Treatment with forskolin or PGE₁ increases phosphorylation of GPIbβ in GPIb/V/IX-transfected cells and platelets, an effect reversed by RAM.1.

K562-GPIb/V/IX, K562-GPIb(βSer166Gly)/V/IX cells, or human platelets were labeled with [³²P]PO₄, then preincubated with 10 μg/mL RAM.1 (+) or rat control IgG (-) and treated with dimethyl sulfoxide (resting), 10 μM PGE₁ (+PGE₁), or 10 μM forskolin (+Fsk) for 10 minutes at 37°C. Following cell lysis, the GPIb/V/IX complex was immunoprecipitated by RAM.1 and proteins were separated on a 7.5%-15% SDS-PAGE gel. Gels were analyzed by autoradiography or using the phosphoimager system. Results are representative of 2 separate experiments. Forskolin or PGE₁ treatment increased the GPIbβ phosphorylation on both platelets and K562-GPIb/V/IX cells whereas RAM.1 switched it off. In contrast, forskolin and PGE₁ had no effect on phosphorylation of the GPIbβ(Ser166Gly) subunit.