Probing platelet factor 4 α‐granule targeting

• Published in: Journal of thrombosis and haemostasis 2004-12, Vol.2 (12), p.2231-2240
• Main Authors: Briquet‐Laugier, V., Lavenu‐Bombled, C., Schmitt, A., Leboeuf, M., Uzan, G., Dubart‐Kupperschmitt, A., Rosa, J.‐P.
• Format: Article
• Language: English
• Subjects: Adrenocorticotropic Hormone - metabolism; Animals; Antigens, CD34 - biosynthesis; Blood Platelet Disorders - blood; Blood Platelet Disorders - congenital; Blotting, Western; Cell Line; Cell Line, Tumor; Cytoplasm - metabolism; Cytoplasmic Granules - metabolism; DNA Primers - chemistry; Fetal Blood - metabolism; green fluorescent protein intracellular targeting; Green Fluorescent Proteins - metabolism; Immunoprecipitation; Lentivirus - genetics; megakaryocyte alpha granule; Megakaryocytes - cytology; Megakaryocytes - metabolism; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Microscopy, Immunoelectron; Platelet Factor 4 - metabolism; Protein Sorting Signals; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins - metabolism; Thrombin - metabolism; Transfection; von Willebrand factor; von Willebrand Factor - metabolism
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/j.1538-7836.2004.01037.x

The storage mechanism of endogenous secretory proteins in megakaryocyte α‐granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well‐known platelet α‐granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4‐GFP), were transfected in the well‐characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno‐electronmicroscopy. In preliminary experiments, GFP, a non‐secretory protein (no signal peptide), localized in the cytoplasm, while PF4‐GFP colocalized with adrenocorticotropin hormone (ACTH)‐containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4‐GFP localized in α‐granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify α‐granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4‐GFP), GFP was not stored into granules in spite of its efficient translocation to the ER‐Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in α‐granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4‐GFP appears as an appropriate probe with which to analyze α‐granule biogenesis and its alterations in the congenital defect gray platelet syndrome.