技术资料

BACKGROUND Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma- and/or platelet-derived factor (F) Va and FXa on the activated platelet surface. While the plasma-derived procofactor,FV,must be proteolytically activated by α-thrombin to FVa to function in prothrombinase,the platelet molecule is released from α-granules in a partially activated state,obviating the need for proteolytic activation. OBJECTIVES The current study was performed to test the hypothesis that subsequent to its endocytosis by megakaryocytes,plasma-derived FV is proteolytically processed to form the platelet-derived pool. METHODS & RESULTS Subsequent to FV endocytosis,a time-dependent increase in FV proteolytic products was observed in megakaryocyte lysates by SDS-PAGE followed by phosphorimaging or western blotting. This cleavage was specific and resulted in the formation of products similar in size to FV/Va present in a platelet lysate as well as to the α-thrombin-activated FVa heavy chain and light chain,and their respective precursors. Other proteolytic products were unique to endocytosed FV. The product/precursor relationships of these fragments were defined using anti-FV heavy and light chain antibodies with defined epitopes. Activity measurements indicated that megakaryocyte-derived FV fragments exhibited substantial FVa cofactor activity that was comparable to platelet-derived FV/Va. CONCLUSIONS Taken together,these observations suggest that prior to its packaging in α-granules endocytosed FV undergoes proteolysis by one or more specific megakaryocyte protease(s) to form the partially activated platelet-derived pool. View Publication

Increasing evidence supports the importance of cell extrinsic regulation in stem cell fate control. Hematopoietic stem cells (HSC) are responsive to local signals from their niche and to systemic feedback from progenitors and mature cells. The Notch ligand Delta-1 (DL1),a key component of the stem cell niche,regulates human hematopoietic lineage development in a dose-dependent manner and has been used clinically for primitive progenitor expansion. How DL1 acts to regulate HSC fate and whether these actions are related to its lineage skewing effects are poorly understood. Here we demonstrate that,although DL1 activates signal transducer and activator of transcription 3 signaling similarly to the gp130-activating cytokine interleukin-6 (IL-6),it has opposite effects on myeloid cell production. Mechanistically,these different outcomes are attributable to a DL1-mediated reduction in membrane (m)-bound IL-6 receptor (R) expression,converting progenitor cells from being directly IL-6 responsive to requiring both IL-6 and soluble (s) IL-6R for activation. Concomitant reduction of both mIL-6R (by DL1 supplementation) and sIL-6R (using dynamically fed cultures) reduced myeloid cell production and led to enhanced outputs of human HSCs. This work describes a new mode of cytokine action in which DL1 changes cytokine receptor distributions on hematopoietic cells,altering feedback networks and their impact on stem cell fate. View Publication

Without effective therapy,chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized,but biologically poorly characterized,accelerated phase (AP). Here,we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts,which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML,we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML,including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients. View Publication

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues,including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury,while poor tissue availability of the protein limits its efficacy. Here,we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine),which selectively activates the tissue-protective EPO receptor,comprising an EPO receptor subunit (EPOR) and the common β-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR,CD131,and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations,STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer,lack of activity in off-target selectivity screening,and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge,STS-E412 is the first nonpeptidyl,selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted. View Publication

An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid,myeloid,and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required,and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells,this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells. View Publication

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels,differentiation intermediates were investigated. ES cells generated progeny expressing CD34,which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7,the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells,and such lymphoid potential was highly enriched in the CD34+ population. Interestingly,the cytotoxic cells expressed the natural killer (NK) cell markers,such as NKR-P1,perforin,and granzymes,classified into two types,one of which showed target specificity of NK cells. Thus,ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells,and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate,on which VEGF may play an important role. View Publication