Figure 5.

Pxn-KD fails to increase tyrosine phosphorylation and calcium mobilization. (A) Washed platelets obtained from control and Pxn-KD experiments were stimulated with 150 ng/mL convulxin for the indicated times. The cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and then immunoblotted with an anti-phosphotyrosine mAb (4G10). The data shown are representative of three independent experiments. (B) Control and Pxn-KD platelets were labeled with GFP-Certified™ FluoForte™ dye. Changes in intracellular calcium levels after stimulation with an indicated concentration of AYPGKF or convulxin were then measured every 30 s. Data are expressed as the relative fluorescence unit (RFU) measured using a microplate spectrofluorometer (excitation, 530 nm; emission, 570 nm). The peak calcium concentration was measured after stimulation (open bars: control platelets; black bars: Pxn-KD platelets). Columns and error bars represent the mean ± s.d. (n = 5–8). Statistical significance was determined by the Student’s t-test. (C) Control and Pxn-KD platelets were pretreated with 1 mmol/L EDTA and/or 20 μmol/L BAPTA-AM for 10 min, and then stimulated with or without 1 mmol/L AYPGKF or 150 ng/mL convulxin. P-selectin expression on GFP-positive platelets was determined by flow cytometry. Columns and error bars represent the mean ± s.d. of P-selectin expression (n = 4).