A Novel Monoclonal Antibody against the Extracellular Domain of GPIbβ Modulates vWF Mediated Platelet Adhesion

Summary

GPIb is disulfide-linked to GPIbα 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 GPIbβ 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 GPIbβ 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.1 bound to GPIbβ deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbβ, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking 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. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbβ in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbβ.

Keywords

GPIb/V/IX complex - von Willebrand factor - adhesion

• References

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