Adenovirus-mediated Expression and Packaging of Tissue-type Plasminogen Activator in Megakaryocytic Cells

Joseph L. Chuang; Raymond R. Schleef

doi:10.1055/s-0037-1615967

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2001; 85(06): 1079-1085
DOI: 10.1055/s-0037-1615967

Review Article

Schattauer GmbH

Adenovirus-mediated Expression and Packaging of Tissue-type Plasminogen Activator in Megakaryocytic Cells

Joseph L. Chuang

Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA, USA

,

Raymond R. Schleef

Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA, USA

› Institutsangaben

Abstract

Summary

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 α-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 α-granules and target PAs directly to fibrin clots. This study was initiated to investigate adenovirus (Ad)-mediated expression and packaging of t-PA into α-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/10⁶ cells at 5 MOI) in comparison to non-or Ad/β-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/β-gal-infected cells. For the isolation of α-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/β-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 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. 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 α-granules.

Abbreviations: Ad, Adenovirus; t-PA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor 1; PMA, phorbol myristate acetate; ELISA, enzyme-linked immunosorbent assay; CM, conditioned media; vWF, von Willebrand factor; ECL, enhanced chemiluminescence; PA, plasminogen activator.

Key words

Tissue-type plasminogen activator - plasminogen activator inhibitor I - platelets - Percoll - megakaryocytes - storage granules - gene transfer - subcellular fractionation - fibrinolysis

Volltext

Referenzen

References
1 George JN. Platelets. Lancet 2000; 355: 1531-9.
2 Zuker-Franklin D. Megakaryocyte and platelet structure. In: Hematology. Hoffman R, Benz WC, Shattil SJ, Furie B, Cohen IR, Silbert CK, McGlave P. eds. New York: Churchill Livingstone; 2000: 1730-40.
3 Smith NM, Pathansali R, Bath PM. Platelets and stroke. Vasc Med 1999; 4: 165-72.
4 Kohler HP, Grant PJ. Plasminogen-activator inhibitor type 1 and coronary artery disease. N E J Med 2000; 342: 1792-801.
5 Devine DV, Carter CJ. Profibrinolytic and antifibrinolytic effects of platelets. Coron Artery Dis 1995; 6: 915-22.
6 Loscalzo J, Pasche B, Ouimet H, Freedman JE. Platelets and plasminogen activation. Thromb Haemost 1995; 74: 291-3.
7 Verstraete M. Third-generation thrombolytic drugs. Am J Med 2000; 109: 52-8.
8 Parmer JR, Miles LA. Targeting of tissue plasminogen activator to the regulated pathway of secretion. Trends Cardiovasc Med 1998; 8: 306-12.
9 Wang DL, Pan YT, Wang JJ, Cheng CH, Liu CY. Demonstration of a functionally active tPA-like plasminogen activator in human platelets. Thromb Haemost 1994; 71: 493-8.
10 Saito H. Megakaryocytic cell lines. Baillieres Clin Haematol 1997; 10: 47-63.
11 Ogura M, Morishima Y, Okumura M, Hotta T, Takamoto S, Ohno R, Hirabayashi N, Nagura H, Saito H. Functional and morphological differentiation induction of a human megakaryoblastic leukemia cell line (MEG-01s) by phorbol diesters. Blood 1988; 72: 49-60.
12 Alessi MC, Chomiki N, Berthier R, Schweitzer A, Fossat C, Juhan-Vague I. Detection of plasminogen activator inhibitor-1 (PAI-1) mRNA in human megakaryocytes by in situ hybridization. Thromb Haemost 1994; 72: 931-6.
13 Wohn K, Kanse SM, Deutsch V, Schmidt T, Eldor A, Preissner KT. The urokinase-receptor (CD87) is expressed in cells of the megakaryoblastic lineage. Thromb Haemost 1997; 77: 540-7.
14 Chuang JL, Schleef R. Recombinant Semliki Forest virus enhanced plasminogen activator inhibitor 1 expression and storage in the megakaryocytic cell line MEG-01. J Cell Biochem. 2001 in press.
15 Takeuchi K, Satoh M, Kuno H, Yoshida T, Kondo H, Takeuchi M. Platelet-like particle formation in the human megakaryoblastic leukaemia cell lines, MEG-01 and MEG-01s. Br J Haematol 1998; 100: 436-44.
16 Takeuchi K, Ogura M, Saito H, Satoh M, Takeuchi M. Production of Platelet-like Particles by a Human Megakaryoblastic Leukemia Cell Line (MEG-01). Exp Cell Res 1991; 193: 223-6.
17 Kullo IJ, Simari RD, Schwartz RS. Vascular gene transfer: From bench to bedside. Arterioscler Thromb Vasc Biol 1999; 19: 196-207.
18 Vassalli G, Dichek DA. Gene therapy for arterial thrombosis. Cardiovasc Res 1997; 35: 459-69.
19 Simmons WL, Rivera KE, Curiel DT, Williams WF, Olman MA. Adenovi-rally mediated gene transfer of functional human tissue-type plasminogen activator to murine lungs. Am J Respir Cell Mol Biol 1998; 18: 307-14.
20 Schleef RR, Higgins DL, Pillemer E, Levitt LJ. Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor. J Clin Invest 1989; 83: 1747-52.
21 Lang IM, Schleef RR. Calcium-dependent stabilization of type 1 plasminogen activator inhibitor within platelet α-granules. J Biol Chem 1996; 271: 2754-61.
22 Loskutoff DJ, Schleef RR. Plasminogen activators and their inhibitors. Methods Enzymol 1988; 163: 293-302.
23 Rabellino EM, Nachman RL, Williams N, Winchester RJ, Ross GD. Human megakaryocytes. I. Characterization of the membrane and cytoplasmic components of isolated marrow megakaryocytes. J Exp Med 1979; 149: 1273-87.
24 Frey BM, Hackett NR, Bergelson JM, Finberg R, Crystal RG, Moore MAS, Rafii S. High-efficiency gene transfer into ex vivo expanded human hematopoietic progenitors and precursor cells by adenovirus vectors. Blood 1998; 91: 2781-92.
25 Jeanneau C, Sultan Y. Tissue plasminogen activator in human megakaryocytes and platelets: immunocytochemical localization, immunoblotting and zymographic analysis. Thromb Haemost 1988; 59: 529-34.
26 Rosnoblet C, Vischer UM, Gerard RD, Irminger J-C, Halban PA, Kruithof EKO. Storage of tissue-type plasminogen activator in Weibel-Palade bodies of human endothelial cells. Arterioscler Throm Vasc Biol 1999; 19: 1796-803.
27 Krasnykh VN, Douglas JT, van Beusechem VW. Genetic targeting of adenoviral vectors. Mol Ther 2000; 1: 391-405.
28 Wilcox DA, Olsen JC, Ishizawa L, Bray PF, French DL, Steeber DA, Bell WR, Griffith M, White II GC. Megakaryocyte-targeted synthesis of the integrin β(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia. Blood 2000; 95: 3645-52.