Abstract

The hereditary bleeding disorder Bernard-Soulier syndrome (BSS) is caused by an absent or dysfunctional GPIb/IX complex. Lopez et al. have reported that three subunits of the complex (GPIbα, GPIbβ and GPIX) are all necessary for efficient translocation to the cell surface. In contrast, Meyer et al. have demonstrated that GPIbα alone can incorporate into the cell surface. We have established a cell culture system with inducible levels of GPIbβ and GPIX. Using these cell lines, we characterize an intrinsic ability of GPIbα to form dimers and the relevance of specific GPIbα sequences for dimer formation and the assembly of a GPIb/IX complex.

A stable CHO cell line was established that expresses GPIbβ and GPIX under the control of Tet-responsive elements. This cell line was then transfected with WT GPIbα and truncated GPIbα mutants. Expression was monitored by Western blot analysis and flow cytometry.

We observed that expression of GPIbα, by itself, targets GPIbα to the cell surface as a dimer albeit at reduced levels. The GPIbα binding domain for filamin (residues 545–605) is essential for forming a dimer but not for assembly of a GPIb/IX complex. We also observed GPIbα dimer formation in a transgenic mouse that expresses human GPIbα and a mouse chimeric GPIb-IX complex suggesting dimer formation can occur in the platelet. To investigate the role of filamin in the dimerization process, we established a stable cell line deficient in filamin by targeting filamin expression with RNAi. We observed GPIbα dimers in the absence of filamin expression. These results illustrate the importance of expressing all three subunits of the GPIb/IX complex, but also document as intrinsic potential within GPIbα for surface expression via intermolecular homodimerization.

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