FIGURE 4.
Reduced platelet aggregation, fibrinogen binding, and P-selectin exposure in GSK3α/β KI platelets. A, washed GSK3α+/+/β+/+ (GSK3α/β WT) and GSK3 αS21A/S21A/βS9A/S9A (GSK3α/β KI) platelets were stimulated with 0.15 unit/ml thrombin for 5 min before extraction in NuPAGE sample buffer and immunoblot analysis with the indicated antibodies. B, platelets were stimulated with 0.15 unit/ml thrombin under stirring conditions and aggregation monitored for 5 min. C and D, a representative aggregation curve is presented for three independent experiments. Washed platelets were incubated with the indicated concentrations of PAR4 agonist AYPGKF-NH2 for 15 min in the presence of FITC-fibrinogen (C) or PE-anti-P-selectin Ab (D). Samples were fixed in 4% paraformaldehyde and analyzed by flow cytometry (C and D). Results are expressed as mean ± S.E. (error bars; n = 6) of percentage of maximal fibrinogen binding to GSK3α/β WT platelets. C and D, significance testing using GraphPad Prism 4.0 determined that different curve fittings were required for GSK3α/β WT and GSK3α/β KI data. E, washed GSK3α/β WT and GSK3α/β KI platelets were lysed in NuPAGE sample buffer and receptor expression levels determined by immunoblotting with the indicated antibodies.