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

GPIb beta is disulfide-linked to GPIb alpha to form GPIb, a platelet receptor for von Willebrand factor (vWF), GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex. the exact function of GPIb beta is not well established due to a lack of suitable ligands or antibodies. 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. Using synthetic peptides. the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flank-Mg region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAMA inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWT matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study 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 GPIb beta.