| Assay Method Information | |
| | Fluorometric ATIII/alphaIIa IC50 Plate Assay at pH 7.8 |
| Description: | PAI-1 inhibitor compounds were dissolved in DMSO to a final concentration of (10-50 mM), depending upon solubility. Compounds were then diluted in physiologic buffer (40 mM HEPES, 100 mM NaCl, 0.05% Tween-20, pH7.8) containing (10% DMSO and a dilution series (from 0 to 1000 μM depending on solubility) was prepared. 80 μL of compound was added per well to a 96-well black, opaque microplate in duplicate. 10 μL of 20 nM recombinant active anti-thrombin III (ATIII) (Molecular Innovations), a PAI-1-related protein, in physiologic buffer, as set out above, was added and the mixture was agitated for 15 minutes at room temperature. 10 μL of 25 nM human α-Thrombin (αIIa) (Haematologic Technologies) was added to each reaction well and the plate was agitated for an additional 30 minutes at room temperature. 100 μL of 100 μM fluorogenic, tripeptide aminomethylcoumarin benzoyl Phe-Val-Arg-AMC substrate was then added and residual αIIa activity was determined based upon cleavage of this substrate. The rate of AMC release by αIIa was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. Controls included PAI-1 and αIIa in the absence of compound and αIIa alone. Percent PAI-1 inhibition is calculated using the following formula: [(αIIa alone-αIIa/PAI-1+ compound)/(αIIa alone-PAI-1/αIIa)]*100%. The IC50 is calculated using Graphit (IC50 0-100%).The invention has been described in terms of particular embodiments found or proposed to comprise preferred modes for the practice of the invention. It will be appreciated by those of ordinary skill in the art that, in light of the disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed. |
| Affinity data for this assay | |
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