Agonist-induced aggregation of Chinese hamster ovary cells coexpressing the human receptors for fibrinogen (integrin αIIbβ3) and the platelet-activating factor: dissociation between adhesion and aggregation by Susana Larrucea, Consuelo González-Manchón, Nora Butta, Elena G. Arias-Salgado, Linnan Shen, Matilde S. Ayuso, and Roberto Parrilla Blood Volume 99(8):2819-2827 April 15, 2002 ©2002 by American Society of Hematology

Detection and characterization of PAFR stably transfected in CHO-αIIbβ3 cells.(A) Cell extracts were subjected to Western blotting analysis using a rabbit polyclonal anti-PAFR. Detection and characterization of PAFR stably transfected in CHO-αIIbβ3 cells.(A) Cell extracts were subjected to Western blotting analysis using a rabbit polyclonal anti-PAFR. The arrow points to a band of the expected size of PAFR that is present in human platelets and CHO-αIIbβ3-PAFR cells, but absent in CHO-αIIbβ3 cells transfected with the void plasmid. (B) and (C) depict the effects of PAF in increasing the intracellular cytosolic levels of free calcium and intracellular pH, and the effect of a PAF antagonist in preventing both effects. Measurement of intracellular calcium and pH was carried out fluorimetrically as described in “Materials and methods.” The data are representative of at least 6 observations made in different cell preparations. Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Effect of PAF on adhesion of CHO-αIIbβ3-PAFR cells to immobilized Fg Effect of PAF on adhesion of CHO-αIIbβ3-PAFR cells to immobilized Fg.(A) Time course of the effect of PAF on adhesion of CHO-αIIbβ3-PAFR cells to immobilized Fg. CHO cells (4 × 105) coexpressing αIIbβ3 and PAFR were added to the wells of microtiter plates c... Effect of PAF on adhesion of CHO-αIIbβ3-PAFR cells to immobilized Fg.(A) Time course of the effect of PAF on adhesion of CHO-αIIbβ3-PAFR cells to immobilized Fg. CHO cells (4 × 105) coexpressing αIIbβ3 and PAFR were added to the wells of microtiter plates coated with 10 μg/mL Fg, incubated for different periods of time at 37°C in the presence or absence of 100 nM PAF, and visualized by phase contrast microscopy. (B) Comparative effect of PAF on adhesion of CHO, CHO-β3-PAFR, CHO-αIIbβ3, and CHO-αIIbβ3-PAFR cell lines to immobilized Fg. Cells were allowed to adhere on Fg-coated wells for 5 minutes, in the absence or presence of 100 nM PAF. Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Effect of PTX and PKC inhibitors on the effect of PAF in stimulating the adherence of CHO-αIIbβ3-PAFR cells to immobilized Fg.(A) CHO-αIIbβ3-PAFR cells were incubated with 200 ng/mL PTX for 2 hours at 37°C and then stimulated with 100 nM PAF for 2 minutes. Effect of PTX and PKC inhibitors on the effect of PAF in stimulating the adherence of CHO-αIIbβ3-PAFR cells to immobilized Fg.(A) CHO-αIIbβ3-PAFR cells were incubated with 200 ng/mL PTX for 2 hours at 37°C and then stimulated with 100 nM PAF for 2 minutes. Cell adhesion to immobilized Fg at 10 minutes was measured as described in “Materials and methods.” To determine the effect of inhibiting PKC on the PAF-stimulated adhesion to immobilized Fg, cells were preincubated at 37°C with the indicated concentration of staurosporine (B) for 1 hour, H7 (C) for 45 minutes, or bisindolylmaleimide-I (BIM-I) for 30 minutes (D). The data are expressed relative to the adhesion of PAF-stimulated cells in the absence of inhibitor and are means ± SD of at least 3 experiments. Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Soluble Fg-dependent aggregation of CHO-αIIbβ3-PAFR cells induced by PAF.Cells (2.5 × 106/mL) were incubated with 1 mg/mL Fg for 15 minutes at room temperature in a 250 μL final volume and then stimulated with 100 nM PAF and plated onto BSA-precoated wells. Soluble Fg-dependent aggregation of CHO-αIIbβ3-PAFR cells induced by PAF.Cells (2.5 × 106/mL) were incubated with 1 mg/mL Fg for 15 minutes at room temperature in a 250 μL final volume and then stimulated with 100 nM PAF and plated onto BSA-precoated wells. Aggregate formation was examined by phase-contrast microscopy as described in “Materials and methods.” Original magnification, × 100. Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Flow cytometric assessment of the PAF-induced aggregation of CHO-αIIbβ3-PAFR cells.Either CHO-αIIbβ3-PAFR (A) or CHO-αIIbβ3 cells (B) (2.5 × 106/mL) were incubated for 15 minutes with soluble Fg (0.5 mg/mL) in the presence or absence of 100 nM PAF and then ... Flow cytometric assessment of the PAF-induced aggregation of CHO-αIIbβ3-PAFR cells.Either CHO-αIIbβ3-PAFR (A) or CHO-αIIbβ3 cells (B) (2.5 × 106/mL) were incubated for 15 minutes with soluble Fg (0.5 mg/mL) in the presence or absence of 100 nM PAF and then fixed with 0.25% paraformaldehyde. Forward (FS) and side (SS) light scatter data of each sample were analyzed by flow cytometry as described in “Materials and methods.” Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Effect of PAF on binding of soluble Fg or PAC1 to CHO-αIIbβ3-PAFR cells.(A) Binding of FITC-Fg to CHO-αIIbβ3-PAFR cells. Effect of PAF on binding of soluble Fg or PAC1 to CHO-αIIbβ3-PAFR cells.(A) Binding of FITC-Fg to CHO-αIIbβ3-PAFR cells. Cells were incubated for 15 minutes with soluble FITC-Fg, in the presence or absence of 100 nM PAF, as described in “Materials and methods.” The data are means of 3 independent experiments performed in duplicate. The insert shows the Scatchard plot of data. (B) The histograms represent the expression of PAC1 binding sites in cells incubated with or without 100 nM PAF. Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Tyrosine phosphorylation induced by PAF in CHO cell lines. Cells (2 Tyrosine phosphorylation induced by PAF in CHO cell lines.Cells (2.5 × 106/mL) were incubated with 1 mg/mL Fg for 15 minutes at room temperature in a 250 μL final volume and then stimulated with 100 nM PAF and plated onto BSA-precoated wells. Tyrosine phosphorylation induced by PAF in CHO cell lines.Cells (2.5 × 106/mL) were incubated with 1 mg/mL Fg for 15 minutes at room temperature in a 250 μL final volume and then stimulated with 100 nM PAF and plated onto BSA-precoated wells. After 15 minutes, cells were lysed and proteins (50 μg) were separated by 6% to 16% SDS-PAGE, transferred to PVDF sheets and probed with antiphosphotyrosine mAb PY99, as described in “Materials and methods.” The arrows point to phosphorylated p100-PPA and MAPK proteins. Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Time course of Fg and/or PAF-induced tyrosine phosphorylation of proteins in CHO-αIIbβ3-PAFR cells.(A) Cells (2.5 × 106/mL) were incubated with 100 nM PAF for 2 minutes at room temperature in a 250 μL final volume and then Fg was added at 1 mg/mL and cells ... Time course of Fg and/or PAF-induced tyrosine phosphorylation of proteins in CHO-αIIbβ3-PAFR cells.(A) Cells (2.5 × 106/mL) were incubated with 100 nM PAF for 2 minutes at room temperature in a 250 μL final volume and then Fg was added at 1 mg/mL and cells were plated onto BSA-precoated wells. (B) Cells were incubated with 1 mg/mL Fg for 15 minutes at room temperature in a 250 μL final volume and then stimulated with 100 nM PAF and plated onto BSA-precoated wells. At the indicated times, cells were lysed and proteins (50 μg) were resolved by SDS-PAGE, transferred to PVDF membranes and probed with antiphosphotyrosine mAb PY99, as described in “Materials and methods.” The arrow points to the phosphorylated MAPK protein. The densitometric analysis was carried out using the public domain program NIH Image (http://rsb.info.nih.gov/nih-image/). Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Differential effects of PAF and PMA on tyrosine phosphorylation of p100-PPA and MAPK in CHO-αIIbβ3-PAFR cells.Cells were incubated with either 100 nM PAF for 2 minutes or 100 nM PMA for 30 minutes. Differential effects of PAF and PMA on tyrosine phosphorylation of p100-PPA and MAPK in CHO-αIIbβ3-PAFR cells.Cells were incubated with either 100 nM PAF for 2 minutes or 100 nM PMA for 30 minutes. Then 1 mg/mL Fg was added and cells were plated onto BSA-precoated wells. After 15 minutes, cells were lysed and proteins (50 μg) were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with antiphosphotyrosine mAb PY99, as described in “Materials and methods.” Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology

Time course of PAF-induced tyrosine phosphorylation of FAK in CHO-αIIbβ3-PAFR cells in the presence of either soluble or solid-phase Fg.In aggregation assays, cells were incubated with 1 mg/mL Fg for 15 minutes at room temperature and then were stimulated w... Time course of PAF-induced tyrosine phosphorylation of FAK in CHO-αIIbβ3-PAFR cells in the presence of either soluble or solid-phase Fg.In aggregation assays, cells were incubated with 1 mg/mL Fg for 15 minutes at room temperature and then were stimulated with 100 nM PAF and plated onto BSA-precoated wells. At the indicated times, cells were lysed and immunoprecipitation of FAK was performed using a polyclonal anti-FAK antibody. Immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine mAb PY99, as described in “Materials and methods.” In adhesion experiments, cells were added to Fg-coated wells and allowed to adhere for 15 minutes. Then 100 nM PAF was added and incubation continued until the indicate times. Cells were lysed and tyrosine phosphorylation of FAK was determined as above. The densitometric analysis was carried out using the public domain program NIH Image (http://rsb.info.nih.gov/nih-image/). Susana Larrucea et al. Blood 2002;99:2819-2827 ©2002 by American Society of Hematology