Recombinant semliki forest virus enhanced plasminogen activator inhibitor 1 expression and storage in the megakaryocytic cell line MEG-01

Platelet plasminogen activator inhibitor I (PAI-1), a trace alpha -granule protein, is a key physiological regulator of fibrinolysis. Because informat ion on the packaging of PAI-1 into alpha -granules during megakaryocytopoie sis may reveal novel approaches for controlling hemostasis, this study inve stigated basal, plasmid-mediated, and alphavirus-mediated PAI-1 packaging i nto alpha -granules-like structures in the megakaryocytic cell line MEG-01. Differentiation of MEG-01 cells with phorbol myristate acetate (PMA) was o bserved to result in a four-fold increase in both secreted and cell-associa ted 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 g ranules share a similar size distribution (350-600 nm) and morphology to pl atelet alpha -granules. PAI-1 (40 ng/mg protein) in isolated MEG-01 storage granules was similar to 10% Of the levels present in isolated platelet alp ha -granules. To elevate PAI-1 production/storage, two expression systems w ere investigated. Experiments with plasmids encoding PAI-1 and beta -galact osidase resulted in low transfection efficiency (0.001%). In contrast, Seml iki 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 express ion could enhance PAI-1 storage 6-9-fold, reaching levels present within pl atelets. To document the ability of PAI-1 to be stored in a rapidly releasa ble form in MEG-01 cells, we isolated platelet-like particles from the medi a conditioned by the cells and examined secretagogue-induced release of PAI -1. Particles from SFV/PAI-1 infected cells display a 5-fold enhanced secre tion of PAI-1 following treatment with ADP in comparison to particles incub ated in the absence of secretagogue. These results suggest that SFV mediate d gene expression in MEG-01 cells provides a useful framework for analyzing the production and storage of alpha -granule proteins. J. Cell. Biochem. 8 2: 277-289, 2001. (C) 2001 Wiley-Liss, Inc.