Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4084-4092
Ex Vivo Cultured Megakaryocytes Express Functional Glycoprotein
IIb-IIIa Receptors and Are Capable of Adenovirus-Mediated Transgene
Expression
Nauder Faraday,
Jeffrey J. Rade,
David C. Johns,
Gopal Khetawat,
Stephen J. Noga,
John F. DiPersio,
Ying Jin,
Janet L. Nichol,
Jeff
S. Haug, and
Paul F. Bray
From the Department of Anesthesiology and Critical Care Medicine, the
Divisions of Cardiology and Hematology, the Department of Medicine, and
the Department of Oncology, Johns Hopkins University School of
Medicine, Baltimore, MD; the Division of BMT and Cell Biology,
Washington University School of Medicine, St Louis, MO; and Amgen, Inc,
Thousand Oaks, CA.
Investigation of the molecular basis of megakaryocyte (MK) and
platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of
transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence
of pegylated recombinant human MK growth and development factor
(PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as
assessed by agonist inducible soluble fibrinogen and PAC1 binding. The
specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa
receptors were similar to those described for blood platelets. The
reversibility and internalization of ligands bound to MK GPIIb-IIIa
also shared similarities with those observed in platelets. Cultured MKs
were transduced with an adenoviral vector encoding green fluorescence
protein (GFP) or 
-galactosidase (-gal). Efficiency of gene
transfer increased with increasing multiplicities of infection and
incubation time, with 45% of MKs expressing GFP 72 hours after viral
infection. Transduced MKs remained capable of agonist induced
GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist
responsive GPIIb-IIIa receptors, (2) are capable of expressing
transgenes, and (3) may prove useful for investigation of the molecular
basis of MK differentiation and GPIIb-IIIa function.
