FIGURE 7.

Role of Src family kinases in CLEC-2-mediated aggregation following multivalent ligands and podoplanin ligation. A, washed platelets (2 × 10⁸/ml) under basal, CLEC-2 mAb (10 μg/ml) or rhodocytin-stimulated (30 nm) conditions had their surface proteins cross-linked with the addition of 1.5 mm Sulfo-EGS cross-linking reagent, with a linker length of 1.6 nm (16 Å) as described under “Experimental Procedures.” Monomeric CLEC-2 was quantified, and the data represent the means and standard error of three independent experiments. **, p < 0.005 (significant difference versus wild type, according to two-tailed Student's t test). mAb, CLEC-2 mAb; Rh, rhodocytin. B, Lyn-deficient washed platelets and their litter-matched wild-type platelets (2 × 10⁸ platelets/ml) were stimulated with 2 μg/ml CLEC-2 mAb for 2 min and then with 10 μg/ml anti-rat Fc antibody for 5 min. Representative aggregation traces of three independent experiments are shown. The addition of the agonists is indicated by an arrowhead. C, Lyn-deficient washed platelets and their litter-matched wild-type platelets (2 × 10⁸ platelets/ml) incubated with or without 20 μm PP2 were stimulated with IgM CLEC-2 antibody. The data represent the means of the time to get 50% of aggregation and standard error of three to six independent experiments. *, p < 0.05 (significant difference versus wild type, according to two-tailed Student's t test). D, Lyn-deficient platelets and their litter-matched control platelets (2 × 10⁸ platelets/ml) incubated with or without 20 μm PP2 were stimulated in plasma with 1.5 × 10⁵ lymphatic endothelial cells (hLEC) or mouse podoplanin expressing CHO cells (mPodoCHO). Platelet aggregation was measured as a change in light transmission, using a lumi-aggregometer. Representative aggregation traces of three independent experiments are shown. The addition of the agonist is indicated by an arrowhead.