To begin the assay using the platelet receptor agonist, Collagen-Related Peptide Cross-Linked, or CRPXL, dilute an appropriate amount of the CRPXL stock in the assay buffer to a concentration suitable to induce maximum platelet aggregation. The resultant solution will be henceforth referred to as the standard. Next, starting from the same highest prepared concentrations, sequentially perform two-fold serial dilutions for both the standard and the test reagent.
Prepare a six-to-eight point dilution series for both the test and the standard in the assay buffer that generates aggregation responses from 100%down to 0%When the platelet-rich plasma, or PRP, is ready to use, add an aliquot of PRP into the light transmission aggregometry cuvette. Allow the samples to warm to 37 degrees Celsius in the holding wells for a couple of minutes. Once appropriately warmed, place the cuvettes in the aggregometer to start recording the traces.
With constant stirring, add the agonist, and record the data. Perform data collection for each prepared dilution of the standard and the test reagent. Use Graphpad Prism, or any suitable software package, to check that the concentration curves for the standard and test are parallel.
Enter the data such that the x-axis contains the concentrations, and the y-axis contains the responses indicated by either the percentage of maximum aggregation or the area under the curve. Next, transform the concentration values to the corresponding logarithms, or log concentration. Select non-linear regression from the analysis functions, and use the equation for dose response stimulation.
Select log agonist versus response variable slope, and use a three or four-way fit, depending on which is best for the data. Determine if the curves generated are parallel by clicking the compare tab and comparing the datasets. Ensure that the slope and asymptotes of the test, and the standard curves, are equivalent.
If these conditions are met, proceed to calculate the potency, or EC50, using non-linear regression curve fitting. Calculate the relative potency by dividing the EC50 value of the standard by that of the test. Adjust the mass per volume concentration of the test reagent to account for the difference in potency with respect to the standard.
The platelet aggregation traces for the standard and the test reagent revealed the difference between the two aggregation responses. At 0.075 micrograms per milliliter, there was a clear response to the standard, while, for the test, the corresponding concentration showed no response. The EC50 values of the standard and test reagent were calculated by plotting the concentration response curve.
