A novel monoclonal antibody against the extracellular domain of GPIb beta modulates vWF mediated platelet adhesion

GPIb beta is disulfide-linked to GPIb alpha to form GPIb, a platelet recept or for von Willebrand factor (vWF), GPIb is in turn non covalently linked t o GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution t o controlling surface expression of the complex. the exact function of GPIb beta is not well established due to a lack of suitable ligands or antibodi es. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIb beta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM. I bound to GPIb beta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIb beta, did not. Usi ng synthetic peptides. the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flank-Mg region. In functional a ssays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and b otrocetin induced vWF binding. RAMA inhibited adhesion of GPIb/V/IX transfe cted K562 cells to a vWT matrix under flow, increased their rolling velocit y and decreased the resistance of cells to detachment at high shear. This s tudy suggests a role of GPIb beta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPI b beta.