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 de vitro fibrin clots and analyzing the effect of beta-amyloid (Aβ) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). Aβ, which forms neurotoxic amyloid aggregates in Alzheimer's disease (AD), has been shown to interact with fibrinogen. This Aβ-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. Aβ-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the Aβ-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of Aβ and Aβ-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of Aβ-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate Aβ-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified Aβ-fibrinogen interaction inhibitor.
Alzheimer’s disease (AD), a neurodegenerative disease leading to cognitive decline in elderly patients, predominately arises from abnormal beta-amyloid (Aβ) expression, aggregation and impaired clearance resulting in neurotoxicity1,2. Despite the well-characterized association between Aβ aggregates and AD3, the precise mechanisms underlying the disease pathology are not well understood4. Increasing evidence suggests that neurovascular deficits play a role in the progression and severity of AD5, as Aβ directly interacts with the components of the circulatory system6. Aβ has a high-affinity interaction with fibrinogen7,8, which also localizes to Aβ deposits in both AD patients and mouse models9,10,11. Furthermore, the Aβ-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 Aβ and fibrinogen13,14. We, therefore, identified several small compounds inhibiting the Aβ-fibrinogen interaction using high throughput screening and medicinal chemistry approaches13,14. To test the efficacy of Aβ-fibrinogen interaction inhibitors, we optimized two methods for the analysis of in vitro fibrin clot formation: clot turbidity assay and scanning electron microscopy (SEM)14.
Clot turbidity assay is a straight-forward and rapid method for monitoring fibrin clot formation using UV-visible spectroscopy. As the fibrin clot forms, light is increasingly scattered and the turbidity of the solution increases. Conversely, when Aβ is present, the structure of the fibrin clot is altered, and the turbidity of the mixture is reduced (Figure 1). The effect of inhibitory compounds can be assessed for the potential to restore clot turbidity from Aβ-induced abnormalities. While the turbidity assay allows for rapid analysis of multiple conditions, it provides limited information on the clot shape and structure. SEM, in which the topography of solid objects is revealed by electron probe, allows for the analysis of the 3D architecture of the clot15,16,17,18 and the assessment of how the presence of Aβ 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 Aβ. It also details the methods to analyze the effect of Aβ on fibrin clot formation and structure. The effectiveness of these two methods for measuring the inhibition of the Aβ-fibrinogen interaction is demonstrated using TDI-2760, a small inhibitory compound14. These methods, both individually and together, allow for rapid and straightforward analysis of in vitro fibrin clot formation.
1. Preparation of Aβ42 and Fibrinogen for Analysis
2. Clot Turbidity Assay
3. Scanning Electron Microscopy
In the in vitro clotting (turbidity) assay, the enzyme thrombin cleaves fibrinogen, resulting in the formation of the fibrin network24. This fibrin clot formation causes scattering of the light passing through the solution, resulting in increased turbidity (Figure 1), plateauing before the end of the reading period (Figure 2, green). When the fibrinogen was incubated in the presence of Aβ42, the 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 Aβ aggregation blockers were synthesized and assessed for their ability to inhibit the Aβ-fibrinogen interaction, identifying the compound TDI-276014. We have used TDI-2760 in our study to block Aβ-fibrinogen interaction. As reported previously, in the presence of TDI-2760, the effect of Aβ was ameliorated, as the turbidity was higher than with Aβ 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 Aβ 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 Aβ 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 Aβ was present, the fibrin threads became thinner, with several sticky clumps/aggregates indicating Aβ-induced structural abnormalities (Figure 3B). Consistent with the turbidity assay results, TDI-2760 partially restored the structure of the fibrin clot from Aβ-induced changes, as fewer clumps were present (Figure 3C). Together with the turbidity assay, SEM reveals the extent and quality of Aβ-induced changes to fibrin clot formation, as well as the effectiveness of an inhibitory compound, TDI-2760.
Figure 1: Schematic representation of fibrin clot analysis by turbidity assay and SEM. The schematic shows the steps involved in fibrin clot analysis and the effect of Aβ on fibrin clot turbidity and structural topography. The scale bars of SEM images (bottom left) are 2 µm and the magnification is 10,000X. Please click here to view a larger version of this figure.
Figure 2: Measurement of the effect Aβ42 on in vitro fibrin clot formation by turbidity assay. (A) Fibrinogen was incubated in the presence and absence of Aβ42. Clot formation was induced by thrombin, resulting in increased turbidity of the solution (green). In the presence of Aβ42, 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 Aβ42. TDI-2760 restored Aβ42-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 µM GPRP and the turbidity measured after adding thrombin. Similar to Aβ42, the turbidity of fibrin clot formation was significantly reduced in the presence of GPRP (blue versus green). Please click here to view a larger version of this figure.
Figure 3: Scanning electron micrographs of Aβ-induced abnormalities to fibrin clot architecture. Scanning electron micrographs of fibrin clot structure obtained from purified fibrinogen (A), fibrinogen + Aβ42 (B), fibrinogen + Aβ42+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 Aβ42 are thinner and abnormally clumped compared to fibrinogen by itself. TDI-2760, which is known to inhibit Aβ42-fibrinogen interaction, partially corrected the Aβ42 induced structural abnormality of the fibrin clot. Scale bar is 1 µm. Please click here to view a larger version of this figure.
The methods described here provide a reproducible and rapid means of assessing fibrin clot formation in vitro. Furthermore, the simplicity of the system makes the interpretation of how Aβ affects the fibrin clot formation and structure relatively straight-forward. In this lab's previous publication, it was shown that these assays can be used to test compounds for their ability to inhibit the Aβ-fibrinogen interaction13,14. Using these two assays, a series of synthesized compounds were analyzed for the ability to inhibit the Aβ-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 Aβ-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 Aβ and test compounds. Under the experimental conditions presented here, Aβ, 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, Aβ is an aggregation-prone peptide and a higher concentration of Aβ solution when kept for a very long time at room temperature or 37 °C (several hours) can form fibrillar aggregates which may result in increased turbidity. If analyzing the effect an Aβ 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 Aβ 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 Aβ 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 Aβ, which certainly affects the oligomerization rate. It is advisable to check the concentration of Aβ solution (BCA method) before incubating for oligomerization. To minimize these variations, we tried using at least 0.1 mg/mL of Aβ 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 Aβ 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 Aβ 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 Aβ 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 Aβ, 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 µM 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 Å/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 "islets" of coating molecules on the material.
While the focus here has been on the Aβ-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.
The authors have nothing to disclose.
Authors thank Masanori Kawasaki, Kazuyoshi Aso, and Michael Foley from Tri-Institutional Therapeutics Discovery Institute (TDI), New York for synthesis of Aβ-fibrinogen interaction inhibitors and their valuable suggestions. Authors also thank members of the Strickland lab for helpful discussion. This work was supported by NIH grant NS104386, the Alzheimer's Drug Discovery Foundation, and Robertson Therapeutic Development Fund for H.A., NIH grant NS50537, the Tri-Institutional Therapeutics Discovery Institute, Alzheimer's Drug Discovery Foundation, Rudin Family Foundation, and John A. Herrmann for S.S.
Fibriogen, Plasminogen-Depleted, Plasma | EMD Millipore | 341578 | keep lid parafilm wrapped to avoid exposure to moisture |
Beta-Amyloid (1-42), Human | Anaspec | AS-20276 | |
Thrombin from human plasma | Sigma-Aldrich | T7009 | |
1,1,1,3,3,3-Hexafluoro-2-propanol, Greater Than or Equal to 99% | Sigma-Aldrich | 105228 | |
DIMETHYL SULFOXIDE (DMSO), STERILE-FILTERED | Sigma-Aldrich | D2438 | |
Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | |
Tris Base | Fischer Scientific | BP152 | |
HEPES | Fischer Scientific | BP310 | |
NaCl | Fischer Scientific | S271 | |
CaCl2 | Fischer Scientific | C70 | |
Filter Syringe, 0.2µM, 25mm | Pall | 4612 | |
Millex Sterile Syringe Filters, 0.1 um, PVDF, 33 mm dia. | Millipore | SLVV033RS | |
Solid 96 Well Plates High Binding Certified Flat Bottom | Fischer Scientific | 21377203 | |
Spectramax Plus384 | Molecular Devices | 89212-396 | |
Centrifuge, 5417R | Eppendorf | 5417R | |
Branson 200 Ultrasonic Cleaner | Fischer Scientific | 15-337-22 | |
Lab Rotator | Thermo Scientific | 2314-1CEQ | |
Spare washers for cover slip holder | Tousimis | 8766-01 | |
Narrow Stem Pipets – Sedi-Pet | Electron Microscopy Sciences (EMS) | 70967-13 | |
Sample holder for CPD (Cover slip holder) | Tousimis | 8766 | |
Mount Holder Box, Pin Type | Electron Microscopy Sciences (EMS) | 76610 | |
Round glass cover slides (12 mm) | Hampton Research | HR3-277 | |
10% Glutaraldehyde | Electron Microscopy Sciences (EMS) | 16120 | |
Ethanol | Decon Labs | 11652 | |
24 well plate | Falcon | 3047 | |
Na Cacodylate | Electron Microscopy Sciences (EMS) | 11652 | |
SEM Stubs, Tapered end pin. | Electron Microscopy Sciences (EMS) | 75192 | |
PELCO Tabs, Carbon Conductive Tabs, 12mm OD | Ted Pella | 16084-1 | |
Autosamdri-815 Critical Point Dryer with Gold/Palladium target | Tousimis | ||
Denton Desk IV Coater | Denton Vacuum | ||
Leo 1550 FE-SEM | Carl Zeiss | ||
Smart SEM Software | Carl Zeiss |