Primary megakaryocytes reveal a role for transcription factor NF-E2 in integrin alpha IIb beta 3 signaling.

Platelet integrin alphaIIbbeta3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of alphaIIbbeta3 signaling remain poorly understood. To better understand this process, we established conditions to study alphaIIbbeta3 signaling in primary murine megakaryocytes. Unlike platelets, these platelet precursors are amenable to genetic manipulation. Cytokine-stimulated bone marrow cultures produced three arbitrary populations of alphaIIbbeta3-expressing cells with increasing size and DNA ploidy: small progenitors, intermediate-size young megakaryocytes, and large mature megakaryocytes. A majority of the large megakaryocytes bound fibrinogen in response to agonists, while almost none of the smaller cells did. Fibrinogen binding to large megakaryocytes was inhibited by Sindbis virus-mediated expression of isolated beta3 integrin cytoplasmic tails. Strikingly, large megakaryocytes from mice deficient in the transcription factor NF-E2 failed to bind fibrinogen in response to agonists, despite normal surface expression of alphaIIbbeta3. Furthermore, while megakaryocytes from wild-type mice spread on immobilized fibrinogen and exhibited filopodia, lamellipodia and Rho-dependent focal adhesions and stress fibers, NF-E2-deficient megakaryocytes adhered poorly. These studies establish that agonist-induced activation of alphaIIbbeta3 is controlled by NF-E2-regulated signaling pathways that mature late in megakaryocyte development and converge at the beta3 cytoplasmic tail. Megakaryocytes provide a physiologically relevant and tractable system for analysis of bidirectional alphaIIbbeta3 signaling.

Mechanisms and consequences of affinity modulation of integrin alpha(V)beta(3) detected with a novel patch-engineered monovalent ligand.

Integrin alpha(V)beta(3) mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which alpha(V)beta(3) is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, alpha(IIb)beta(3), has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of alpha(V)beta(3), a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single alpha(V) integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated alpha(V)beta(3), but not to alpha(IIb)beta(3), in receptor and cell binding assays. alpha(V)beta(3) affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K(d) = 2.4 microM), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K(d) = 80 nM), with no change in receptor number. In contrast, alpha(V)beta(3) in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of beta(3) cytoplasmic tails. Up-regulation of alpha(V)beta(3) affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that alpha(V)beta(3) is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.

[The hemoprotective effect of platelet factor 4 (PF4) and tetrapeptide AcSDKP].

OBJECTIVE: To study the effects of platelet factor 4(PF4) and tetrapeptide N-acetyl-Ser-Asp-Lys-Pro(AcSDKP) on hemopoietic progenitors in mice treated with 5-Fluorouracil (5-FU). METHODS: Mice were injected with PF4 (40 micrograms/kg) or AcSDKP (4 micrograms/kg) twice at 6 h intervals, and 20 h after the second injection they were given one injection of 5-FU (150 mg/kg), and the high proliferative potential-colony forming cell (HPP-CFC), burst-forming unit erythroid (BFU-E), colony forming unit megakaryocyte (CFU-MK), and megakaryocyte (MK) were examined 6, 8 and 13 days later. RESULTS: The administration of PF4 or AcSDKP resulted in a significant increase of the number of HPP-CFC on days 6-8 and BFU-E and CFU-GM on day 8 when compared to 5-FU alone. Furthermore, PF4 was found to increase significantly the number of CFU-MK and MK on day 8, which was not observed with AcSDKP. CONCLUSION: PF4 or AcSDKP accelerate the recovery in vivo of HPP-CFC, CFU-GM and BFU-E after 5-FU treatment but their effect may be different on megakaryocytic progenitors. Both molecules may have a hemoprotective effect against chemotherapeutic agents.

The tetrapeptide AcSDKP reduces the sensitivity of murine CFU-MK and CFU-GM progenitors to aracytine in vitro and in vivo.

The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been described as an inhibitor of CFU-S entry into DNA synthesis; as a result, its administration can protect mice against lethal doses of cytosine arabinoside (Ara-C). In the present study, we tested the protective effect of AcSDKP on CFU-MK and CFU-GM progenitor cells in mice treated at lower doses of Ara-C more relevant to human clinical situations. Firstly, we report for the first time that in vitro pre-incubation of murine BM MNC with AcSDKP at concentrations of 10(-10) and 10(-9) M for 48 h decreased CFU-MK, in parallel to CFU-GM, progenitor growth. This resulted in an increase of recovery of these progenitors after exposure to Ara-C. Secondly, we tested the effect of AcSDKP on progenitor cells in vivo in different conditions in Ara-C treated mice. We show that the administration of AcSDKP before starting Ara-C treatment resulted in a significant increase in progenitor CFU-GM, CFU-MK and mature MK numbers, 6 and 8 days after the first Ara-C injection. Interestingly, no difference was observed whether AcSDKP was started 24 or 48 h before Ara-C. In a protocol in which AcSDKP was administered for 8 days starting 48 h before Ara-C treatment, the dose did not appear to be critical at least within the range tested (4 vs. 40 micrograms/injection). In addition, the administration of AcSDKP at 64 micrograms/kg per injection for 5 days and stopping it 3 days before the end of Ara-C treatment, i.e. five instead of eight applications, further increased its protective effect. Thus our results demonstrate protective effect of AcSDKP for progenitors during a fractionated protocol of Ara-C treatment and indicates an importance of the dose and the schedule of administration of AcSDKP in designing future clinical trials.

Quantitation of megakaryocytopoiesis by computerized automatic in culture image analysis.

A computer-assisted automatic image procedure was karyocytopoiesis in culture. This analysis system was based on acetylcholinesterase staining, a specific staining for murine bone marrow megakaryocytes, and an image capturing instrument with a computer program. Two kinds of routine megakaryocyte culture methods were used, the plasma clot and the serum-free agar systems. A comparison between manual counting and the instrument was made. The image analysis software was able to distinguish between megakaryocytes (MK) at different stages of maturation. The results show that this analysis system can simultaneously detect not only the number of megakaryocytes and their colonies in each dish, but also the surface area of individual megakaryocytes. In addition, this analysis system functions automatically 24 hours a day and the results obtained are reproducible. Using this system, we have confirmed previous observations that thrombopoietin (TPO) and heparin stimulate both proliferation and maturation of megakaryocytes. In addition, we found that platelet factor 4 (PF-4) significantly reduced the number of megakaryocytes but not their cell surface area, whereas TGFbeta1 decreased both number and surface area of megakaryocytes, suggesting that PF4 and TGFbeta1 negatively regulate megakaryocytopoiesis by different mechanisms. We noticed that megakaryocytes grown under agar culture conditions regularly had an increased size in comparison with those grown in a plasma clot system, which may be an indication that the plasma clot culture media contains an inhibitor(s) of megakaryocyte maturation. Our data indicate that this image analysis system, in addition to its automatic and reproducible features, is more efficient and allows detection of more parameters than routine manual microscopic detection.

A 13-24 C-terminal peptide related to PF4 accelerates hematopoietic recovery of progenitor cells in vivo in mice treated with 5-fluorouracil.

We have recently reported that platelet factor 4 (PF4), a megakaryocyte-platelet protein, is a potent inhibitor of human and murine megakaryocytopoiesis. In addition, PF4 accelerated the recovery of the marrow precursor cells in 5-fluorouracil (5-FU) treated mice. We show in this study that a slight modification of the C-terminal peptide related to PF4 (C13-24DE), which was previously reported as the carboxy terminal region of PF4 implicated in PF4 inhibitory activity, is also able to significantly increase murine high proliferating-potential-colony forming cells (HPP-CFC), colony-forming-unit megakaryocyte (CFU-MK) and colony-forming unit granulocyte-macrophage (CFU-GM) progenitor number, eight days after 5-FU administration, when it was given intraperitoneally twice a day (200 micrograms/kg/inj) prior to 5-FU administration (150 mg/kg). Furthermore, the C13-24DE pretreatment enhanced both the number and the diameter of single megakaryocyte (MK) by day 8. These data indicate that the C13-24DE peptide related to PF4 accelerated the in vivo recovery of stem cells, progenitors (CFU-GM, CFU-MK) and single MK after 5-FU treatment and may have a hemoprotective effect against chemotherapeutic agents.

In vivo effect of platelet factor 4 (PF4) and tetrapeptide AcSDKP on haemopoiesis of mice treated with 5-fluorouracil.

In vivo effects of platelet factor 4 (PF4) and tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) on haemopoietic progenitors were studied in mice treated with 5-fluorouracil (5-FU). The mice were injected with PF4 (40 micrograms/kg) or AcSDKP (4 micrograms/kg) twice at 6 h intervals, and 20 h after the second injection they were given one injection of 5-FU (150 mg/kg). 6, 8 and 13 d later the high proliferative potential-colony forming cell (HPP-CFC), burst-forming unit erythroid (BFU-E), colony forming unit granulocyte-macrophage (CFU-GM) colony forming unit megakaryocyte (CFU-MK), and megakaryocytes (MK) were examined. The results showed that the administration of PF4 or AcSKDP resulted in a significant increase in the number of HPP-CFC on days 6-8 and BFU-E and CFU-GM on day 8 when compared to 5-FU alone. Furthermore, PF4 was found to increase significantly the number of CFU-MK and MK on day 8, which was not observed with AcSDKP. However, both molecules had no obvious effect on peripheral blood cells. These data indicate that PF4 or AcSDKP accelerate the recovery in vivo of HPP-CFC, CFU-GM and BFU-E after 5-FU treatment but their effect may be different on megakaryocytic progenitors and suggests that both molecules may have a haemoprotective effect against chemotherapeutic agents.

Carboxy-terminal peptides (C1-24 and C13-24 but not C1-13) of platelet factor 4 inhibit murine megakaryocytopoiesis, an activity which is neutralized by heparin.

Negative regulation of megakaryocytopoiesis is a complex process involving various cytokines. One of these cytokines is platelet factor 4 (PF4), a megakaryocyte/platelet specific protein. PF4 and a carboxy-terminal peptide related to PF4 have been reported to inhibit human and murine megakaryocytopoiesis. The growth of several megakaryoblastic cell lines: human erythroleukaemia cell line (HEL). Meg-01 and Dami, was also inhibited by PF4 and a 13-24 carboxy-terminal peptide related to PF4. We report that peptides corresponding to the 1-24 and 13-24 but not 1-13 carboxy-terminal region of PF4 inhibit murine megakaryocytopoiesis both in vivo (5 micrograms/inj) and in vitro (2.5 and 5 micrograms/ml). Moreover, such an inhibitory activity of PF4-related peptides is abrogated by heparin (5 IU/dish). These overall data indicate that carboxy-terminal PF4-related peptides retain the inhibitory effect of PF4 on both murine single MK and CFU-MK in vivo and in vitro by acting on an early stage of megakaryocytopoiesis and strongly suggest that the inhibitory activity of the multi-functional PF4 might be localized in a short carboxy-terminal region which might include, in part, the PF4 heparin binding domain.

[Platelet factor 4, reversible inhibitor of megakaryocytogenesis, protector of megakaryocytes during chemotherapy]. / Facteur 4 plaquettaire, inhibiteur réversible de la mégacaryocytogénèse, protecteur des mégacaryocytes au cours de la chimiothérapie.

Development of megakaryocyte (MK) from CD34+ cord blood (CB) cells in both plasma clot culture and liquid culture was significantly inhibited by human platelet factor 4 (PF4) and human transforming growth factor beta 1 (TGF beta 1). Inhibition of cell growth by PF4 was reversible judging from the fact that the CD34+ cells preincubated with PF4 could regenerate colonies after washing and replating into the cultures. By contrast, TGF beta 1-pretreated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF4 in liquid culture caused an increase in the number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells, and exhibited greater capacity to form MK colonies than control after the treatment of 5-FU. In vivo in mice, twice injections of PF4 at 40 micrograms/kg resulted in a significant increase in the number of colony-forming cells with high proliferative potential (HPP-CFC) and colony-forming unit-megakaryocyte (CFU-MK) in bone marrow. In exponentially growing human erythroleukemia cells (HEL), the addition of PF4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis. Different from PF4, TGF beta 1 blocked more cells in G1 phase. These results demonstrate that PF4 and TGF beta 1 inhibit MK development from CD34+ CB cells by different mechanisms and suggest that PF4, unlike TGF beta 1, exerts its inhibitory effect on cell growth in a reversible and S phase-specific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity.