Abstract Number: PB0987
Meeting: ISTH 2021 Congress
Background: Phosphatidylinositol-4-phosphate (PI4P) is a phosphoinositide found mostly at the Golgi apparatus and partially at the plasma membrane (PM). At the PM, PI4P can contribute to the polyanionic lipid pool or can serve for the formation of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 is primarily localized at the PM where it regulates actin reorganization. The staining method utilizing antibodies for visualizing these phosphoinositides on the PM has been well established in HeLa cells (Hammond et al 2009. Biochem J) but not in blood cells such as human platelets (hPLTs). When using the established protocol, we observed that the majority of activated hPLTs did not stain while the resting hPLTs and control HEK293T cells displayed phosphoinositide staining.
Aims: We tested if changes in the percentage of the permeabilizing agent, the time of the permeabilization, or different permeabilizing agents would improve the success rate of the phosphoinositide staining in hPLTs.
Methods: hPLTs were fixed as rested or spread on glass, permeabilized with different permeabilizing agents (triton, tween, digitonin, saponin) for different times (5, 30, 45 minutes, and one hour) and immunostained for PI4P or PI(4,5)P2. HEK293T cells were used as controls.
Results: hPLTs showed the best staining results for PI4P and PI(4,5)P2 when permeabilized for a shorter period (5 min) with 0,5% saponin while unpermeabilized cells did not exhibit any staining. In contrast, HEK293T cells were stained in all tested conditions. PI4P and PI(4,5)P2 were confined to the PM, and the phosphoinositide signals could be modulated with specific inhibitors of PI4 kinase and PI(4,5)P2 phosphatase.
Conclusions: Our data suggest that PI4P and PI(4,5)P2 at the PM of hPLTs are more sensitive to extraction by permeabilizing agents than in HEK293T cells when PLTs undergo spreading, but not in their resting state. Further studies are underway to investigate the exact role of these lipids in platelet activation.
To cite this abstract in AMA style:Bura A, Đurić I, Čabrijan S, Jurak Begonja A. PI4P and PI(4,5)P2 Immunofluorescence Staining Optimization in Human Platelets [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/pi4p-and-pi45p2-immunofluorescence-staining-optimization-in-human-platelets/. Accessed October 1, 2023.
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