Name: analysis of &#946;-amyloid-induced abnormalities on fibrin clot structure by spectroscopy and scanning electron microscopy
Uploaded: 2018-11-30T14:30:00
Description: 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

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. Fi...

<ol>
	<li>Selkoe, D. J., Schenk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+molecular+understanding+predicts+amyloid-based+therapeutics.">Alzheimer's disease: molecular understanding predicts amyloid-based therapeutics.</a> Annual Review of Pharmacology and Toxicology. 43, 545-584 (2003).</li><li>Kurz, A., Perneczky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+clearance+as+a+treatment+target+against+Alzheimer's+disease.">Amyloid clearance as a treatment target against Alzheimer's disease.</a> Journal of Alzheimer's Disease. 24, 61-73 (2011).</li><li>Benilova, I., Karran, E., De Strooper, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+toxic+Abeta+oligomer+and+Alzheimer's+disease:+an+emperor+in+need+of+clothes.">The toxic Abeta oligomer and Alzheimer's disease: an emperor in need of clothes.</a> Nature Neuroscience. 15 (3), 349-357 (2012).</li><li>Karran, E., Hardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critique+of+the+drug+discovery+and+phase+3+clinical+programs+targeting+the+amyloid+hypothesis+for+Alzheimer+disease.">A critique of the drug discovery and phase 3 clinical programs targeting the amyloid hypothesis for Alzheimer disease.</a> Annals of Neurology. 76 (2), 185-205 (2014).</li><li>Yoshikawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+cerebral+blood+flow+in+Alzheimer+disease+and+vascular+dementia.">Heterogeneity of cerebral blood flow in Alzheimer disease and vascular dementia.</a> AJNR, American Journal of Neuroradiology. 24 (7), 1341-1347 (2003).</li><li>Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+will+out:+vascular+contributions+to+Alzheimer's+disease.">Blood will out: vascular contributions to Alzheimer's disease.</a> The Journal of Clinical Investigation. 128 (2), 556-563 (2018).</li><li>Ahn, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+peptide+beta-amyloid+interacts+with+fibrinogen+and+induces+its+oligomerization.">Alzheimer's disease peptide beta-amyloid interacts with fibrinogen and induces its oligomerization.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (50), 21812-21817 (2010).</li><li>Zamolodchikov, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+structural+analysis+of+the+interaction+between+beta-amyloid+and+fibrinogen.">Biochemical and structural analysis of the interaction between beta-amyloid and fibrinogen.</a> Blood. 128 (8), 1144-1151 (2016).</li><li>Cortes-Canteli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+beta-amyloid+association+alters+thrombosis+and+fibrinolysis:+a+possible+contributing+factor+to+Alzheimer's+disease.">Fibrinogen and beta-amyloid association alters thrombosis and fibrinolysis: a possible contributing factor to Alzheimer's disease.</a> Neuron. 66 (5), 695-709 (2010).</li><li>Cortes-Canteli, M., Mattei, L., Richards, A. T., Norris, E. H., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+deposited+in+the+Alzheimer's+disease+brain+promotes+neuronal+degeneration.">Fibrin deposited in the Alzheimer's disease brain promotes neuronal degeneration.</a> Neurobiology of Aging. 36 (2), 608-617 (2015).</li><li>Liao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+characterization+of+postmortem+amyloid+plaques+isolated+by+laser+capture+microdissection.">Proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection.</a> The Journal of Biological Chemistry. 279 (35), 37061-37068 (2004).</li><li>Zamolodchikov, D., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abeta+delays+fibrin+clot+lysis+by+altering+fibrin+structure+and+attenuating+plasminogen+binding+to+fibrin.">Abeta delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin.</a> Blood. 119 (14), 3342-3351 (2012).</li><li>Ahn, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Abeta-fibrinogen+interaction+inhibitor+rescues+altered+thrombosis+and+cognitive+decline+in+Alzheimer's+disease+mice.">A novel Abeta-fibrinogen interaction inhibitor rescues altered thrombosis and cognitive decline in Alzheimer's disease mice.</a> The Journal of Experimental Medicine. 211 (6), 1049-1062 (2014).</li><li>Singh, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminopyrimidine+Class+Aggregation+Inhibitor+Effectively+Blocks+Abeta-Fibrinogen+Interaction+and+Abeta-Induced+Contact+System+Activation.">Aminopyrimidine Class Aggregation Inhibitor Effectively Blocks Abeta-Fibrinogen Interaction and Abeta-Induced Contact System Activation.</a> Biochemistry. 57 (8), 1399-1409 (2018).</li><li>Pretorius, E., Mbotwe, S., Bester, J., Robinson, C. J., Kell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+induction+of+anomalous+and+amyloidogenic+blood+clotting+by+molecular+amplification+of+highly+substoichiometric+levels+of+bacterial+lipopolysaccharide.">Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide.</a> Journal of the Royal Society Interface. 13 (122), (2016).</li><li>Veklich, Y., Francis, C. W., White, J., Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+studies+of+fibrinolysis+by+electron+microscopy.">Structural studies of fibrinolysis by electron microscopy.</a> Blood. 92 (12), 4721-4729 (1998).</li><li>Weisel, J. W., Nagaswami, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+modeling+of+fibrin+polymerization+kinetics+correlated+with+electron+microscope+and+turbidity+observations:+clot+structure+and+assembly+are+kinetically+controlled.">Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled.</a> Biophysical Journal. 63 (1), 111-128 (1992).</li><li>Chernysh, I. N., Nagaswami, C., Purohit, P. K., Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+clots+are+equilibrium+polymers+that+can+be+remodeled+without+proteolytic+digestion.">Fibrin clots are equilibrium polymers that can be remodeled without proteolytic digestion.</a> Scientific Reports. 2, 879 (2012).</li><li>Klunk, W. E., Jacob, R. F., Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+amyloid+beta-peptide+(Abeta)+aggregation+using+the+Congo+red-Abeta+(CR-abeta)+spectrophotometric+assay.">Quantifying amyloid beta-peptide (Abeta) aggregation using the Congo red-Abeta (CR-abeta) spectrophotometric assay.</a> Analytical Biochemistry. 266 (1), 66-76 (1999).</li><li>Zhao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+amyloid+formation+by+turbidity+assay-seeing+through+the+cloud.">Measurement of amyloid formation by turbidity assay-seeing through the cloud.</a> Biophysical Reviews. 8 (4), 445-471 (2016).</li><li>Bester, J., Soma, P., Kell, D. B., Pretorius, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+and+ultrastructural+characteristics+of+whole+blood+and+plasma+in+Alzheimer-type+dementia,+and+the+possible+role+of+bacterial+lipopolysaccharides+(LPS).">Viscoelastic and ultrastructural characteristics of whole blood and plasma in Alzheimer-type dementia, and the possible role of bacterial lipopolysaccharides (LPS).</a> Oncotarget. 6 (34), 35284-35303 (2015).</li><li>Pretorius, E., Page, M. J., Mbotwe, S., Kell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-binding+protein+(LBP)+can+reverse+the+amyloid+state+of+fibrin+seen+or+induced+in+Parkinson's+disease.">Lipopolysaccharide-binding protein (LBP) can reverse the amyloid state of fibrin seen or induced in Parkinson's disease.</a> PLoS One. 13 (3), e0192121 (2018).</li><li>Kell, D. B., Pretorius, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteins+behaving+badly.+Substoichiometric+molecular+control+and+amplification+of+the+initiation+and+nature+of+amyloid+fibril+formation:+lessons+from+and+for+blood+clotting.">Proteins behaving badly. Substoichiometric molecular control and amplification of the initiation and nature of amyloid fibril formation: lessons from and for blood clotting.</a> Progress in Biophysics and Molecular Biology. 123, 16-41 (2017).</li><li>Wolberg, A. S., Campbell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombin+generation,+fibrin+clot+formation+and+hemostasis.">Thrombin generation, fibrin clot formation and hemostasis.</a> Transfusion and Apheresis Science. 38 (1), 15-23 (2008).</li><li>Soon, A. S., Lee, C. S., Barker, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+fibrin+matrix+properties+via+knob:hole+affinity+interactions+using+peptide-PEG+conjugates.">Modulation of fibrin matrix properties via knob:hole affinity interactions using peptide-PEG conjugates.</a> Biomaterials. 32 (19), 4406-4414 (2011).</li></ol>

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 a...

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>CaCl2</td>
			<td>Fischer Scientific</td>
			<td>C70</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Syringe, 0.2&#181;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 with Gold/Palladium target</td>
			<td>Tousimis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Denton Desk IV Coater</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, ...

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 &amp;#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.

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 ...

The main advantage of this plate-based turbidity assay is that it is quick and allows for several amyloid-beta fibrinogen interaction inhibitors to be screened at once without sophisticated setup or requirements. The head compounds that are in the fibrin turbidity assay can be further evaluated for their ability to restore A-beta-induced structural abnormality in the fibrin clots analyzed by scanning electron microscopy. Visual demonstration of clot preparation for scanning electron microscopy analysis is critical as any variations in the preparation can contribute to variations within the experimental results.The focus of this demonstration here is on A-beta fibrinogen interactions. This protocol can be readily modified to analyze other interactions with other proteins and the compounds with the fibrin clot. Begin by adding 1.5 micromolar of freshly prepared fibrinogen in 200 microliters of clot formation buffer to each A-beta negative 42 containing and control wells and incubate the plate on a rotating platform at room temperature.After 30 minutes simultaneously add 30 microliters of freshly prepared thrombin solution directly into the center of fibrinogen-containing well to initiate clot formation and immediately read the absorbance of the in vitro clots at 350 nanometers, repeating the measurement every 30 to 60 seconds over the course of 10 minutes. To evaluate the effect of A-beta fibrinogen interaction inhibitors on fibrin clot structure, first use forceps to place clean, 12 millimeter siliconized glass circle cover slips to individual wells of a 12 well plate and add 80 microliters of fibrinogen onto each cover slip, gently spreading the solution so that they are evenly distributed. Add 20 microliters of thrombin solution to each well to initiate clot formation.Cover the plate for a 30 to 60 minute incubation at room temperature, then gently submerge each clot in two milliliters of ice-cold sodium cacodylate buffer for two minutes, covered, at room temperature two times, using a one milliliter pipette to carefully remove the buffer after each wash. After the last wash, check the status of the clot formation on the cover slip and fix the clots in two to three milliliters of ice-cold 2%glutaraldehyde on ice for 30 minutes. At the end of the incubation, gently remove the glutaraldehyde from each well and wash the clots with fresh sodium cacodylate buffer as demonstrated on ice.To hydrate the fixed clots in a graded series of five minute ice-cold ethanol washes on ice without completely removing the ethanol between the washes to keep the clots protected from air exposure. During the last 100%ethanol wash, transfer the cover slips into a critical point dryer sample holder, placing at least 1 washer between each cover slip and place the holder into a critical point dryer chamber filled with ethanol. After a 30 minute drying cycle, use carbon tape to mount the cover slips on individual scanning electron microscopy stubs and transfer the samples to the sputter coating chamber of a vacuum sputter coater.Then sputter coat less than 20 nanometers of gold palladium or other conductive materials onto the samples for 25 seconds at four angstroms per second and image the samples on a scanning electron microscope equipped with a type two secondary electrons detector at four kilovolts. Fibrin clot formation causes scattering of the light passing through the solution, resulting in an increased turbidity that plateaus at the end of the reading period. When the fibrinogen is incubated in the presence of A-beta 42 the turbidity of the solution decreases with the curve reaching a maximum height of roughly half that of fibrinogen alone.In the presence of an A-beta 42 interaction blocker the effect of beta-amyloid is ameliorated and the turbidity is higher than that with A-beta alone. The effect of the blocker does not appear due to background turbidity as the compound does not change the fibrin clot turbidity when A-beta is absent. Further, clot formation in the presence of GPRP, a peptide known to interfere with fibrin polymerization, demonstrates a significantly reduced turbidity compared to the fibrin clot formation in the absence of the inhibitor.Fibrinogen generated in the presence of thrombin and calcium chloride only form a fibrin mesh with elongated and intercalated threads of fibrin as well as larger bundles. When A-beta is present, the fibrin threads became thinner with several sticky clumps in aggregates, indicating A-beta induced structural abnormalities. Consistent with the turbidity assay results, clot formation in the presence of an A-beta fibrinogen interaction blocker partially restores the structure of the fibrin clot from the A-beta-induced changes as fewer clumps are observed with this treatment.The clot turbidity assay and scanning electron microscope analysis protocols have been optimized for evaluating several A-beta fibrinogen interaction inhibitors in a quick and reproducible manner. And soon data will provide valuable information about fibrin clot formation that can be applied to further studies both in vitro and in vivo.

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

Research

JoVE Journal

Biochemistry

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 &#197;, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each.

Negative stain EM is a powerful technique for visualizing macromolecular structure, but different staining techniques can produce varying results in a sample dependent manner. Here several negative staining approaches are described in detail to provide an initial workflow for tackling the visualization of challenging systems.

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.