Dephosphorylation of platelet substrates by LMW-PTP. Protein-tyrosine phosphorylation was induced by stimulation of washed platelets with 1 U/mL thrombin or 100 ng/mL convulxin or by FcγRIIA clustering, obtained by addition of 2 μg/mL IV.3 monoclonal antibody and 30 μg/mL sheep antimouse F(ab′)2 fragments, as indicated on the top. Identical aliquots of the platelet lysates from stimulated platelets, but also from nonstimulated platelets (Bas), were incubated in the absence (−) or in the presence (+) of 5 U/mL purified LMW-PTP at 30°C for 5 minutes, as indicated on the bottom. Upon protein separation on a 10% to 20% acrylamide gradient gel, the level of protein-tyrosine phosphorylation was analyzed by immunoblotting with antiphosphotyrosine antibody. In the lane corresponding to the samples stimulated with convulxin, the phosphorylation of the protein in the low range of molecular masses, where FcR γ-chain migrates, is reported as a separated panel, because a prolonged exposure has been necessary to detect the reported band. Since platelet stimulation by FcγRIIA clustering involves addition of IV.3 monoclonal antibody, but also of a higher amounts of F(ab′)2 fragments, cross-reactivity with the peroxidase-conjugated secondary antibody is expected when whole platelet lysates are tested with antiphosphotyrosine monoclonal antibody (A). Therefore, in panel B, a corresponding immunoblot where the primary antibody was omitted is reported as a control, and shows that the major band at about 20 kDa represents probably the single chains of the antibody and F(ab′)2 fragments added to the platelet samples. All the reported results are representative of at least 3 different experiments.
