The development of megakaryocytes (MKs) from their marrow precursors is one of the least understood aspects of hematopoiesis. Current models suggest that earlyacting MK colony-stimulating factors, such as interleukin (IL) 3 or c-kit ligand, are required for expansion of hematopoietic progenitors into cells capable of responding to lateacting MK potentiators, including IL-6 and IL-li. Recently, the Mpl ligand, or thrombopoietin (Tpo), has been shown to display both MK colony-stimulating factor and potentiator activities, at potencies far greater than that of other cytokines. In light of these findings, we tested the hypothesis that Tpo is absolutely necessary for MK development. In this report we demonstrate that neutralizing the biological activity of Tpo eliminates MK formation in response to c-kit ligand, IL-6, and IL-11, alone and in combination, but that these reagents only partially reduce MK formation in the presence of combinations of cytokines including IL-3. However, despite the capacity of IL-3 to support the proliferation and initial stages of MK differentiation, elimination of Tpo prevents the full maturation of IL-3-induced MK. These data indicate that two populations of MK progenitors can be identified: one that is responsive to IL-3 but can fully develop only in the presence of Tpo and a second that is dependent on Tpo for both proliferation and differentiation. Thus, our results strongly suggest that Tpo is the primary regulator of MK development and platelet production.The generation of megakaryocytes (MKs) is a complex process dependent on the interaction of hematopoietic progenitor cells, cytokines, and stromal elements (1-3). Committed MK progenitor cells must undergo a series of mitotic divisions, shift to endomitotic replication, express specific membrane glycoproteins, and undergo cytoplasmic maturation in preparation for platelet shedding. Models of erythropoiesis and granulopoiesis have been established that stress the importance of both early-acting and late-acting cytokines or hormones for completion of the erythroid and granulocytic developmental programs (4-6). A number of investigators have shown that interleukin (IL) 3 and c-kit ligand (KL) can support the production of MK colonies from their progenitors [MK colony-forming units (CFU-MK)] in semisolid medium and of individual MKs in suspension culture (7-10). Moreover, although IL-6, IL-11, and leukemia inhibitory factor are not reported to support MK formation alone, these cytokines augment the MK response to IL-3 or KL (11)(12)(13)(14). Recently, we (15) and other groups (16, 17) have cloned and expressed the ligand for the c-Mpl receptor. Based on its capacity to induce increases in the size, ploidy, and maturation of MKs, its inverse relationship with platelet levels, and its ability to increase platelet production manyfold (18, 19), we proposed that the Mpl ligand is identical to thrombopoietin (Tpo) (20, 21).The publication costs of this article were defrayed in part by page charge payment. This article must therefore...

Significance The myeloproliferative neoplasms (MPNs) are a group of hematological malignancies characterized by increased numbers of myeloid blood cells, such as platelets, erythrocytes, and neutrophils. The main causes of illness and death in patients with MPNs are arterial and venous clotting and also, conversely, bleeding complications. However, the causes of these conditions are poorly understood. In this paper, we use a mouse model of MPNs to determine the cell types responsible for abnormal clotting in MPNs. We demonstrate that endothelial cells, the cell type that lines all blood vessels, have a significant role to play in MPN bleeding complications, potentially identifying a new cellular target for MPN therapies.

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short- term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.

IntroductionFocal adhesion kinase (FAK) is an essential nonreceptor protein tyrosine kinase that is expressed ubiquitously and is conserved in mammals and lower eukaryotic organisms. [1][2][3][4] The principal FAK stimulus is integrin engagement (Guan and Shalloway 5 and reviewed in Parsons 6 ), although its direct interaction with, and activation via, platelet-derived and epidermal growth factor receptors suggests that it may also function downstream of other membrane-bound growth factor and cytokine receptors. 7 Autophosphorylation of FAK Tyr397 occurs rapidly following integrin activation, and the resulting phosphorylated residue acts as a docking site for the SH2 domains of Src-family kinases. 8,9 FAK catalytic activity is increased by subsequent Src-mediated phosphorylation of FAK residues Tyr576 and Tyr577, 10 followed by Tyr861 and Tyr925, which act as binding sites for the SH3 domain of p130CAS and the SH2 domain of the adaptor protein GRB2, respectively. 11,12 Tyr925, located in the focal adhesion targeting domain, also mediates interactions with integrin-associated proteins such as talin and paxillin, 13 thereby recruiting FAK to focal adhesion sites.Activated FAK modulates the activity of a broad range of downstream signaling proteins, including phosphoinositide 3-kinase (PI3-K) 14 and phospholipase C (PLC)-␥, 15 as well as a number of small GTPases such as Ras, Rac, and Rho (reviewed in 16 ). Extensive studies indicate that FAK is essential for normal cell migration. FAK-deficient cells migrate poorly in response to chemokines; form an increased number of prominent "immature" focal adhesions, apparently due to decreased focal adhesion turnover; and do not spread normally on extracellular matrices (ECMs). 17,18 Megakaryopoiesis and platelet production are tightly regulated by a number of growth factors and cytokines to maintain a normal number of circulating platelets. The principal regulator of megakaryopoiesis is thrombopoietin (Tpo), 19 although other fac 1 tors such as interleukin-3 and stem-cell factor work in synergy with Tpo during the earlier stages of megakaryocytic progenitor cell expansion. 20 It has become apparent that the microenvironments in which megakaryocytes function are also of critical importance to megakaryopoiesis. 21,22 Indeed, direct cell-cell and cell-ECM interactions have been demonstrated to influence megakaryocyte differentiation and proplatelet formation. [23][24][25] Although FAK is important in regulating cell spreading and migration in response to integrin-ECM interactions, the role of FAK in megakaryopoiesis remains unclear, partly because Fak deletion in mice is lethal at embryonic day 8.5, before the onset of significant definitive hematopoiesis. Although the role of proline-rich tyrosine kinase-2 (PYK2), which shares sequence homology and similar characteristics with FAK, has been characterized in megakaryocytes, 8,[26][27][28] its localization and dependence on intracellular calcium make the 2 proteins functionally different. 29 FAK activation in platelets require...

No abstract