Purification and partial characterization of a megakaryocyte colony-stimulating factor from human plasma.

Abstract

Human plasma obtained from patients with hypomegakaryocytic thrombocytopenia contains a factor that promotes megakaryocyte colony formation by normal human marrow cells. This megakaryocyte colony-stimulating factor was purified from such a plasma specimen. A four-step purification scheme which included ammonium sulfate precipitation, diethylaminoethylSepharose chromatography, affinity chromatography on wheat germ lectin-Sepharose 6MB, and reverse-phase high performance liquid chromatography resulted in a recovery of 16.6% of the initial biological activity and an increase in specific activity by 3,489-fold. The purified protein produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified megakaryocyte colony-stimulating factor was capable of promoting megakaryocyte colony formation at a concentration of 7.6 X iO-M. Megakaryocyte colony-stimulating factor was shown to be a glycoprotein and had an apparent 46,000 mol wt. Deglycosylation of megakaryocyte colony-stimulating factor by treatment with trifluoromethanesulfonate resulted in the loss of its ability to promote megakaryocyte colony formation. Megakaryocyte colony-stimulating factor appears to be an important regulator of in vitro human megakaryocytopoiesis at the level of the colony-forming unit megakaryocyte and may be of importance physiologically.

Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia.

“…Measurable levels of MGDF in serum collected from patients undergoing myeloablative therapy and transplantation demonstrate the physiological relevance of this molecule. These data support the concept that all of the megakaryopoietic and thrombopoietic activities present in plasma, sera, or urine from models of bone marrow aplasia or thrombocytopenia (2,11,(17)(18)(19)(20) can be substantially, if not wholly, accounted for by the cytokine MGDF.…”

“…The hematopoietic factor(s) directing the processes of megakaryocyte development and platelet formation have been the object of intense study for nearly 40 yr (1,2). Traditionally, regulation of this lineage has been attributed to separate factors controlling growth (megakaryocyte colony-stimulating factors, Meg-CSF') versus differentiation (thrombopoietin, TPO) (3)(4)(5)(6)(7)(8)(9)(10).…”

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

“…With this tool, the existence of several positive, though not necessarily lineage specific, growth-enhancing molecules has been demonstrated. Such proliferation and/or maturation promoting activities include megakaryocyte colony-stimulating factor (Meg-CSF)l (2), granulocyte-macrophage colony-stimulating factor (GM-CSF) (3), IL 3 (4), thrombopoietin (TPO) (5,6), megakaryocyte stimulatory factor (MSF) (7,8), and erythropoietin (9).…”

Recent developments in ex vivo platelet production

“…A subsequent study presented evidences that platelets produced from this system functioned in a similar manner to human-isolated platelets (Choi et al 1995). Since then, the successful purification of TPO from serum (Kato et al 1995) or plasma (Hoffman et al 1985) and the synthesis of recombinant TPO (Bartley et al 1994;Lok et al 1994) have driven extensive studies on optimizing HSCs as platelet-producing cells in vitro.…”