PB0850 - Lowe syndrome in platelets: OCRL controls the reorganization of actin and tubulin filaments during platelet activation

Background: Lowe syndrome (LS) is a rare X-linked disorder characterized by a triade of symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe protein (OCRL) which is a 5-phosphatase that dephosphorylates phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to phosphatidylinositol-4-monophosphate (PI4P). PI(4,5)P2 regulates actin nucleation and reorganization which is a crucial step during platelet (PLT) activation. Moreover, it has been shown that LS patients have occasional bleeding problems that led us to investigate the role of OCRL in PLTs.

Aims: Our aim is to determine the molecular mechanism by which OCRL regulates PLT cytoskeletal rearrangements.

Methods: We pharmacologically inhibited OCRL and used confocal and electron microscopy to visualize PLTs, Western blot for the analysis of PLT signalling pathways, and flow cytometry to investigate PLT activation markers and F-actin levels.

Results: We show that OCRL inhibition increases PI(4,5)P2 levels in PLTs and impairs PLT spreading on fibrinogen, resulting in the extensive formation of actin nodules which colocalize with proteins implicated in actin dynamics (ARP2/3, Vinculin, SNX9). These nodules also colocalize with pTyr showing they are sites of active signalling. Furthermore, OCRL inhibition decreases the levels of MLC phosphorylation upon stimulation with thrombin and TRAP-6, although without change in net F-actin. Interestingly, OCRL inhibition also impairs the disassembly of the microtubular coil during PLT spreading and it increases the levels of acetylated tubulin. Altered cytoskeletal changes did not change P-selectin surface expression or αIIbβIII activation upon platelet activation. Finally, we show that the kinetics of GPIbα surface downregulation is impaired upon OCRL inhibition.

Conclusion(s): OCRL controls actin and tubulin reorganization as well as GPIbα downregulation in PLTs but has no effect on PLT degranulation or integrin activation.