Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis.
(1/1747)
The identification of stromal cell-derived factor (SDF)-1alpha as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1alpha during megakaryocytopoiesis. CD34(+) cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41(+) cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34(+) cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41(+) cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1alpha acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1alpha is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1alpha induced the migration of colony forming unit-megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1alpha as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1-related thrombocytopenia. (+info)
The Megakaryocyte/Platelet-specific enhancer of the alpha2beta1 integrin gene: two tandem AP1 sites and the mitogen-activated protein kinase signaling cascade.
(2/1747)
The alpha2beta1 integrin, a collagen receptor on platelets and megakaryocytes, is required for normal platelet function. Transcriptional regulation of the alpha2 integrin gene in cells undergoing megakaryocytic differentiation requires a core promoter between bp -30 and -92, a silencer between bp -92 and -351, and megakaryocytic enhancers in the distal 5' flank. We have now identified a 229-bp region of the distal 5' flank of the alpha2 integrin gene required for high-level enhancer activity in cells with megakaryocytic features. Two tandem AP1 binding sites with dyad symmetry are required for enhancer activity and for DNA-protein complex formation with members of the c-fos/c-jun family. The requirement for AP1 activation suggested a role for the mitogen-activated protein kinase (MAPK) signaling pathway in regulating alpha2 integrin gene expression. Inhibition of the MAP kinase cascade with PD98059, a specific inhibitor of MAPK kinase 1, prevented the expression of the alpha2 integrin subunit in cells induced to become megakaryocytic. We provide a model of megakaryocytic differentiation in which expression of the alpha2 integrin gene requires signaling via the MAP kinase pathway to activate two tandem AP1 binding sites in the alpha2 integrin enhancer. (+info)
Differential expression and phosphorylation of CTCF, a c-myc transcriptional regulator, during differentiation of human myeloid cells.
(3/1747)
CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels. (+info)
Depolarization-evoked Ca2+ release in a non-excitable cell, the rat megakaryocyte.
(4/1747)
1. The effect of membrane potential on [Ca2+]i in rat megakaryocytes was studied using simultaneous whole-cell patch clamp and fura-2 fluorescence recordings. 2. Depolarization from -75 to 0 mV had no effect on [Ca2+]i in unstimulated cells, but evoked one or more spikes of Ca2+ increase (peak increase: 714 +/- 95 nM) during activation of metabotropic purinoceptors by 1 microM ADP. 3. The depolarization-evoked Ca2+ increase was present in Ca2+-free medium and also following removal of Na+. Thus depolarization mobilizes Ca2+ from an intracellular store without a requirement for altered Na+-Ca2+ exchange activity. 4. Intracellular dialysis with heparin blocked the depolarization-evoked Ca2+ increase, indicating a role for functional IP3 receptors. 5. Under current clamp, ADP caused the membrane potential to fluctuate between -43 +/- 1 and -76 +/- 1 mV. Under voltage clamp, depolarization from -75 to -45 mV evoked a transient [Ca2+]i increase (398 +/- 91 nM) during exposure to ADP. 6. We conclude that during stimulation of metabotropic purinoceptors, membrane depolarization over the physiological range can stimulate Ca2+ release from intracellular stores in the rat megakaryocyte, a non-excitable cell type. This may represent an important mechanism by which electrogenic influences can control patterns of [Ca2+]i increase. (+info)
Influence of monoclonal antiplatelet glycoprotein antibodies on in vitro human megakaryocyte colony formation and proplatelet formation.
(5/1747)
The influence of antiplatelet glycoprotein (GP) antibodies on megakaryocytopoiesis in patients with idiopathic or immune thrombocytopenic purpura (ITP) has been well studied. However, the influence of GP antibodies on proplatelet formation is poorly understood. Here we investigated whether in vitro human megakaryocyte colony formation and proplatelet formation are affected by various monoclonal antiplatelet GP antibodies (MoAb). The megakaryocyte colony formation inhibition assay was performed by methylcellulose culture with modifications, using peripheral blood nonadherent mononuclear cells. The proplatelet formation inhibition assay was performed by megakaryocytes derived from CD34(+) cells, stimulated with thrombopoietin + stem cell factor, which were then incubated with antiplatelet GP MoAb for 24 or 48 hours. Anti-GP-Ibalpha MoAb (CD42b; HIP1) slightly inhibited megakaryocyte colony formation (P < .05). and strongly inhibited proplatelet formation (after 24 hours incubation, P < .0002; after 48 hours incubation, P < .0007). Anti-GP-IIb MoAb (CD41; 5B12) inhibited only proplatelet formation (only after 24 hours incubation, P <. 03). Anti-integrin alphavbeta3 MoAb (CD51/CD61; 23C6) only slightly inhibited colony size (P < .05). However, anti-GP-IIIa MoAb (CD61; Y2/51) did not inhibit either colony formation or proplatelet formation. These results suggest that antiplatelet GP MoAbs have differing effects on in vitro megakaryocyte colony formation and proplatelet formation. (+info)
A role for changes in platelet production in the cause of acute coronary syndromes.
(6/1747)
Platelets are heterogeneous with respect to their size, density, and reactivity. Large platelets are more active hemostatically, and platelet volume has been found to be increased both in patients with unstable angina and with myocardial infarction. Furthermore, platelet volume is a predictor of a further ischemic event and death when measured after myocardial infarction. Platelets which are anucleate cells with no DNA are derived from their precursor, the megakaryocyte. Therefore, it is suggested that changes in platelet size are determined at thrombopoiesis in the megakaryocyte and that those changes might precede acute cardiac events. Understanding of the signaling system that controls platelet production may also further elucidate the cascade of events leading to acute vascular occlusion in some patients. (+info)
A model for studying megakaryocyte development and biology.
(7/1747)
The limited current understanding of megakaryocyte-lineage development and megakaryocyte biology is in large part because of a paucity of useful systems in which to conduct experiments. To overcome this problem, we have developed a transgenic mouse that uses the GP-Ibalpha regulatory sequences to achieve megakaryocyte-lineage restricted expression of an avian retroviral receptor. Through the transgenic avian receptor, avian retroviruses can efficiently and selectively infect megakaryocyte-lineage cells in vitro and in vivo. Serial infections can be performed to introduce and express multiple genes in the same cell. We have used this system to generate and characterize a pure population of primary CD41-positive megakaryocyte progenitors. (+info)
Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin.
(8/1747)
Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin -- PAR1, PAR3, and PAR4 -- are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 account for activation of human platelets by thrombin, or whether PAR3 or still other receptors contribute, is unknown. We have examined the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein were detected in human platelets. Activation of either receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone by antagonist, blocking antibody, or desensitization blocked platelet activation by 1 nM thrombin but only modestly attenuated platelet activation by 30 nM thrombin. Inhibition of PAR4 alone using a blocking antibody had little effect at either thrombin concentration. Strikingly, simultaneous inhibition of both PAR1 and PAR4 virtually ablated platelet secretion and aggregation, even at 30 nM thrombin. These observations suggest that PAR1 and PAR4 account for most, if not all, thrombin signaling in platelets and that antagonists that block these receptors might be useful antithrombotic agents. (+info)