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 α‐granule protein, is a key physiological regulator of fibrinolysis. Because information on the packaging of PAI‐1 into α‐granules during megakaryocytopoiesis may reveal novel approaches for controlling hemostasis, this study investigated basal, plasmid‐mediated, and alphavirus‐mediated PAI‐1 packaging into α‐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 α‐granules. PAI‐1 (40 ng/mg protein) in isolated MEG‐01 storage granules was ∼10% of the levels present in isolated platelet α‐granules. To elevate PAI‐1 production/storage, two expression systems were investigated. Experiments with plasmids encoding PAI‐1 and β‐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/106 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 α‐granule proteins. J. Cell. Biochem. 82: 277–289, 2001. © 2001 Wiley‐Liss, Inc.