CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Robert Brian Jenkins, W. L. Nichols, K. G. Mann, L. A. Solberg

Research output: Contribution to journalArticle

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Abstract

Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with ≥95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1-1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1-1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1-1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.

Original languageEnglish (US)
Pages (from-to)682-688
Number of pages7
JournalBlood
Volume67
Issue number3
StatePublished - 1986

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Platelet Glycoprotein GPIIb-IIIa Complex
Megakaryocytes
Sepharose
Bone
Proteins
Resins
Methionine
Bone Marrow
Monoclonal Antibodies
Plasma (human)
Integrin beta3
Platelet Membrane Glycoproteins
Factor X
Electrophoretic mobility
Methylcellulose
Macrophages
Electrophoresis
Autoradiography
Bone Marrow Cells
Detergents

ASJC Scopus subject areas

  • Hematology

Cite this

Jenkins, R. B., Nichols, W. L., Mann, K. G., & Solberg, L. A. (1986). CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa. Blood, 67(3), 682-688.

CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa. / Jenkins, Robert Brian; Nichols, W. L.; Mann, K. G.; Solberg, L. A.

In: Blood, Vol. 67, No. 3, 1986, p. 682-688.

Research output: Contribution to journalArticle

Jenkins, RB, Nichols, WL, Mann, KG & Solberg, LA 1986, 'CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa', Blood, vol. 67, no. 3, pp. 682-688.
Jenkins RB, Nichols WL, Mann KG, Solberg LA. CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa. Blood. 1986;67(3):682-688.
Jenkins, Robert Brian ; Nichols, W. L. ; Mann, K. G. ; Solberg, L. A. / CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa. In: Blood. 1986 ; Vol. 67, No. 3. pp. 682-688.
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abstract = "Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with ≥95{\%} viability and containing 70{\%} to 90{\%} megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1-1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1-1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1-1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.",
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