engleski

Platelets in Lowe syndrome: how OCRL contributes to the cytoskeletal rearrangements during platelet activation

Lowe syndrome (LS) is a rare X-linked disorder characterized by symptoms that affect three specific organs: the brain, the eyes, and the kidneys. LS patients suffer from central hypotonia, mental retardation, glaucoma, congenital cataracts, and low molecular weight proteinuria that leads to renal Fanconi syndrome. 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 is localized at the plasma membrane and has many roles in the cell including the regulation of actin nucleation and reorganization. This is also a crucial step during the activation of the smallest blood cells, platelets (PLT), which upon vessel wall injury adhere to the exposed molecules of extracellular matrix, activate, aggregate, and with the help of fibrinogen form a clot. In addition to the plethora of LS symptoms, it has been shown that occasionally LS patients have bleeding problems, especially if undergoing surgeries. This led us to investigate the molecular mechanism by which OCRL could modulate PLT function. We show by immunocytochemistry that OCRL inhibition with the YU142670 inhibitor increases PI(4, 5)P2 levels in PLTs suggesting its specificity. Furthermore, OCRL inhibition impairs PLT spreading on fibrinogen, resulting in the extensive formation of actin nodules as opposed to the actin stress fibres of control PLTs. These actin nodules colocalize with proteins implicated in actin dynamics (ARP2/3, vinculin, SNX9) and with phospho-tyrosines showing they are sites of active signalling. Although OCRL-inhibited PLTs form actin nodules, the flow cytometry analysis revealed no change in the net F-actin levels. Moreover, Western Blot analysis showed that OCRL inhibition decreases the levels of myosin light chain (MLC) phosphorylation upon stimulation with PLT activators, thrombin and TRAP-6. Interestingly, OCRL inhibition also impairs the disassembly of the microtubular coil needed for full PLT spreading. Impaired cytoskeletal rearrangements were further confirmed by electron microscopy. The effect of OCRL inhibition was specific to the cytoskeleton since PLT could release their granules and activate integrins after stimulation of thrombin or collagen receptors. We conclude that OCRL reorganizes actin and tubulin cytoskeleton during PLT spreading but has no impact on PLT degranulation or integrin activation. These results could also explain the mild bleeding problems that affect LS patients.

OCRL, PI(4, 5)P2, platelets, Lowe syndrome

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