Effect of -thrombin and PMA on p0p 1-5 binding to mouse platelets.

Platelets were first incubated with 0.2 U/mL α-thrombin or 50 ng/mL PMA for 10 minutes at RT and subsequently stained with FITC-labeled mAbs for 15 minutes at RT (A) or vice versa (B). Samples were analyzed on a FACScan. The results shown are representative of 6 individual experiments. (C) Immunoprecipitation with p0p 1-5 and control IgG1 from NP-40 lysates of surface-biotinylated resting platelets (lanes 1, 3, 5, 7, 9, 11) or supernatant of PMA-activated surface-biotinylated platelets (lanes 2, 4, 6, 8, 10, 12; 10 minutes, at 15 000g). Proteins were separated by 9% to 15% SDS-PAGE under reducing conditions, transferred to a PVDF-membrane, and detected by streptavidin-HRP/ECL. (D) Detection of GPIbβ and the membrane-anchored truncated 20-kd remainder of GPIbα (t-GPIbα). Surface-biotinylated platelets were stimulated with PMA (50 ng/mL) for 10 minutes at RT, washed twice, and lysed with NP-40. Immunoprecipitates of p0p 1, p0p 2, and p0p 3 (control) were separated under reducing and nonreducing conditions and blotted onto a PVDF-membrane, and biotinylated proteins were detected by streptavidin-HRP/ECL.