Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex.

Glycoprotein (GP) Ib/V/IX complex-dependent platelet adhesion to von Willebrand factor (VWF) is supported by the 45-kd N-terminal extracellular domain of the GPIb alpha subunit. Recent results with an adhesion blocking antibody (RAM.1) against GPIb beta, which is disulfide linked to GPIb alpha, have suggested a novel function of this subunit in regulating VWF-mediated platelet adhesion, possibly involving its intracellular face. A putative cooperation between the GPIb alpha and GPIb beta cytoplasmic domains was investigated by measuring the adhesion under flow to immobilized VWF of K562 and Chinese hamster ovary (CHO) cells transfected with GPIb/(V)/IX containing mutations in this region. Adhesion of cells carrying a glycine substitution of the GPIb beta Ser166 phosphorylation site was 50% lower than normal and became insensitive to inhibition by RAM.1. In contrast, forskolin or PGE(1) treatment increased both the phosphorylation of GPIb beta and adhesion of control cells, both effects being reversed by RAM.1, but had no influence on cells expressing the Ser166Gly mutation. A role of the GPIb alpha intracellular domain was also apparent as the VWF-dependent adhesion of cells containing deletions of the entire (Delta 518-610) or portions (Delta 535-568, Delta 569-610) of the GPIb alpha cytoplasmic tail was insensitive to RAM.1 inhibition. Cells carrying progressive 11 amino acid deletions spanning the GPIb alpha 535-590 region were equally unresponsive to RAM.1, with the exception of those containing GPIb alpha Delta 569-579, which behaved like control cells. These findings support a role of the GPIb beta intracellular domain in controlling the adhesive properties of the GPIb/V/IX complex through phosphorylation of GPIb beta Ser166 and point to the existence of cross-talk between the GPIb beta and GPIb alpha intracellular domains.