title: Recombinant Semliki Forest virus enhanced plasminogen activator inhibitor 1 expression and storage in the megakaryocytic cell line MEG-01.
abstract: Platelet plasminogen activator inhibitor I (PAI-1), a trace alpha-granule protein, is a key physiological regulator of fibrinolysis. Because information on the packaging of PAI-1 into alpha-granules during megakaryocytopoiesis may reveal novel approaches for controlling hemostasis, this study investigated basal, plasmid-mediated, and alphavirus-mediated PAI-1 packaging into alpha-granules-like structures in the megakaryocytic cell line MEG-01. Differentiation of MEG-01 cells with phorbol myristate acetate (PMA) was observed to result in a four-fold increase in both secreted and cell-associated PAI-1 antigen over a four day period. Subcellular fractionation of PMA-treated MEG-01 cells on 45% self-forming Percoll gradients was employed to separate low density membrane and Golgi-rich fractions from a high density granule-containing region. A subsequent 30-60% pre-formed Percoll gradient was employed to remove contaminating lysosomes from the PAI-1/glycoprotein IIbIIIa-containing granules. Electron microscopy showed that these MEG-01 granules share a similar size distribution (350-600 nm) and morphology to platelet alpha-granules. PAI-1 (40 ng/mg protein) in isolated MEG-01 storage granules was approximately 10% of the levels present in isolated platelet alpha-granules. To elevate PAI-1 production/storage, two expression systems were investigated. Experiments with plasmids encoding PAI-1 and beta-galactosidase resulted in low transfection efficiency (0.001%). In contrast, Semliki Forest virus (SFV)-mediated gene transfer increased cellular PAI-1 by 31-fold (1,200 ng/10(6) cells at 10 MOI) in comparison to mock-infected cells. Pulse-chase experiments demonstrated that SFV/PAI-1 mediated gene expression could enhance PAI-1 storage 6-9-fold, reaching levels present within platelets. To document the ability of PAI-1 to be stored in a rapidly releasable form in MEG-01 cells, we isolated platelet-like particles from the media conditioned by the cells and examined secretagogue-induced release of PAI-1. Particles from SFV/PAI-1 infected cells display a 5-fold enhanced secretion of PAI-1 following treatment with ADP in comparison to particles incubated in the absence of secretagogue. These results suggest that SFV mediated gene expression in MEG-01 cells provides a useful framework for analyzing the production and storage of alpha-granule proteins.
authors.affiliation: 92037, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Chuang
authors.forename: R R
authors.initials: RR
authors.lastname: Schleef
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003594:Cytoplasmic Granules / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics* / Q000502:physiology; D005966:Glucuronidase / Q000032:analysis; D007668:Kidney; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology*; D008647:Mesocricetus; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D019704:Protein Disulfide-Isomerases / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012672:Semliki forest virus / Q000235:genetics* / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis; D014841:von Willebrand Factor / Q000032:analysis
chemical_list: D015415:Biomarkers; D017395:Plasminogen Activator Inhibitor 1; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D000244:Adenosine Diphosphate; D001616:beta-Galactosidase; D005966:Glucuronidase; D019704:Protein Disulfide-Isomerases; D013755:Tetradecanoylphorbol Acetate