 U/mL) can be used.
   6. Cover the 12-well plate with a plastic lid and leave it at room temperature for 30-60 min.
   7. While clotting reaction is running, prepare dehydration and fixation solutions. Prepare sodium cacodylate buffer (0.1 M, pH 7.4). Dilute 10% glutaraldehyde stock solution in sodium cacodylate buffer (0.1 M, pH 7.4) to make a 2% working solution of glutaraldehyde. Keep all these solutions on ice. Freshly diluted glutaraldehyde (2%) should be used within 1 week, when properly stored in a refrigerator at 4 °C.
   8. Dilute absolute ethanol (100%) in double distilled water (80%, 50%, and 20% ethanol). Keep these ethanol solutions and absolute ethanol (100%) on ice.
      ​CAUTION: Use care when handling sodium cacodylate and glutaraldehyde, perform these steps in a chemical hood.
   9. After 30 min, gently wash the clots with ice-cold sodium cacodylate buffer (0.1 M) twice. Add 2 mL of the buffer to each well such that the clot is completely submerged. Cover the well plate with a lid and leave it for 2 min at room temperature. After 2 min, gently remove the buffer using a 1 mL pipette or narrow stem transfer pipette. Repeat once.
   10. Fix the clots with ice-cold glutaraldehyde (2%). Keeping the well plate on ice, add around 2-3 mL of 2% glutaraldehyde to each well. Make sure the clots are completely submerged. Cover and leave the plate on ice for 30 min.
   11. After 30 min, gently remove the glutaraldehyde from each well and wash the clots using sodium cacodylate buffer (0.1 M) as described above (2 min, twice). Keep the well plate on ice.
2. Serial dehydration of clot and critical point drying
   1. Dehydrate the clots in a graded series of ice-cold ethanol washes prepared in Step 3.1.8 (20%, 50%, 80%, 100%, and again100%) for 5 min each. For each ethanol series, add 2-3 mL, making sure the clots are submerged. Cover and incubate on ice.
   2. After each ethanol step, remove the ethanol solution using a 1 mL pipette or disposable pipette dropper. Do not remove the ethanol completely. Make sure the clot surface is not exposed to air.
      NOTE: Dehydration in absolute ethanol (100%) should be performed twice.
   3. While keeping the sample submerged in the final 100% ethanol, transfer to a critical point dryer (CPD). Use a CPD sample holder or a cover slip holder with a washer for transferring the slides into the CPD chamber. Place at least one washer between each slide.
   4. Take out the cover slide from the CPD chamber and mount it on an SEM stub using carbon tape.
3. Sputter coating and imaging
   1. Transfer all the samples with SEM stub to the sputter coating chamber.
   2. Sputter coat less than 20 nm of gold/palladium or other conductive materials, such as carbon, using a vacuum sputter coater.
      NOTE: We have used 18 nm of gold/palladium coating. The sputter coating was performed for 45 s and the coating speed was 4 Å/ s. Sputter coated samples are stable when kept properly in a dry environment at room temperature for few weeks. SEM analysis can be performed anytime.
   3. Acquire images on a scanning electron microscope equipped with the SE2 detector at 4 kV.
      NOTE: The image pixel size in this image set range from 13 nm- 31 nm.

Results

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, the t...

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

List of materials used in this article

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	Tousimis
Denton Desk IV Coater with Gold/Palladium target	Denton Vacuum
Leo 1550 FE-SEM	Carl Zeiss
Smart SEM Software	Carl Zeiss