Authors thank Masanori Kawasaki, Kazuyoshi Aso, and Michael Foley from Tri-Institutional Therapeutics Discovery Institute (TDI), New York for synthesis of A&#946;-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&#39;s Drug Discovery Foundation, and Robertson Therapeutic Development Fund for H.A., NIH grant NS50537, the Tri-Institutional Therapeutics Discovery Institute, Alzheimer&#39;s Drug Discovery Foundation, Rudin Family Foundation, and John A. Herrmann for S.S.

1. Preparation of A&#946;42 and Fibrinogen for Analysis
<ol>
	<li>Prepare monomeric A&#946;42 from lyophilized powder
	<ol>
		<li>Warm A&#946;42 powder to room temperature and spin down at 1,500 x g for 30 s.</li>
		<li>Add 100 &#181;L of ice-cold hexafluoroisopropanol (HFIP) per 0.5 mg of A&#946;42 powder and incubate for 30 min on ice. 
		CAUTION: Use care when handling HFIP and perform all steps in a chemical hood.</li>
		<li>Prepare 20 &#181;L aliquots and let the films air dry in a chemical hood for 2-3 h. Films can be stored at -20 &#176;C.</li>
		<li>Reconstitute monomeric A&#946;42 film in 10 &#181;L of fresh dimethyl sulfoxide (DMSO) by agitation in a bath sonicator for 10 min at room temperature.</li>
		<li>Add 190 &#181;L of 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.4) and pipette up and down gently.</li>
		<li>Remove the protein aggregates by centrifugation at 20,817 x g at 4 &#176;C for 20 min.</li>
		<li>Incubate the supernatant at 4 &#176;C for overnight and next day centrifuge the solution at 20,817 x g at 4 &#176;C for 20 min to discard any further protein aggregates.</li>
		<li>Measure A&#946;42 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 &#181;L of each standard to the wells of a multi-well plate in triplicate. Dilute A&#946; samples 1:4 and add 10 &#181;L to the wells in triplicate. Mix BCA solutions A and B and add 200 &#181;L to each well. Incubate for 30 min at 37 &#176;C and read on a plate reader at 562 nm. Keep the remaining A&#946; solution on ice and use this preparation for turbidity assay and SEM.</li>
	</ol>
	</li>
	<li>Prepare Fibrinogen solution
	<ol>
		<li>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).</li>
		<li>Incubate in a 37 &#176;C water bath for 10 min.</li>
		<li>Filter solution through a 0.2 &#181;m syringe filter and store at 4 &#176;C for 30 min. Take out the solution from 4 &#176;C and filter again through 0.1 &#181;m syringe filter to remove fibrinogen aggregates or pre-existing fibrin.</li>
		<li>Measure the fibrinogen concentration by BCA assay. Follow the instructions from Step 1.1.8. Keep the remaining fibrinogen solution at 4 &#176;C and use this preparation for turbidity assay and SEM.</li>
	</ol>
	</li>
</ol>
2. Clot Turbidity Assay
<ol>
	<li>To each experimental well of the 96-well plate, add 20 &#181;L of 30 &#181;M A&#946;42 solution from Step 1.1.8 so that its final concentration is 3.0 &#181;M in 200 &#181;L 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 A&#946;42 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 &#181;L.</li>
	<li>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 A&#946;42 and mix well. 
	NOTE: A&#946;42 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.</li>
	<li>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 A&#946;42 containing and control wells of the 96-well plate. Adjust the volume of the fibrinogen solution so that its final concentration becomes 1.5 &#181;M in total 200 &#181;L 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 &#181;L while incubation.</li>
	<li>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.</li>
	<li>After 30 min of incubation, simultaneously add 30 &#181;L 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.</li>
	<li>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.</li>
</ol>
3. Scanning Electron Microscopy
<ol>
	<li>Preparation of clot, fixation, and washing
	<ol>
		<li>Place clean siliconized glass circle cover slides (12 mm) into a 12-well or 24-well plate using forceps.</li>
		<li>Prepare fibrinogen in clot formation buffer. See protocol step 1.2 for details.</li>
		<li>To evaluate the effect of A&#946;-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 A&#946; and inhibitors.</li>
		<li>Pipette 80 &#181;L 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.</li>
		<li>Dilute thrombin stock (50 U/mL) into 20 mM HEPES buffered saline to a final concentration of 2.5 U/mL and add 20 &#181;L 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.</li>
		<li>Cover the 12-well plate with a plastic lid and leave it at room temperature for 30-60 min.</li>
		<li>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 &#176;C.</li>
		<li>Dilute absolute ethanol (100%) in double distilled water (80%, 50%, and 20% ethanol). Keep these ethanol solutions and absolute ethanol (100%) on ice. 
		&#8203;CAUTION: Use care when handling sodium cacodylate and glutaraldehyde, perform these steps in a chemical hood.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Serial dehydration of clot and critical point drying
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Take out the cover slide from the CPD chamber and mount it on an SEM stub using carbon tape.</li>
	</ol>
	</li>
	<li>Sputter coating and imaging
	<ol>
		<li>Transfer all the samples with SEM stub to the sputter coating chamber.</li>
		<li>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 &#197;/ 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 A&#946; affects the fibrin clot formation and structure relatively straight-forward. In this lab&#39;s previous publication, it was shown that these assays can be used to test compounds for their ability to inhibit the A&#946;-fibrinogen interaction13,14. Using these two assays, a series of synthesized compounds were analyzed for the ability to inhibit the A&#946;-fibrinogen interaction. Actually, the clot turbidity assay can be used to narrow the pool of hit compounds to those which have therapeutic potential, as not all compounds that can inhibit the A&#946;-fibrinogen interaction can also restore fibrin clot formation.
There are a few aspects of the turbidity assay that may require troubleshooting or limit the uses of this technique. The major limitation is that there can be some variations between the experiments that may complicate the interpretation of the results. Some of this variation is to be due to the thrombin activity. To troubleshoot for thrombin activity, users can test multiple concentrations of thrombin for clot-formation activity prior to beginning experimentation with A&#946; and test compounds. Under the experimental conditions presented here, A&#946;, in the absence of fibrinogen, does not increase the turbidity of the solution above background levels indicating that observed turbidity curves are due to fibrin polymerization. However, A&#946; is an aggregation-prone peptide and a higher concentration of A&#946; solution when kept for a very long time at room temperature or 37 &#176;C (several hours) can form fibrillar aggregates which may result in increased turbidity. If analyzing the effect an A&#946; solution that has been stored for long periods of time at room temperature or warmer, the aggregation status of the solution can be determined by transmission electron microscopy. In the protocol described here, freshly prepared A&#946; and fibrinogen solutions are used, which should eliminate aggregation issues. However, to ensure the quality of these preparations they have been assessed by transmission electron microscopy. Additionally, when using a new lot of commercial A&#946; peptide, the extent of oligomerization and quantity of oligomers should be assessed by transmission electron microscopy. Because there might be lot to lot variation in peptide quality and ratio of preformed aggregates and monomeric A&#946;, which certainly affects the oligomerization rate. It is advisable to check the concentration of A&#946; solution (BCA method) before incubating for oligomerization. To minimize these variations, we tried using at least 0.1 mg/mL of A&#946; solution for oligomer formation reaction.
Because this assay is also sensitive to small environmental changes such as temperature and motion, turbidity readings from different experiments should not be analyzed together. This means that the number of conditions that can be compared is limited by the number of wells that thrombin can be simultaneously added to with a multichannel pipette (i.e., 12). Users should also be aware that viscosity or color in the buffers and test compounds can lead to a high background turbidity, obscuring the signal from the fibrin clot and making interpretation of the experiment difficult. However, if all of the controls are included in each experiment, it should be possible to readily determine if background signal is altering the turbidity absorbance.
The turbidity assay provides information about whether formation of the fibrin clot is hindered by A&#946; and furthermore if inhibitor compounds can alleviate this effect. However, it does not reveal how the structure of the fibrin clot is altered in response to A&#946; and/or hit compounds. This question can be addressed by SEM, as this allows for direct visualization of the clot architecture. SEM is a well-established technique and is routinely used for visualizing clot structure from purified fibrinogen or from plasma. However, the traditional clot preparation process for SEM analysis can be complicated and time-consuming. Furthermore, small differences in protocols between different research groups can make it challenging to replicate results24. To address these issues this optimized protocol was developed, with which the effect of A&#946; to induce thinner fibrin strands and clumps of protein can be observed (Figure 3). As seen with the turbidity assay, TDI-2760 alleviates the effect of A&#946;, restoring the typical structure of fibrin bundles (Figure 3).
There are a few steps in the SEM sample preparation, which may also require troubleshooting. When using very low concentrations of fibrinogen (0.5 &#181;M or less), the clots may not be firmly attached to the glass slide and can be damaged/washed away during the fixation/washing steps. If using low concentrations of fibrinogen, the clot formation time, between the addition of thrombin and fixation, can be increased up to several hours, as this may increase the clot stability. Also, users should avoid using high strength phosphate buffer or phosphate buffered saline for both the turbidity and SEM assays, as phosphate ions may interfere with calcium-mediated clot formation process. If using any other high salt containing buffer for clot formation and SEM analysis, after fixation, washing steps should also be performed with cold ddH2O.
SEM analysis of non-conductive samples such as fibrin clots requires coating with charged particles such as, gold, palladium, silver or carbon. Coating thickness is an important factor in SEM imaging, as it is possible that a very thick metal coating can mask the ultra-structure surface topography of the fibrin clot. In this protocol, thin gold/palladium coating (less than 20 nm) are used for sputter coating. To achieve less than 20 nm thickness, the sputtering was done for 45 s with a coating rate of 4 &#197;/s. This thin coating (18 nm) does not appear to mask the surface features of fibrin assembly. However, if concerned about masking the ultra-structure of the clot, carbon coating can be used as an alternative to gold/palladium, as it will leave less &#34;islets&#34; of coating molecules on the material.
While the focus here has been on the A&#946;-fibrinogen interaction, this protocol can be readily modified to analyze the interaction of other proteins or compounds with the fibrin clot. Following these instructions, investigators should be able to reproduce in vitro fibrin clot formation and perform analysis with these streamlined clot-turbidity assay and SEM protocols. As shown here with the previously published inhibitor compound TDI-2760, these methods provide valuable information regarding fibrin clot formation that can be applied to further studies both in vitro and in vivo.

Alzheimer's disease (AD), a neurodegenerative disease leading to cognitive decline in elderly patients, predominately arises from abnormal beta-amyloid (A&#946;) expression, aggregation and impaired clearance resulting in neurotoxicity1,2. Despite the well-characterized association between A&#946; 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 A&#946; directly interacts with the components of the circulatory system6. A&#946; has a high-affinity interaction with fibrinogen7,8, which also localizes to A&#946; deposits in both AD patients and mouse models9,10,11. Furthermore, the A&#946;-fibrinogen interaction induces abnormal fibrin-clot formation and structure, as well as resistance to fibrinolysis9,12. One therapeutic possibility in treating AD, is alleviating circulatory deficits by inhibiting the interaction between A&#946; and fibrinogen13,14. We, therefore, identified several small compounds inhibiting the A&#946;-fibrinogen interaction using high throughput screening and medicinal chemistry approaches13,14. To test the efficacy of A&#946;-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 A&#946; 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 A&#946;-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 clot15,16,17,18 and the assessment of how the presence of A&#946; and/or inhibitory compounds alters that structure9,14. Both spectrometry and SEM are classical laboratory techniques that have been used for various purposes, for example, spectrophotometry is used for monitoring amyloid aggregation19,20. Similarly, SEM is also used to analyze fibrin clot formed from the plasma of Alzheimer's, Parkinson's and thromboembolic stroke patients21,22,23. 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 A&#946;. It also details the methods to analyze the effect of A&#946; on fibrin clot formation and structure. The effectiveness of these two methods for measuring the inhibition of the A&#946;-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.

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

analysis of &#946;-amyloid-induced abnormalities on fibrin clot structure by spectroscopy and scanning electron microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-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 A&#946;-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 A&#946; and A&#946;-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 A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-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 A&#946;-fibrinogen interaction inhibitor.

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 A&#946;42, the turbidity of the solution decreased, with the curve reaching a maximum height of roughly half that of fibrinogen alone (Figure 2A, blue). In a recent publication, a series of A&#946; aggregation blockers were synthesized and assessed for their ability to inhibit the A&#946;-fibrinogen interaction, identifying the compound TDI-276014. We have used TDI-2760 in our study to block A&#946;-fibrinogen interaction. As reported previously, in the presence of TDI-2760, the effect of A&#946; was ameliorated, as the turbidity was higher than with A&#946; alone (Figure 2A, red). The effect of TDI-2760 does not appear to be due to background turbidity as the compound did not change the fibrin clot turbidity when A&#946; was absent (Figure 2A, purple). The in vitro clotting turbidity assay described here is a rapid and simple method by which fibrin-clot formation and factors that may attenuate that process can be observed. GPRP, which is known to interfere with fibrin polymerization via interfering with fibrin monomer knob-hole interactions18,25 can be used as a positive control for the turbidity assay (Figure 2B). Consistent with the earlier reports25, with the presence of GPRP peptide, the fibrin clot turbidity was significantly reduced as compared to the fibrin clot formation with its absence (Figure 2B, blue vs green).
Following the same protocol and conditions as the turbidity assay described above, fibrin clots were prepared in the presence and absence of A&#946; and/or TDI-2760. The clots were then processed for electron microscopy by fixation, dehydration, critical point drying, and gold-sputter coating (Figure 1). Fibrinogen in the presence of only thrombin and CaCl2 formed a fibrin mesh, with elongated and intercalated threads of fibrin as well as larger bundles (Figure 3A). When A&#946; was present, the fibrin threads became thinner, with several sticky clumps/aggregates indicating A&#946;-induced structural abnormalities (Figure 3B). Consistent with the turbidity assay results, TDI-2760 partially restored the structure of the fibrin clot from A&#946;-induced changes, as fewer clumps were present (Figure 3C). Together with the turbidity assay, SEM reveals the extent and quality of A&#946;-induced changes to fibrin clot formation, as well as the effectiveness of an inhibitory compound, TDI-2760.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58475/58475fig1.jpg" /> 
Figure 1: Schematic representation of fibrin clot analysis by turbidity assay and SEM. The&#160;schematic shows the steps involved in fibrin clot analysis and the effect of A&#946; on fibrin clot turbidity and structural topography. The scale bars of SEM images (bottom left) are 2 &#181;m and the magnification is 10,000X. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58475/58475fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58475/58475fig2.jpg" /> 
Figure 2: Measurement of the effect A&#946;42 on in vitro fibrin clot formation by turbidity assay. (A) Fibrinogen was incubated in the presence and absence of A&#946;42. Clot formation was induced by thrombin, resulting in increased turbidity of the solution (green). In the presence of A&#946;42, the fibrin clot was abnormally structured, resulting in decreased turbidity (blue). The compound TDI-2760 or DMSO were incubated with the fibrinogen solution, both with and without A&#946;42. TDI-2760 restored A&#946;42-induced decrease of turbidity (red) without altering the normal clot formation (purple). (B) The turbidity of a known fibrinolysis inhibitor, GPRP was also measured as a positive control for this assay. Fibrinogen was incubated in the presence and absence of 1.5 &#181;M GPRP and the turbidity measured after adding thrombin. Similar to A&#946;42, the turbidity of fibrin clot formation was significantly reduced in the presence of GPRP (blue versus green). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58475/58475fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58475/58475fig3.jpg" /> 
Figure 3: Scanning electron micrographs of A&#946;-induced abnormalities to fibrin clot architecture. Scanning electron micrographs of fibrin clot structure obtained from purified fibrinogen (A), fibrinogen + A&#946;42 (B), fibrinogen + A&#946;42+TDI-2760 (C). Clot formation was initiated by adding thrombin and CaCl2 to the mixture. The structural analysis revealed that clots formed in the presence of A&#946;42 are thinner and abnormally clumped compared to fibrinogen by itself. TDI-2760, which is known to inhibit A&#946;42-fibrinogen interaction, partially corrected the A&#946;42 induced structural abnormality of the fibrin clot. Scale bar is 1 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58475/58475fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Î²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy at JoVE.com

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

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 (A&#946;) protein on clot formation and ...

analysis-amyloid-induced-abnormalities-on-fibrin-clot-structure

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.1K Views.  Rockefeller University. Het belangrijkste voordeel van deze op plaat gebaseerde troebelheidstest is dat het snel is en het mogelijk maakt om verschillende amyloïde-bètafibrinogen-interactieremmers in één keer te screenen zonder geavanceerde setup of vereisten. De hoofdverbindingen die zich in de fibrin troebelheidstest bevinden, kunnen verder worden geëvalueerd op hun vermogen om a-bèta-geïnduceerde structurele afwijking in de fibrinstols te herstellen die worden geanalyseerd door elektronenmicroscopie te sca...

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

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

Medicine

Characterizing Spherulites with Polarization-Sensitive Two-Photon Microscopy

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

Author Spotlight: Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy 

This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid fibrils.

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue ...

Chemistry

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Neuroscience

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Analyzing Amyloid Structures in a Tissue Section Using Fluorescence Lifetime Imaging Microscopy

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/56279/imaging-amyloid-tissues-stained-with-luminescent-conjugated'>Source: Nystr&#246;m, S., et al.,&#160;Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging. J. Vis. Exp.&#160;(2017).</a>This video demonstrates analyzing amyloid structures with fluorescence lifetime imaging microscopy or FLIM, using hFTAA dye to reveal compact cores as blue/green and unstable peripheries as red/yellow in color-coded images, reflecting amyloid organization.

Source: Nystr&#246;m, S., et al.,&#160;Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy ...

JoVE Encyclopedia of Experiments

Neuroimaging

Neurodegeneration & Disease Monitoring

UV-Visible Spectroscopy for Monitoring Fibrin Clot Formation

Source: Singh, P. K. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58475">Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy.</a>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the use of a UV-visible spectrophotometer for monitoring insoluble fibrin clot formation as a function of the change in the solution turbidity. The UV-visible spectrophotometer measures the solution&#8217;s absorbance over time, which represents the turbidity change due to the insoluble fibrin clot formation.

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

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