Characterization of Ca-TSP and EDTA-TSP by competitive immunoassay with MoAb D4.6. EDTA-TSP was prepared similarly to Ca-TSP (see Materials and Methods), except that 5 mmol/L EDTA was added to the activated platelet suspension after aggregation occurred, before the separation of the supernatant from platelets. EDTA-TSP was then purified from EDTA-containing supernatant by the same procedure as described for Ca-TSP using Ca²⁺ -containing buffers. The microtiter plate was coated with 10 μg/mL TSP containing 3 mmol/L EDTA at 4°C for overnight. After incubation of Ca-TSP or EDTA-TSP in either 2 mmol/L Ca²⁺ or 6 mmol/L EDTA for 1 hour at 37°C the TSP was diluted into solutions containing MoAb D4.6 with 2 mmol/L Ca²⁺ or 3 mmol/L EDTA. After 1 hour, the mixtures were added to the wells coated with TSP to determine the amount of antibody free to bind to the coated TSP. After washing four times, antibody bound was detected by the color reaction of p-nitrophenylphosphate with alkaline phosphatase coupled to a secondary antibody as described in Materials and Methods. The absorbance in the absence of competing TSP was taken as 100%. Competition by TSP is the reciprocal of antibody bound to plate, expressed as percent of maximum. Shown here are the competition curves using TSP (means ± SE of four separate experiments) that had been incubated for 1 hour in either 2 mmol/L Ca²⁺ (□) or 6 mmol/L EDTA (○). It shows that Ca-TSP can be converted to the EDTA-conformation, but that the reverse does not occur.