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

Abstract

Platelets release large quantities of plasminogen activator inhibitor 1 (PAI-1) that plays an important role in maintaining the integrity of fibrin-rich thrombi. In addition, tissue-type plasminogen activator (t-PA), a key physiological regulator of fibrinolysis, has been detected in platelet alpha-granules at low abundance. This information raises the possibility of enhancing t-PA expression in megakaryocytes as a means to enhance the fibrinolytic properties of platelet alpha-granules and target PAs directly to fibrin clots. This study was initiated to investigate adenovirus (Ad)-mediated expression and packaging of t-PA into alpha-granules-like structures in the megakaryocytic cell line MEG-01. Ad/t-PA infection of phorbol myristate acetate (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 indicates 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 alpha-granule-like structures, MEG-01 cell homogenates were fractionated by differential centrifugation and two consecutive Percoll density gradients. Fibrin autography of storage granules revealed a prominent lytic zone at Mr 66 kD comigrating with 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-infected cells. To document the ability of t-PA to be stored in a rapidly-releasable 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 clasical secretagogue calcium ionphore 23187, ADP, or thrombin. Confocal immunofluoresence microscopy analysis indicates that Ad/t-PA mediated productive expression of t-PA in murine megakaryocytes. These data provide support for the concept of increasing the expression of t-PA in megakaryocytes as a means to alter the hemostatic properties of alpha-granules.

• Authors: J L Chuang, R R Schleef
• Journal: Thrombosis and haemostasis (Thromb Haemost) Vol. 85 Issue 6 Pg. 1079-85 (Jun 2001) ISSN: 0340-6245 [Print] Germany
• PMID: 11434688 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Fibrinolytic Agents
• Plasminogen Activator Inhibitor 1
• Platelet Glycoprotein GPIIb-IIIa Complex
• Tissue Plasminogen Activator
• Tetradecanoylphorbol Acetate

Topics

• Adenoviridae (genetics)
• Animals
• Bone Marrow Cells (cytology)
• Cell Differentiation (drug effects)
• Cytoplasmic Granules (metabolism)
• Fibrinolytic Agents (metabolism)
• Megakaryocytes (metabolism, ultrastructure)
• Mice
• Microscopy, Fluorescence
• Plasminogen Activator Inhibitor 1 (metabolism)
• Platelet Glycoprotein GPIIb-IIIa Complex (analysis)
• Tetradecanoylphorbol Acetate (pharmacology)
• Tissue Plasminogen Activator (drug effects, genetics, metabolism)
• Transduction, Genetic