Fig. 1.
Fig. 1. Aggregation-dependent and -independent mechanisms of induction of PECAM-1 tyrosine phosphorylation. / PECAM-1 was immunoprecipitated from human platelets under resting conditions or after stimulation with TRAP (7 μM); (A) collagen (10 μg/mL) (0-6 minutes); (B) CRP (2 μg/mL) (0-6 minutes); and (C) no stimulation, 2 μg/mL CRP alone for 2 minutes, 2 μg/mL CRP + 0.5 mM RGDW, 2 μg/mL CRP + 20 μg/mL c7E3 Fab (Reopro), or 2 μg/mL CRP + 500 nM Aggrastat. All αIIbβ3 blockers were pre-incubated with washed platelets for 10 minutes before CRP stimulation for 2 minutes at 37°C with stirring. Proteins were separated on SDS-PAGE and immunoblotted for anti-phosphotyrosine content using an HRP-conjugated 4G10 anti-phosphotyrosine antibody. The presence of PECAM-1 antigen was confirmed by reprobing with polyclonal anti–PECAM-1 antibody, SEW16.

Aggregation-dependent and -independent mechanisms of induction of PECAM-1 tyrosine phosphorylation.

PECAM-1 was immunoprecipitated from human platelets under resting conditions or after stimulation with TRAP (7 μM); (A) collagen (10 μg/mL) (0-6 minutes); (B) CRP (2 μg/mL) (0-6 minutes); and (C) no stimulation, 2 μg/mL CRP alone for 2 minutes, 2 μg/mL CRP + 0.5 mM RGDW, 2 μg/mL CRP + 20 μg/mL c7E3 Fab (Reopro), or 2 μg/mL CRP + 500 nM Aggrastat. All αIIbβ3 blockers were pre-incubated with washed platelets for 10 minutes before CRP stimulation for 2 minutes at 37°C with stirring. Proteins were separated on SDS-PAGE and immunoblotted for anti-phosphotyrosine content using an HRP-conjugated 4G10 anti-phosphotyrosine antibody. The presence of PECAM-1 antigen was confirmed by reprobing with polyclonal anti–PECAM-1 antibody, SEW16.

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