GPIb-filamin A interaction regulates megakaryocyte localization and budding during platelet biogenesis

Glycoprotein Ib alpha (GPIb alpha) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIb alpha (hGPIb alpha WT) or a flnA-binding mutant (hGPIb alpha FW) and lacking endogenous mouse GPIb alpha. Mice expressing the mutant GPIb alpha transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIb alpha FW mice. The most striking abnormalities in hGPIb alpha FW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIb alpha interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIb alpha corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIb alpha is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud