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.
