Interferon-α2b–induced thrombocytopenia is caused by inhibition of platelet production but not proliferation and endomitosis in human megakaryocytes

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 542-550 ◽  
Author(s):  
Akiko Yamane ◽  
Takanori Nakamura ◽  
Hidenori Suzuki ◽  
Mamoru Ito ◽  
Yasuyuki Ohnishi ◽  
...  
Keyword(s):  
Human Platelets ◽  
Human Interferon ◽  
Inhibitory Effects ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Nog Mice ◽  
Thrombopoietin Mimetic

AbstractHuman interferon (IFN)–α is the standard therapy for chronic hepatitis C to prevent its progression to liver cirrhosis and hepatocellular carcinoma. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to dose reduction or treatment discontinuation. However, there is little information on how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α did not inhibit colony formation of megakaryocytes from human CD34+ hematopoietic stem cells. IFN-α did not inhibit endomitosis but did inhibit cytoplasmic maturation of megakaryocytes and platelet production in vitro. IFN-α suppressed the expression of transcription factors regulating late-stage megakaryopoiesis, such as GATA-1, p45NF-E2, MafG. IFN-α also significantly reduced the number of human platelets but not megakaryocytes, and did not inhibit endomitosis of human megakaryocytes in immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice transplanted with human CD34+ cells (hu-NOG). We also demonstrated that a novel thrombopoietin mimetic, NIP-004, was effective for treating IFN-α–induced thrombocytopenia in hu-NOG mice. From ultrastructural study, IFN-α inhibited the maturation of demarcation membranes in megakaryocytes, although NIP-004 prevented the inhibitory effects of IFN-α. These results defined the pathogenesis of IFN-α–induced thrombocytopenia and suggested possible future clinical applications for thrombopoietin mimetics.

Interferon-α2b-Induced Thrombocytopenia Is Caused by Inhibition of Cytoplasmic Maturation and Platelet Production, but Not Proliferation and Endomitosis in Human Megakaryocytes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2195-2195
Author(s):  
Akiko Yamane ◽  
Takanori Nakamura ◽  
Mamoru Ito ◽  
Yasuyuki Ohnishi ◽  
Yasuo Ikeda ◽  
...  
Keyword(s):  
Liver Cirrhosis ◽  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Human Platelets ◽  
Platelet Production ◽  
Human Cd34 ◽  
Nog Mice ◽  
Cytoplasmic Maturation

Abstract Human interferon (IFN)-α is a standard treatment for the patients with chronic hepatitis C. It is well known that chronic hepatitis C can transform into liver cirrhosis and hepatocellular carcinoma, and the risk of progression to liver cirrhosis is around 40% in 5 years. Thus, it is important for chronic hepatitis C patients to receive IFN-α treatment to prevent its malignant transformation. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to a dose reduction or discontinuation of IFN-α therapy. However, there is little information how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α does not inhibit colony formation of megakaryocytes (CFU-MK) from human CD34-positive hematopoietic stem cells. IFN-α also does not inhibit endomitosis (DNA duplication without cytokinesis), but inhibits cytoplasmic maturation of megakaryocytes and platelet production in vitro. The inhibitory effects of IFN-α on the development of demarcation membrane in human megakaryocytes were visualized by staining primary megakaryocytes with di-8-ANEPPS dye under a confocal laser microscope. Loss of platelet production by IFN-α was analyzed by measuring proplatelet formation (PPF) and counting the human platelets produced in the supernatant of human primary megakaryocytes by flow cytometry. The expression of transcription factors regulating late stage megakaryopoiesis such as GATA-1, p45 NF-E2 and MafG was suppressed by IFN-α. To confirm these in vitro observations in vivo system, we transplanted human CD34-positive hematopoietic cells into the immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice (hu-NOG) after 2.4 Gy irradiation. Recombinant human(rh) IFN-α significantly reduced the number of human platelets but did not decrease the number of human megakaryocytes in hu-NOG mice. rhIFN-α did not change murine platelet counts in hu-NOG mice, as IFN-α has species specificity. The DNA ploidy of human megakaryocytes in the bone marrow of hu-NOG mice was not altered after treatement with rhIFN-α. We also confirmed the life time of human platelets was not shortend by rhIFN-α in hu-NOG mice, indicating IFN-α does not promote the clearance of human platelets. We have also confirmed that a novel thrombopoietin mimetics, NIP-004, prevented rhIFN-α-induced thrombotytopenia and increased the number of polyploid human megakaryocytes in hu-NOG mice, suggesting NIP-004 might be useful for the patients with hepatitis C to avoid IFN-α-induced thrombocytopenia. In conclusion, these results clarified that IFN-α induces thrombocytopenia by inhibiting the cytoplasmic maturation of megakaryocytes but not proliferation and endomitosis in human megakaryocytes.

Abstract P019: Exosomes from Human CD34+ Cells: Critical Mediators of Proangiogenic Paracrine Effect of EPCs

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Susmita Sahoo ◽  
Sol Misener ◽  
Tina Thorne ◽  
Meredith Millay ◽  
Kathryn M Schultz ◽  
...  
Keyword(s):  
Cell Transplantation ◽  
Human Peripheral Blood ◽  
Limb Ischemia ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Paracrine Effect ◽  
Human Cd34 ◽  
Cd34 Cells

Local transplantation of human CD34+ hematopoietic stem cells has been shown to promote neovascularization in pre-clinical studies in models of myocardial and limb ischemia. In early phase clinical trials, transplantation of CD34+ cells has been associated with reduced angina, improved exercise time and reduced amputation rates. Several studies have suggested that paracrine effects by these pro-angiogenic cells mediate the effects induced by cell transplantation. We hypothesized that CD34+ cells secrete exosomes (Exo), which mediate at least a part of the therapeutic function of the cells. Methods and Results: We isolated Exo from the conditioned media of adult human peripheral blood (PB) CD34+ cells. The angiogenic and therapeutic potency of CD34+ Exo was compared with the intact CD34+ cells and also with PB mononuclear cell (MNC) Exo. Exo from both CD34+ cells and MNC are 50–90nm in size, have cup shaped morphology, and carry known Exo-marker proteins such as CD63, TSG101 and Annexin V as shown by electron microscopy, Western blot and flow cytometry. Compared to CD34+ cells or MNC Exo, CD34+ Exo significantly induces in vitro angiogenic activities such as viability, proliferation and tube formation of HUVECs on matrigel- in a dose dependent manner. In vivo, CD34+ Exo stimulated significant neovascularization in mouse corneal angiogenesis assay (14±4 mm v MNC Exo, 4±1 mm, p<0.01) and incorporation of endothelial (CD31+) cells in mouse matrigel-plug assay (6±1.7% v CD34+ cells, 2±0.8%, p<0.01). Finally, in a mouse model of hind limb ischemia (HLI), CD34+ Exo significantly improved perfusion (ratio: 1.01±0.04 v 0.57±0.1, P<0.05), increased capillary density (1.8±0.3/HPF v 0.9±0.1/HPF, p<0.001) and prevented ischemic leg amputation (16% v 100%), as compared with MNC Exo. Conclusions: These data demonstrate that CD34+ Exo induce angiogenic activity and ischemic tissue repair in the absence of CD34+ cells, and suggest that Exo represent important mediators of the therapeutic effects associated with CD34+ cell therapy. We speculate that Exo derived from CD34+ cells may represent a significant component of the paracrine effect of progenitor-cell transplantation for therapeutic angiogenesis.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

A Novel, Small Non-Peptidyl Butyzamide Activates Human Thrombopoietin Receptor and Promotes Megakaryopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2201-2201 ◽  
Author(s):  
Wataru Nogami ◽  
Hiroshi Yoshida ◽  
Kenzo Koizumi ◽  
Hajime Yamada ◽  
Kenji Abe ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Fetal Liver ◽  
Human Platelets ◽  
Stable Transfectants ◽  
Human Cd34 ◽  
Thrombopoietin Receptor ◽  
Cd34 Cells ◽  
Agonistic Activity ◽  
Nog Mice ◽  
Orally Bioavailable

Abstract Butyzamide is a novel non-peptidyl molecule which has agonistic activity to the thrombopoietin (TPO) receptor Mpl. Butyzamide promotes the proliferation of murine pro B cell line Ba/F3 expressing human Mpl (hMpl), and induces phosphorylation of JAK2, STAT3, STAT5 and MAPK. Interestingly, butyzamide does not promote the proliferation of Ba/F3 cells expressing murine Mpl (mMpl). To elucidate the mechanisms for this species specificity, we created stable transfectants such as Ba/F3-hMpl(H499L) and Ba/F3-mMpl(L490H) cells. Butyzamide induced the proliferation of Ba/F3-mMpl(L490H) but not Ba/F3-hMpl(H499L) cells, indicating that a histidine residue in the transmembrane domain of human Mpl is critical for butyzamide-induced signaling. Butyzamide induced the colony-forming unit-megakaryocyte and polyploid megakayocytes from human CD34+ hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was orally administered to immunodeficient NOD/Shi-scid/IL-2Rγcnull (NOG) mice transplanted with human fetal liver-derived CD34+ cells, the human platelet count increased by 6.2- and 22.9-fold at the doses of 10 and 50 mg/kg for 20 days, respectively. Butyzamide also increased the number of reticulated human platelets and human matured megakaryocytes in NOG mice. These results indicate that butyzamide is an orally bioavailable Mpl activator and suggest its potential for clinical development as a therapeutic agent in patients with thrombocytopenia.

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor–negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1735-1738 ◽  
Author(s):  
Stephanie Bridenbaugh ◽  
Linda Kenins ◽  
Emilie Bouliong-Pillai ◽  
Christian P. Kalberer ◽  
Elena Shklovskaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
Cd8 Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8+ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8+ cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8α+CD3ϵ+ T-cell receptor– negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34+ cells. In vitro, cFCs cocultured with CD34+ cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8+CD3+TCR− cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 1885-1893 ◽  
Author(s):  
Kathleen Freson ◽  
Karen Peeters ◽  
Rita De Vos ◽  
Christine Wittevrongel ◽  
Chantal Thys ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Adenylyl Cyclase ◽  
Independent Manner ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Therapeutic Implications ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Megakaryocyte Differentiation

Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase–activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34+ cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34+ cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

scholarly journals In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4040-4048 ◽  
Author(s):  
M Rosenzweig ◽  
DF Marks ◽  
H Zhu ◽  
D Hempel ◽  
KG Mansfield ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
T Cell Differentiation ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

In Vitro and In Vivo Induction of Human Hematopoietic Stem Cell Migration by Extracellular UTP.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1730-1730
Author(s):  
Lara Rossi ◽  
Rossella Manfredini ◽  
Francesco Bertolini ◽  
Davide Ferrari ◽  
Miriam Fogli ◽  
...  
Keyword(s):  
Cell Types ◽  
P2y Receptors ◽  
Extracellular Nucleotides ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor

Abstract Regulatory mechanisms governing homing and engraftment of hematopoietic stem cells (HSCs) involve a complex interplay between chemokines, cytokines, growth factors and adhesion molecules in the intricate architecture of bone marrow (BM) microenvironment. HSCs express P2Y and P2X receptors for extracellular nucleotides, which activation by ATP and UTP has been recently demonstrated (Lemoli et al. Blood. 2004) to produce potent stimulatory effects on HSCs. Moreover extracellular nucleotides are emerging as key factors of flogosis phenomena and related chemotactic responses of several cell types, such as dendritic cells, monocytes and endothelial cells. In this study we investigated the biologic activity of extracellular ATP and UTP and their capacity to cooperatively promote SDF-1 (stromal cell-derived factor-1)-stimulated cell chemotaxis. Low concentrations of UTP (10uM) significantly improved, in vitro, HSCs migration. Moreover, UTP inhibits CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin filaments. Furthermore, in vivo competitive repopulation assays showed that preincubation with UTP significantly improved the homing efficiency of human CD34+ HSCs in nonobese diabetic/severe combined immunodeficient mice. Inhibition assays with Pertussis Toxin from B. Pertussis blocked SDF-1- and UTP-dependent chemotactic responses, suggesting that Gαi proteins may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and subsequent in vitro inhibition assays with Toxin B from C. Difficile suggest that RhoGTPase Rac2 and his downstream effectors ROCK1 and ROCK2 are involved in the UTP-promoted, SDF-1-dependent HSCs migration. Taken together, our data suggest that UTP may physiologically modulate HSC migration and homing to the BM, in concert with the chemotactic peptide SDF-1, via the activation of converging signaling transduction pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases.

Bortezomib Induces Thrombocytopenia by Inhibiting Proplatelet Formation but Not Proliferation and Endomitosis in Human Primary Megakaryocytes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 87-87
Author(s):  
Wataru Nogami ◽  
Akiko Yamane ◽  
Takanori Nakamura ◽  
Eri Matsuki ◽  
Yasuo Ikeda ◽  
...  
Keyword(s):  
Colony Formation ◽  
Proteasome Inhibitors ◽  
Human Platelets ◽  
Cytotoxic Agents ◽  
In Vitro Assays ◽  
Inhibitory Effects ◽  
Hematopoietic Stem ◽  
Proplatelet Formation ◽  
Releasing Process

Abstract The proteasome inhibitor bortezomib has therapeutic activity in patients with multiple myeloma. The most common adverse event from its application is thrombocytopenia, which has kinetics that differ from those induced by other cytotoxic agents. After treatment with bortezomib, platelet counts usually decrease within a couple of days but rapidly recover toward baseline during the rest periods between each cycle. The lowest count of platelets in each cycle does not worsen during the 8 courses of bortezomib treatment. Furthermore, bortezomib does not induce any cytotoxic injury in megakaryocyte in the murine model. Therefore, we postulated that bortezomib-induced thrombocytopenia is caused by inhibition of the platelet releasing process without megakaryocyte toxicity. In vitro assays using human bone marrow-derived CD34-positive hematopoietic stem cells revealed that bortezomib did not inhibit colony formation and endomitosis of human primary megakaryocytes in the presence of recombinant human thrombopoietin (rhTPO). As proplatelet formation (PPF) is often used as the indicator of the platelet releasing process in vitro, we evaluated the inhibitory effects of bortezomib for PPF. Seven days after culture of human CD34-postive cells with 10 ng/ml rhTPO, mature megakaryocytes were enriched by discontinuous bovine serum albumin gradients (purity>90%). The enriched mature megakaryocytes were treated with various concentrations of bortezomib for a further 4 days and the percentage of megakaryocytes bearing PPF was calculated under a microscope. Bortezomib dose-dependently inhibited PPF from mature megakaryocytes. Other proteasome inhibitors such as lactacystin and MG132 also demonstrated inhibitory effects on PPF without inhibiting colony formation of megakaryocytes. Since the inhibition of transcriptional factor NF-kB activity is one of the major pathways of proteasome inhibitors, we evaluated the effects of NF-kB inhibitors such as (−)-DHMEQ and Bay11-7082. Both of these inhibitors also demonstrated inhibitory effects on PPF but did not inhibit the colony formation of megakaryocytes. To exclude the direct effects of bortezomib on human platelets, we analyzed the effects of bortezomib for the activation of caspase-3 and mitochondrial potential in human platelets. We found that bortezomib did not directly induce apoptosis in human platelets. Our results demonstrate that bortezomib induces thrombocytopenia by inhibiting PPF but does not affect proliferation of megakaryocytes, endomitosis and platelet apoptosis. We believe this is the first report using human primary megakaryocytes to clarify the pathogenesis of thrombocytopenia caused by bortezomib therapy.

Effectiveness of a Novel Humanized VB22B Minibody against Human TPO Receptor Compared with Recombinant TPO and Eltrombopag Using Primary Human CD34+ Cells and Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4559-4559
Author(s):  
Eri Matsuki ◽  
Akiko Yamane ◽  
Shinichiro Okamoto ◽  
Yoshitaka Miyakawa
Keyword(s):  
Bone Marrow ◽  
Platelet Aggregation ◽  
Colony Formation ◽  
Human Platelets ◽  
Receptor Agonists ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proplatelet Formation

Abstract Abstract 4559 Thrombopoietin (TPO) is a cytokine produced primarily by the liver and kidney that regulates platelet production by stimulating proliferation and differentiation of hematopoietic stem cells, megakaryocytic progenitor cells and megakaryocytes via activation of its receptor, c-Mpl. Recently, TPO receptor agonists such as eltrombopag and romiplostim have been approved for chronic ITP. huVB22B was created as a novel humanized form of murine sc(Fv) 2VB22B minibody (BLOOD, 2005) which activates human c-Mpl by CDR grafting. The advent of these various TPO receptor agonists prompted us to consider the differences in their mechanisms of action, efficacy or potency. However, to date, there has been no in vivo or in vitro study directly comparing the effects of different TPO receptor agonists. In this study, we compared the efficacy of huVB22B on CFU-GM, CFU-E, CFU-Megakaryocyte (CFU-MK), megakaryocyte maturation (DNA ploidy and proplatelet formation) with those of recombinant human TPO (rhTPO) and eltrombopag. Primary human CD34+ bone marrow cells were cultured with various concentrations of rhTPO, huVB22B and eltrombopag using methylcellulose based media. In serum-free condition, 0.286 nM rhTPO, 0.182 nM huVB22B and 17.7 mcM eltrombopag demonstrated almost equivalent efficacy of megakaryocyte colony formation. At these concentrations, all agents demonstrated similar in vitro efficacy for colony formation of CFU-GM and CFU-E, proplatelet formation and nuclear maturation of megakaryocytes. In preliminary results, huVB22B induced maturation of CFU-MK earlier than rhTPO and eltrombopag, suggesting that huVB22B might have some potential to increase human platelets faster than other agents in vivo. This is compatible with the observation that huVB22B induced tyrosine phosphorylation of STAT3, STAT5 and JAK2 faster and stronger than rhTPO and eltrombopag in human primary platelets. Both rhTPO and huVB22B enhanced low-dose ADP and collagen-induced human platelet aggregation in vitro. In contrast, eltrombopag did not enhance ADP or collagen-induced platelet aggregation, although it induced activation of JAK-STAT pathway in human platelets. Contrary to the fact that huVB22B induces phosphorylation of intracellular signaling molecules faster and stronger than rhTPO in human platelets, the priming effect by huVB22B on platelet aggregation was much weaker than rhTPO. In conclusion, we confirmed that newly created huVB22B minibody induced colony formation of CFU-MK, CFU-E, CFU-GM and maturation of megakaryocytes from human bone marrow-derived CD34+ cells in vitro. The differences among TPO receptor agonists observed in our study would lead to further understanding of the basic biology of megakaryopoiesis and the action of TPO receptor agonists. Disclosures: Okamoto: Alexion: Research Funding. Miyakawa:GlaxoSmithKline: Consultancy.

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