Recombinant Semliki Forest virus enhanced plasminogen activator inhibitor 1 expression and storage in the megakaryocytic cell line MEG-01.

摘要: Platelet plasminogen activator inhibitor I (PAI-1), a trace alpha-granule protein, is key physiological regulator of fibrinolysis. Because information on the packaging PAI-1 into alpha-granules during megakaryocytopoiesis may reveal novel approaches for controlling hemostasis, this study investigated basal, plasmid-mediated, and alphavirus-mediated alpha-granules-like structures in megakaryocytic cell line MEG-01. Differentiation MEG-01 cells with phorbol myristate acetate (PMA) was observed to result four-fold increase both secreted cell-associated antigen over four day period. Subcellular fractionation PMA-treated 45% self-forming Percoll gradients employed separate low density membrane Golgi-rich fractions from high granule-containing region. A subsequent 30-60% pre-formed gradient remove contaminating lysosomes PAI-1/glycoprotein IIbIIIa-containing granules. Electron microscopy showed that these granules share similar size distribution (350-600 nm) morphology platelet alpha-granules. (40 ng/mg protein) isolated storage approximately 10% levels present To elevate production/storage, two expression systems were investigated. Experiments plasmids encoding beta-galactosidase resulted transfection efficiency (0.001%). In contrast, Semliki Forest virus (SFV)-mediated gene transfer increased cellular by 31-fold (1,200 ng/10(6) at 10 MOI) comparison mock-infected cells. Pulse-chase experiments demonstrated SFV/PAI-1 mediated could enhance 6-9-fold, reaching within platelets. document ability be stored rapidly releasable form cells, we platelet-like particles media conditioned examined secretagogue-induced release PAI-1. Particles infected display 5-fold enhanced secretion following treatment ADP incubated absence secretagogue. These results suggest SFV provides useful framework analyzing production proteins.