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OCRL controls actin reorganization in spreading and activation of human platelets

Phosphatidylinositol-4, 5-bisphosphate [PI(4, 5)P2] is one of seven different phosphoinositides mostly localized at the plasma membrane. Its main functions are the regulation of endocytosis and actin reorganization. OCRL is a phosphatase that dephosphorylates PI(4, 5)P2 resulting in the formation of PI4P. Mutations in the OCRL cause Lowe syndrome which is characterized with congenital cataracts, central hypotonia and renal proximal tubular dysfunction. Furthermore, an increased risk of bleedings was shown in some patients with Lowe syndrome. Platelets (PLTs) have a major role in hemostasis by detecting vessel wall injury. They recognize extracellular matrix proteins, become activated, aggregate, and adhere to the injury to prevent bleeding. We hypothesize that the loss of OCRL function leads to the changes in actin dynamics resulting in PLT dysfunction. Here, we show that pharmacological inhibition of OCRL in human PLTs spread on fibrinogen leads to an inhibition of full PLT activation which was shown by the reduced PLT spread area and the formation of actin-rich structures, called actin nodules. The characterization of actin nodules revealed that they partially colocalize with vinculin and SNX9 and substantially with ARP2/3 complex, as well as with phosphotyrosine suggesting the impairment of the cytoskeletal reorganization during PLT activation. To investigate the role of OCRL- inhibition on signaling pathways we inhibited OCRL in PLTs and then activated them with thrombin. The Western blot analysis revealed reduced phosphorylation levels of myosin light chain (MLC), but not p38 or ERK. Furthermore, flow cytometry analysis showed that P-selectin expression was not affected in thrombin-stimulated platelets if OCRL was inhibited. Our data suggest that OCRL modulates actin cytoskeleton rearrangements, but not granule release, during platelet activation and thus contributes to their proper function.

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

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