Adenovirus-mediated expression and packaging of tissue-type plasminogen activator in megakaryocytic cells

Platelets release large quantities of plasminogen activator inhibitor 1 (PA I-1) that plays an important role in maintaining the integrity of fibrin-ri ch thrombi. In addition, tissue-type plasminogen activator (t-PA). a key ph ysiological regulator of fibrinolysis, has been detected in platelet cu-gra nules at low abundance. This information raises the possibility of enhancin g t-PA expression in megakaryocytes as a means to enhance the fibrinolytic properties of platelet alpha -granules and target PAs directly to fibrin cl ots, This study was initiated to investigate adenovirus (Ad)-mediated expre ssion acid packaging of t-PA into alpha -granules-like structures in the me gakaryocytic cell line MEG-01. Adit-PA infection of phorbol myristate aceta te (PMA)-differentiated MEG-01 cells increased cellular t-PA levels by 120 fold (1580 +/- 130 ng/10(6) cells at 5 MOI) in comparison to non-or Ad/beta -gal-infected cells, Fluorescence-activated cell sorter (FACS) analysis in dicates that Ad/t-PA-infected cells yielded a homogenous shift in the t-PA staining profile with a 4-fold shift in mean fluorescence in comparison to non- or Ad/beta -gal-infected cells. For the isolation of a-granule-like st ructures, MEG-01 cell homogenates were Fractionated by differential centrif ugation and two consecutive Percoll density gradients, Fibrin autography of storage granules revealed a prominent lytic zone at Mr 66 kD comigrating w ith free t-PA, Quantitative analyses indicate that a 16-fold elevation in t -PA antigen within storage granules in comparison to non- or Ad/beta -gal-i nfected cells. To document the ability of t-PA to be stored in a rapidly-re leasable form in these cells, we isolated platelet-like particles from the supernatant of differentiated cells and determined that particles from Ad/t -PA-infected cells display a 4-8 fold enhanced secretion of t-PA following treatment with the classical secretagogue calcium ionophore 23187, ADP. or thrombin. Confocal immunofluorescence microscopy analysis indicates that Ad /t-PA mediated productive expression of t-PA in murine megakaryocytes, Thes e data provide support for the concept of increasing the expression oft-PA in megakaryocytes as a means to alter the hemostatic properties of alpha -g ranules.