Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells

• Published in: Journal of cellular and molecular medicine 2012-08, Vol.16 (8), p.1731-1738
• Main Authors: Faille, Dorothée, El‐Assaad, Fatima, Mitchell, Andrew J., Alessi, Marie‐Christine, Chimini, Giovanna, Fusai, Thierry, Grau, Georges E., Combes, Valéry
• Format: Article
• Language: English
• Subjects: Amiloride; Blood Platelets - cytology; Blood Platelets - metabolism; Brain; Brain - cytology; Calcein; Calcium (extracellular); Calcium influx; Cell adhesion molecules; Cell-Derived Microparticles - metabolism; Cellular Biology; Chelation; Cholesterol; Confocal microscopy; Cytochalasin D; Endocytosis; Endoplasmic reticulum; Endosomes; Endosomes - metabolism; Endothelial cells; Endothelial Cells - cytology; Endothelial Cells - metabolism; Flow cytometry; Fluoresceins - metabolism; Humans; Immunology; inflammation; Intercellular adhesion molecule 1; Internalization; Intracellular; Intracellular Space - metabolism; Laboratories; Life Sciences; Lysosomes; Lysosomes - metabolism; macropinocytosis; Membrane Fusion; Methyl-β-Cyclodextrin; Microparticles; Microscopy; Na+/H+-exchanging ATPase; Original; Phagocytosis; Phenotypes; Platelets; Software; Subcellular Fractions - metabolism; Trypsin; Tumor necrosis factor-TNF; Vascular cell adhesion molecule 1
• ISSN: 1582-1838; 1582-4934
• DOI: 10.1111/j.1582-4934.2011.01434.x

Platelet‐derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67‐labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker‐labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein‐loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca2+ chelation by EDTA, suggesting a partial role for receptor‐mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule‐1 and vascular cell adhesion molecule‐1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet‐derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin‐dependent phagocytosis. Platelet‐derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl‐β‐cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na+/H+ exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.