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

ABSTRACT: Glycoprotein Ibα (GPIbα) 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) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW 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-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα 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 GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.

Cerebral vasculature exhibits dose-dependent sensitivity to thrombocytopenia that is limited to fetal/neonatal life.

Whether increasing platelet counts in fetal and neonatal alloimmune thrombocytopenia (FNAIT) is effective at preventing intracerebral hemorrhage (ICH) has been a subject of debate. The crux of the matter has been whether thrombocytopenia is the major driver of ICH in diseases such as FNAIT. We recently demonstrated in mice that severe thrombocytopenia was sufficient to drive ICH in utero and in early neonatal life. It remains unclear what degree of thrombocytopenia is required to drive ICH and for how long after birth thrombocytopenia can cause ICH. By inducing a thrombocytopenic range, we demonstrate that there is a large buffer zone of mild thrombocytopenia that does not result in ICH, that ICH becomes probabilistic at 40% of the normal platelet number, and that ICH becomes fully penetrant below 10% of the normal platelet number. We also demonstrate that although the neonatal mouse is susceptible to thrombocytopenia-induced ICH, this sensitivity is rapidly lost between postnatal days 7 and 14. These findings provide important insights into the risk of in utero ICH with varying degrees of thrombocytopenia and into defining the developmental high-risk period for thrombocytopenia-driven ICH in a mouse model of FNAIT.