Intrinsic Impairment of Platelet Production in Persistent/Chronic ITP

Abstract Abstract 624 Persistent/chronic immune thrombocytopenias (PC-ITP) are acquired thrombocytopenias characterized by a platelet count less than 100 G/L, lasting respectively more than 3 months/one year. It is a diagnosis of exclusion, and other causes of acquired thrombocytopenia such as neoplasms, infections, autoimmune diseases or drugs must have been ruled out. It is commonly admitted that the pathogenesis of ITP associates the presence of antiplatelet antibodies that bind to mature platelets, leading to their elimination by macrophages through Fc-mediated recognition mechanisms mainly in the spleen or liver, or both. These antibodies are also thought to bind to megakaryocytes impairing platelet production. Newly available TPO receptor agonists are highly effective in chronic ITP patients, with a dramatic increase in platelet count, which suggests a crucial role of altered megakaryopoiesis in this disorder. Because antiplatelet antibodies are only found in 20% of PC-ITP, we hypothesized that some of these patients might have an intrinsic defect in megakaryopoiesis rather than an immune-mediated dysmegakaryopoiesis. We thus analyzed in vitro megakaryocyte differentiation and proplatelet formation in 9 PC-ITP patients, 4 acute ITP patients (A-ITP), and 9 healthy controls (CTRL). All PC-ITP patients had ITP criteria regarding last international consensus (Neunert C et al, Blood 2011), with a slowly progressing thrombocytopenia (76 G/L, range: 29–97) lasting more than 3 months (median: 17 months; range: 7–36 months), no anti-platelet antibodies and normal medullar density and number of megakaryocytes. All A-ITP patients also matched ITP criteria. All samples were taken at the time of diagnosis, before any treatment was administered. They were compared with 9 controls including patients undergoing valvular replacement or healthy bone marrow donors with normal blood count. To analyze whether there was a defect of megakaryopoiesis that was cell-intrinsic, we isolated CD34 positive cells from the bone marrow and analyzed in vitro megakaryocytic differentiation with TPO-mimetic romiplostim. Proliferation was measured at days 3, 6 and 10 and compared in the 3 groups by a proliferation coefficient. Membrane maturation was assessed at day 6 and 10 by flow cytometry (FC) after CD41-FITC and CD42-PE staining. Megakaryocytic ploidy was measured at day 10 by FC after propidium iodure and CD41 staining. At day 8, large mature megakaryocytes were isolated after discontinuous HSA density gradient and proplatelets forming megakaryocytes were counted from between days 9 and 13. Late mature megakayocytes (day 12–13) were observed with a confocal microscope to qualitatively analyze proplatelet formation. We did not observe any difference between A-ITP or PC-ITP patients and controls in term of proliferation, ploidy, or expression of surface differentiation markers (CD41, CD42). In contrast, PC-ITP-derived megakaryocytes showed a defect in proplatelet formation, as only 12% of large, mature megakaryocytes were able to form proplatelets in liquid culture at day 11 vs 37% in CTRL (p=0,046) and 39% in A-ITP (p=0,03), 11% at day 12 vs 43,2% in CTRL (p=0,024) and 46% in A-ITP (p=0,0002), and 10% at day 13 vs 44,5% in CTRL (p=0,015) and 46% in A-ITP (p=0,03). Besides, we observed that proplatelet-forming megakaryocytes from ITP patients had less proplatelets per megakaryocyte and less bifurcation per proplatelet. In conclusion, our study shows that megakaryocytes from patients with persistent/chronic ITP have an intrinsic defect in megakaryocyte development that is independent from the medullar environment. This defect affects proplatelet formation and further investigations are now needed to better describe mechanisms underlying proplatelet alteration in this disease. Disclosures: Viallard: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Physiological Levels of Jak2 V617F Result in Enhanced Megakaryocyte Differentiation, Proplatelet Formation and Platelet Reactivity.

Blood ◽

10.1182/blood.v114.22.226.226 ◽

2009 ◽

Vol 114 (22) ◽

pp. 226-226

Author(s):

Cedric J Ghevaert ◽

Juan Li ◽

Sonia Severin ◽

Jocelyn Auger ◽

Steve Watson ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Platelet Reactivity ◽

Jak2 V617f ◽

Signalling Pathways ◽

Red Cell ◽

Thrombotic Risk ◽

Platelet Production ◽

Bone Marrow Histology

Abstract Abstract 226 The major clinical challenge in treating patients with myeloproliferative diseases (MPD) such as essential thrombocythaemia (ET) and polycythaemia vera (PV) is to prevent thrombotic events. Despite adequate red cell and platelet count control by means of venesection or cytoreductive therapy and the use of anti-platelet agents, patients with ET and PV have a three-fold risk-increase of cardiovascular and cerebral ischaemic events. A V617F mutation in the Janus kinase 2 (Jak2) that causes constitutive activation of Jak2 has been shown to be present in >90% and ∼50% of PV and ET patients, respectively. The effect of this activation on the biology of primary megakaryocytic cells is not known and neither is it clear whether the thrombotic risk conferred by this mutation reflects the increase in red cell/platelet counts, an intrinsic increase in platelet reactivity or both. Studies in humans are hampered by the co-existence of normal and clonal haematopoiesis with significant clonal and phenotypic heterogeneity between patients. Chimaeric mouse models based on retroviral transduction of Jak2 V617F have marked overexpression of the mutant Jak2 and show a PV phenotype with normal platelet counts. Recently, Jak2 V617F transgenic mice models have been published but the transgenes have multiple (and variable) insertions and are therefore subject to position effects with varying degrees of expression of the mutant Jak2 compared to the wild-type allele. We have generated a Cre-inducible mouse model where the human Jak2 V617F gene has been knocked into one allele of the mouse endogenous Jak2 gene. Upon Cre induction, the mice exhibit an ET phenotype (platelet count increased by approximately 30% but with a normal haematocrit) that is stable for over 26 weeks. Bone marrow histology shows classical features of ET with increased numbers of megakaryocytes (MKs) and clusters with no fibrosis. Quantitative RT-PCR shows stable expression of Jak2 V617F in the MKs at a similar level to that of endogenous mouse Jak2 therefore reflecting the physiological situation found in human ET patients. In liquid cultures, bone marrow-derived MKs show increased ploidy in response to suboptimal concentrations of thrombopoietin (TPO) in keeping with the increased number of MKs found in the bone marrow histology. Crucially, in vitro proplatelet formation in mutant MKs was increased two-fold compared to MKs derived from litter-match control animals showing that platelet production may not only relate to the increased number of MKs but to intrinsic differences in MK biology and platelet production. Platelet aggregation studies and P-selectin expression in response to an array of agonists was not significantly different between mutant and controls but in vitro laminar flow assays show increased thrombus formation on collagen. Although Jak2V617F expression was down-regulated at protein level in mature MKs and platelets, analysis of downstream signalling pathways showed alteration of the phosphorylation status of Src kinases. This mouse model therefore provides a unique opportunity to understand the biological mechanism of increased platelet production and thrombotic risk in ET patients as well as unravelling the signalling pathways downstream of the Jak2 V617F in primary cells, which will be crucial in the context of specific therapeutic Jak2 inhibitors currently in clinical development. This work was supported by the British Heart Foundation Disclosures: No relevant conflicts of interest to declare.

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Development Of 3D Models For Studying Megakaryopoiesis In Vitro

Blood ◽

10.1182/blood.v122.21.3696.3696 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3696-3696

Author(s):

Lindsay Wray ◽

Christian A Di Buduo ◽

David L. Kaplan ◽

Alessandra Balduini

Keyword(s):

Mechanical Properties ◽

Bone Marrow ◽

Extracellular Matrix ◽

Endothelial Cells ◽

Peripheral Blood ◽

Platelet Production ◽

Vascular Niche ◽

Proplatelet Formation ◽

Bone Marrow Vascular Niche

Abstract Introduction Silk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. Results In order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. Conclusions The goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures: No relevant conflicts of interest to declare.

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A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice

Blood ◽

10.1182/blood-2006-10-050740 ◽

2007 ◽

Vol 109 (10) ◽

pp. 4237-4244 ◽

Cited By ~ 60

Author(s):

Tarvinder S. Dhanjal ◽

Caroline Pendaries ◽

Ewan A. Ross ◽

Mark K. Larson ◽

Majd B. Protty ◽

...

Keyword(s):

Bone Marrow ◽

Spatial Localization ◽

Matrix Proteins ◽

Platelet Production ◽

Stromal Microenvironment ◽

Proplatelet Formation ◽

Stromal Cell Derived Factor ◽

Deficient Mice

Abstract During thrombopoiesis, maturing megakaryocytes (MKs) migrate within the complex bone marrow stromal microenvironment from the proliferative osteoblastic niche to the capillary-rich vascular niche where proplatelet formation and platelet release occurs. This physiologic process involves proliferation, differentiation, migration, and maturation of MKs before platelet production occurs. In this study, we report a role for the glycoprotein PECAM-1 in thrombopoiesis. We show that following induced thrombocytopenia, recovery of the peripheral platelet count is impaired in PECAM-1–deficient mice. Whereas MK maturation, proplatelet formation, and platelet production under in vitro conditions were unaffected, we identified a migration defect in PECAM-1–deficient MKs in response to a gradient of stromal cell–derived factor 1 (SDF1), a major chemokine regulating MK migration within the bone marrow. This defect could be explained by defective PECAM-1−/− MK polarization of the SDF1 receptor CXCR4 and an increase in adhesion to immobilized bone marrow matrix proteins that can be explained by an increase in integrin activation. The defect of migration and polarization was confirmed in vivo with demonstration of altered spatial localization of MKs within the bone marrow in PECAM-1–deficient mice, following immune-induced thrombocytopenia. This study identifies a novel role for PECAM-1 in regulating MK migration and thrombopoiesis.

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Bortezomib Induced Thrombocytopenia Might Be Due to the Inhibition of Proplatelet Formation of Megakaryocyte.

Blood ◽

10.1182/blood.v116.21.3696.3696 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3696-3696

Author(s):

Kazunori Murai ◽

Shugo Kowata ◽

Akiko Abo ◽

Tatsuo Oyake ◽

Kenichi Nomura ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Clinical Studies ◽

Bone Marrow Cells ◽

Platelet Production ◽

Platelet Counts ◽

Proplatelet Formation ◽

Marrow Cells

Abstract Abstract 3696 Background: Bortezomib is potent and reversible proteasome inhibitor that has been extensively used for multiple myeloma. Several clinical studies demonstrated that overall response rates using bortezomib alone to relapsed or refractory patients with multiple myeloma were 33 to 50%. The most common grade 3 adverse event was a cyclic thrombocytopenia, which was reported in 20–30% of patients in several clinical studies. The mechanism by which bortezomib causes thrombocytopenia remains unknown. In this study, we evaluated the effect of bortezomib on megakaryocytic progenitor cells, megakaryocytopoiesis, megakaryocyte and platelet production in mice. Method: All animal procedures were approved by the Institutional Animal Care and Use Committee in Iwate Medical University. Male ddY at 8 weeks of age mice were used in all experiments. In vivo experiments: (a) The mice received 2.5 mg/kg bortezomib via tail-vein injection. Blood was obtained and the following experiments were carried out at day 2, 4, 6, 8, 10 after intravenous injection (n=9, each group). Complete blood counts were measured. Reticulated platelet (RP) was analyzed by flow cytometry using thiazole orange (TO) to evaluate platelet kinetics. Plasma TPO level were measured by ELISA. Bone marrow megakaryocyte's number and morphology from femur in bortezomib- and control-treated mice were observed by microscopy. Femur was fixed in 10% buffered formalin, decalcificated, embedded in paraffin and stained for Hematoxylin-Eosin (H-E). (b) Bortezomib (2.5 mg/kg) was administrated via tail-vein to mice. After 24hr, bone marrow cells were cultured in MegaCult®-C at 5% CO2 and 20% O2for 7 days. The megakaryocytic colonies (CFU-Megs-in vivo) were counted. In vitro experiments: (c) Bone marrow cells, obtained from non-treated mice, were cultured at 37°C in 5% CO2and 20% O2 for 7 days with bortezomib (0.01, 0.1, 1, 10, 100 ng/ml). CFU-Megs were counted (CFU-Megs-in vitro). (d) Proplatelet formation: Murine megakarocytes were partially purified from bone marrow using BSA gradient. They were plated in 96 micro-well culture plates (300 megakaryocyte)well) and cultured in IMDM in duplicates, supplemented with 1 × ITS-G (Life technologies) and each concentration of bortezomib (0.01, 0.1, 1, 10, 100 ng/ml), at 37°C in 5% CO2and 20% O2. After 24 hr incubation, the megakaryocytes with proplatelets in each well were counted. Results: (a) Control mice did not have any significant change in platelet counts, % reticulated platelets and plasma TPO levels at days 0, 2, 4, 6, 8, 10. While, bortezomib treated mice (2.5mg/kg) had a significant reduction in platelet counts at day 2 (470 ± 210 × 109/L. P<0.001), at day 4 (667 ± 118 × 109/L, P<0.001). The platelet counts returned to normal value at day 6 (903 ± 548 × 109/L) and day 10 (1122 ± 187 × 109/L). RP (%) began to increase at day 6 (8.8 ± 4.0 %). Plasma TPO levels tend to increase at day 4. Means megakaryocytes's number in one field of femur was similar in between bortezomib non-treated and –treated mice. The megakaryocytes were similar in morphology at each day, too. (b) CFU-Megs-in vivo were similar in number between bortezomib non-treated and –treated mice (38.0 ± 6.1 vs 34.5 ± 5.6 per 1 × 105 bone marrow cells respectively). (c) CFU-Megs-in vitro were not decreased significantly at 0.001 to 1 ng/ml and decreased significantly (p<0.01) at 10 and 100 ng/ml of bortezomib. (d) Proplatelet formation (PPF) were decreased significantly at 0.01, 0.1, 1, 10, 100 ng/ml bortezomib (0 mg)ml: 25.2 ± 4.8%, 0.01ng/ml: 23.8 ± 4.9%, 0.1 ng/ml: 18.4 ± 3.1% p<0.01, 1 ng/ml: 13.2 ± 3.8% p<0.001, 10 ng/ml: 13.3 ± 2.1% p<0.001, 100ng/ml: 5.9 ± 1.4 % p<0.001). Discussion & Conclusion: Bortezomib did not adversely affect on megakaryocytic prognitors nor megakaryocytes. It did inhibit PPF, that is, the step of platelet production, even when bortezomib plasma concentration levels have gone down. Plasma TPO level showed an inverse relationship against circulating platelet counts. Based on the evidence in which Cmax of plasma bortezomib concentration was under 100 ng/ml in bortezomib-injected mice (2.5mg/kg), bortezomib induced thrombocytopenia might be due to the inhibition of proplatelet formation of megakaryocyte. Disclosures: No relevant conflicts of interest to declare.

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The Y14-p53 Regulatory Circuit in Megakaryocyte Differentiation and Thrombocytopenia

10.1101/2020.11.03.367508 ◽

2020 ◽

Author(s):

Chun-Hao Su ◽

Wei-Ju Liao ◽

Wei-Chi Ke ◽

Ruey-Bing Yang ◽

Woan-Yuh Tarn

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Ex Vivo ◽

Critical Role ◽

Platelet Production ◽

Noncoding Regions ◽

Regulatory Circuit ◽

Hel Cells ◽

Absent Radius ◽

Megakaryocyte Differentiation

SUMMARYThrombocytopenia-absent radius syndrome is caused by a deletion in chromosome 1q21.1 in trans with RBM8A mutations in the noncoding regions. We generated megakaryocyte-specific Rbm8a knockout (Rbm8aKOMK) mice that exhibited marked thrombocytopenia, internal hemorrhage, and splenomegaly, indicating a disorder of platelet production. Rbm8aKOMK mice accumulated immature megakaryocytes in the bone marrow and spleen. Depletion of Y14/RBM8A in human erythroleukemia (HEL) cells inhibited phorbol ester-induced polyploidy and downregulated the signaling pathways associated with megakaryocyte maturation. Accordingly, Rbm8aKOMK mice had reduced expression of surface glycoproteins on platelets and impaired coagulation. Moreover, p53 level was increased in Y14-depleted HEL cells and Rbm8aKOMK megakaryocytes. Treatment with a p53 inhibitor restored ex vivo differentiation of Rbm8aKOMK megakaryocytes and unexpectedly activated Y14 expression in HEL cells. Knockout of Trp53 in part restored the platelet count of Rbm8aKOMK mice. These results indicate that the Y14-p53 circuit plays a critical role in megakaryocyte differentiation and platelet production.

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Effects of Cytokines on Platelet Production From Blood and Marrow CD34+ Cells

Blood ◽

10.1182/blood.v91.3.830 ◽

1998 ◽

Vol 91 (3) ◽

pp. 830-843 ◽

Cited By ~ 96

Author(s):

Françoise Norol ◽

Natacha Vitrat ◽

Elisabeth Cramer ◽

Josette Guichard ◽

Samuel A. Burstein ◽

...

Keyword(s):

Cell Population ◽

Culture System ◽

Simple Liquid ◽

Similar Response ◽

Platelet Production ◽

Liquid Culture System ◽

Cd34 Cells ◽

Proplatelet Formation ◽

And Function

Abstract The late stages of megakaryocytopoiesis, consisting of the terminal processes of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by flow cytometry. A truncated form of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatelet-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34+ cells gave rise to a threefold augmentation of platelets compared with marrow CD34+ cells. This finding was related to the higher proliferative capacity of the former population because the proportion of proplatelet-displaying MKs was similar for both types of CD34+ cells. The production of platelets per MK from CD34+ cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with the degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34+CD41+ progenitors and was associated with a twofold to threefold increment in platelet production per MK. As shown using this CD34+ CD41+ cell population, PEG-rHuMGDF was required throughout the culture period to potently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisingly, when used in combination, these cytokines elicited a degree of platelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibited by non-MK cells which contaminate the cultures when the entire CD34+ cell population is used. Cultured platelets derived from PEG-rHuMGDF– or cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the effects of cytokines and the role of polyploidization on platelet production and function.

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Platelet Alterations Caused by Antiplatelet Antibodies in the Circulation

10.1055/s-0039-1682707 ◽

1977 ◽

Author(s):

T. Nagasawa ◽

B.K. Kim ◽

M.G. Baldini

Keyword(s):

Platelet Count ◽

Specific Activity ◽

Rabbit Model ◽

Antiplatelet Antibodies ◽

Large Numbers ◽

Severe Reduction ◽

Series Of Experiments ◽

Specific Activities

It is known that antiplatelet antibodies cause loss of platelet cytoplasmic and granular contents in vitro. It is, however, unknown whether similar platelet changes occur in vivo, in the circulation, leading to destruction and phagocytosis of platelets in the R.E. system. To study this possibility a rabbit model was devised. Severe and stable thrombocytopenia was first produced in rabbits by one intravenous injection of Adriamycin. Large numbers of allogenic platelets labeled in vitro with 51Cr and 14C-serotonin were then infused to raise the circulating platelet count to 180-250 × 103/mm3. A dilute heteroimmune antiplatelet serum prepared in the guinea pig was infused intravenously and platelet samples were collected four times during the subsequent 30 minutes to 24 hours. Platelet hexokinase and β-glucuronidase, 14C-serotonin and 51Cr were measured. Within the first 60 min the specific activity of 51Cr in platelets decreased by 21%, 14C-serotonin declined by 30%, hexokinase by 5% and β-glucuronidase by 29%. During the subsequent 24 hours only 51Cr and hexokinase registered a mild decrease but 51C-serotonin and β-glucuronidase remained essentially unchanged. In a second series of experiments the effect of platelet alloantibodies was studied in rabbits previously immunized with allogenic platelets. The decline in the specific activities of the enzymes and 14C-serotonin was similar to that observed in animals treated with heteroimmune sera but loss of 51Cr was more severe. These results demonstrate that the platelets remaining in the circulation after the disappearance of the immediate effect of hetero- or alloantibodies were qualitatively altered with a severe reduction of their granular and cytoplasmic contents.

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RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival

Blood ◽

10.1182/blood.2019003251 ◽

2020 ◽

Vol 136 (15) ◽

pp. 1773-1782 ◽

Cited By ~ 1

Author(s):

Daniel DeHelian ◽

Shuchi Gupta ◽

Jie Wu ◽

Chelsea Thorsheim ◽

Brian Estevez ◽

...

Keyword(s):

Platelet Count ◽

G Protein ◽

Platelet Activation ◽

Platelet Reactivity ◽

Rgs Proteins ◽

Platelet Recovery ◽

Platelet Production ◽

Platelet Survival

Abstract G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

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In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation-and chemotherapy- induced bone marrow suppression

Blood ◽

10.1182/blood.v88.3.778.778 ◽

1996 ◽

Vol 88 (3) ◽

pp. 778-784 ◽

Cited By ~ 36

Author(s):

A Ohwada ◽

S Rafii ◽

MA Moore ◽

RG Crystal

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Adenovirus Vector ◽

Bone Marrow Suppression ◽

Single Administration ◽

Platelet Recovery ◽

Platelet Production ◽

Life Threatening ◽

Bone Abnormalities

Abstract Thrombopoietin (TPO, c-mpl ligand) has emerged as a major hematopoietic cytokine stimulating megakaryocyte proliferation, endomitosis, and platelet production. This study shows that a single administration of an adenovirus (Ad) vector encoding TPO (AdCMV.TPO) abrogates thrombocytopenia induced in mice by carboplatin and irradiation. Normal Balb/c mice receiving the vector had increased platelet counts peaking at 7 days and returning to baseline by day 15. Mice rendered pancytopenic with 500 rads and 1.2 mg of carboplatin had a nadir platelet count of five percent of the baseline. Mice receiving AdCMV.TPO 3 days before receiving irradiation and chemotherapy achieved a platelet nadir fourfold higher, and had significant reduction in duration of thrombocytopenia, than mice receiving the control Ad vector. Introduction of AdCMV.TPO the same day of chemotherapy and irradiation was equally effective in acceleration of platelet recovery, but administration of AdCMV.TPO 3 days after chemotherapy-radiation had little effect on platelet recovery. At 30 days after therapy bone marrow and spleen of mice treated with AdCMV.TPO were populated with a large number of polyploid megakaryocytes, but there was no evidence of circulating megakaryocytes in the liver or lungs and no pathologic bone abnormalities such as osteosclerosis or myelofibrosis. These observations suggest that an Ad vector may be an excellent delivery system to provide adequate TPO production to maintain platelet levels in circumstances associated with life-threatening thrombocytopenia.

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ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Blood ◽

10.1182/blood-2009-03-209221 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3216-3226 ◽

Cited By ~ 45

Author(s):

Aisha V. Sauer ◽

Emanuela Mrak ◽

Raisa Jofra Hernandez ◽

Elena Zacchi ◽

Francesco Cavani ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Severe Combined Immunodeficiency ◽

Intrinsic Defect ◽

Combined Immunodeficiency ◽

Hematopoietic Stem ◽

Enzyme Replacement ◽

Bone Phenotype

Abstract Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

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