PLC activation in fyn^−/−platelets.

Pleckstrin phosphorylation (an indicator of PKC activation) and phosphatidic acid (PA) production (a metabolite of diacylglycerol) were measured in [³²P]-labeled platelets in the presence of 1 mmol/L EGTA and 10 μmol/L indomethacin. (A) The autoradiographs show pleckstrin phosphorylation stimulated by a range of concentrations of CRP (0.3-3.0 μg/mL) and thrombin (0.1-1.0 U/mL) in wild-type andfyn^−/−platelets. The results are representative of 3 to 5 experiments. (B) Autoradiographs show phosphatidic acid production stimulated by a range of concentrations of CRP (0.3-3.0 μ/mL) and thrombin (0.1-1.0 U/mL) in wild-type andfyn^−/−platelets. The results are representative of 3 to 5 experiments. Wild-type andfyn^−/−samples were run in parallel, and the same exposure times are shown. A lighter exposure of the autoradiogram from representative samples of the more powerful agonist thrombin is shown to enable differentiation between PA levels stimulated by different concentrations of thrombin. (C) Ca²⁺ mobilization was measured in Fura-2–labeled murine platelets in the presence of 1 mmol/L EGTA. Wild-type and fyn^−/−platelets (i) and wild-type and lyn^−/−platelets (ii) were stimulated by 0.3 μg/mL CRP over 200 seconds. The arrow indicates the point of addition of an agonist. Results are representative of 5 experiments.