title: AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl.
abstract: Thrombopoietin (TPO) plays a pivotal role in megakaryopoiesis. TPO initiates its biological effects by binding to its receptor Mpl. A recombinant protein consisting of a carrier Fc domain linked to multiple Mpl-binding domains was constructed, and is called AMG531. To define the biological activity of AMG531, we examined the ability of AMG531 to support CFU-Meg growth and to promote megakaryocyte maturation in vitro. AMG531 stimulates CFU-Meg growth in a dose-dependent manner, and acts in concert with erythropoietin, stem cell factor, interleukin-3, and interleukin-6 to enhance CFU-Meg growth, similar to parallel experiments with TPO. AMG531-stimulated serum-free liquid cultures support the development of mature polyploid megakaryocytes with a predominant DNA content of 32 N and 64 N, identical to that of parallel TPO-stimulated cultures. Competitive binding experiments show that AMG531 effectively competes with 125I-TPO for binding to BaF3-Mpl cells or normal platelets. Treatment of BaF3-Mpl cells with AMG531 or with TPO resulted in rapid tyrosine phosphorylation of Mpl, JAK2, and STAT5. These results indicate that AMG531 is a potent stimulant of megakarypoiesis in vitro, and provide support for its further characterization in vivo.
authors.affiliation: Division of Hematology, University of Washington, Seattle, WA 98144, USA, vcbroudy@u.washington.edu
authors.forename: Virginia C
authors.initials: VC
authors.lastname: Broudy
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Lin
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000166:cytology; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology*; D002454:Cell Differentiation; D016207:Cytokines / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006410:Hematopoiesis; D006801:Humans; D053614:Janus Kinase 2; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008894:Milk Proteins; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000378:metabolism*; D011961:Receptors, Fc; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D050799:STAT5 Transcription Factor; D013234:Stem Cells / Q000378:metabolism; D013926:Thrombopoietin; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D016207:Cytokines; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D011961:Receptors, Fc; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; C082218:MPL protein, human; D013926:Thrombopoietin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; C488777:romiplostim