Early Endosomal GTPase Rab5 (Ras-Related Protein in Brain 5) Regulates GPIbß (Glycoprotein Ib Subunit ß) Trafficking and Platelet Production In Vitro.

OBJECTIVE: Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 (Ras-related protein in brain 5) regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3-monophosphate production important for binding of effector proteins during early-to-late endosomal/lysosomal maturation. Approach and Results: To investigate the role of Rab5 in megakaryocytes, we expressed GFP (green fluorescent protein)-coupled Rab5 wild type and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived megakaryocytes. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing megakaryocytes, when compared with GFP or Rab5 wild type. Moreover, trafficking of GPIbß (glycoprotein Ib subunit beta), a subunit of major megakaryocytes receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbß was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 wild type megakaryocytes, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbß plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in late endosomes/lysosomes. Localization of GPIbß in enlarged endosomes was phosphatidylinositol 3-monophosphate dependent. CONCLUSIONS: Taken together, our results demonstrate that Rab5-dependent endocytosis plays an important role in megakaryocytes receptor trafficking, membrane formation, and thrombopoiesis.

Methylation of dual-specificity phosphatase 4 controls cell differentiation.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Role of Rho-GTPases in megakaryopoiesis.

Megakaryocytes (MKs) are the bone marrow (BM) cells that generate blood platelets by a process that requires: i) polyploidization responsible for the increased MK size and ii) cytoplasmic organization leading to extension of long pseudopods, called proplatelets, through the endothelial barrier to allow platelet release into blood. Low level of localized RHOA activation prevents actomyosin accumulation at the cleavage furrow and participates in MK polyploidization. In the platelet production, RHOA and CDC42 play opposite, but complementary roles. RHOA inhibits both proplatelet formation and MK exit from BM, whereas CDC42 drives the development of the demarcation membranes and MK migration in BM. Moreover, the RhoA or Cdc42 MK specific knock-out in mice and the genetic alterations in their down-stream effectors in human induce a thrombocytopenia demonstrating their key roles in platelet production. A better knowledge of Rho-GTPase signalling is thus necessary to develop therapies for diseases associated with platelet production defects.Abbreviations: AKT: Protein Kinase BARHGEF2: Rho/Rac Guanine Nucleotide Exchange Factor 2ARP2/3: Actin related protein 2/3BM: Bone marrowCDC42: Cell division control protein 42 homologCFU-MK: Colony-forming-unit megakaryocyteCIP4: Cdc42-interacting protein 4mDIA: DiaphanousDIAPH1; Protein diaphanous homolog 1ECT2: Epithelial Cell Transforming Sequence 2FLNA: Filamin AGAP: GTPase-activating proteins or GTPase-accelerating proteinsGDI: GDP Dissociation InhibitorGEF: Guanine nucleotide exchange factorHDAC: Histone deacetylaseLIMK: LIM KinaseMAL: Megakaryoblastic leukaemiaMARCKS: Myristoylated alanine-rich C-kinase substrateMKL: Megakaryoblastic leukaemiaMLC: Myosin light chainMRTF: Myocardin Related Transcription FactorOTT: One-Twenty Two ProteinPACSIN2: Protein Kinase C And Casein Kinase Substrate In Neurons 2PAK: P21-Activated KinasePDK: Pyruvate Dehydrogenase kinasePI3K: Phosphoinositide 3-kinasePKC: Protein kinase CPTPRJ: Protein tyrosine phosphatase receptor type JRAC: Ras-related C3 botulinum toxin substrate 1RBM15: RNA Binding Motif Protein 15RHO: Ras homologousROCK: Rho-associated protein kinaseSCAR: Suppressor of cAMP receptorSRF: Serum response factorSRC: SarcTAZ: Transcriptional coactivator with PDZ motifTUBB1: Tubulin ß1VEGF: Vascular endothelial growth factorWAS: Wiskott Aldrich syndromeWASP: Wiskott Aldrich syndrome proteinWAVE: WASP-family verprolin-homologous proteinWIP: WASP-interacting proteinYAP: Yes-associated protein.

Platelet Dysfunction and Thrombosis in JAK2V617F-Mutated Primary Myelofibrotic Mice.

OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.

The role of AGK in thrombocytopoiesis and possible therapeutic strategies.

Abnormal megakaryocyte development and platelet production lead to thrombocytopenia or thrombocythemia and increase the risk of hemorrhage or thrombosis. Acylglycerol kinase (AGK) is a mitochondrial membrane kinase that catalyzes the formation of phosphatidic acid and lysophosphatidic acid. Mutation of AGK has been described as the major cause of Sengers syndrome, and the patients with Sengers syndrome have been reported to exhibit thrombocytopenia. In this study, we found that megakaryocyte/platelet-specific AGK-deficient mice developed thrombocytopenia and splenomegaly, mainly caused by inefficient bone marrow thrombocytopoiesis and excessive extramedullary hematopoiesis, but not by apoptosis of circulating platelets. It has been reported that the G126E mutation arrests the kinase activity of AGK. The AGK G126E mutation did not affect peripheral platelet counts or megakaryocyte differentiation, suggesting that the involvement of AGK in megakaryocyte development and platelet biogenesis was not dependent on its kinase activity. The Mpl/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (Stat3) pathway is the major signaling pathway regulating megakaryocyte development. Our study confirmed that AGK can bind to JAK2 in megakaryocytes/platelets. More interestingly, we found that the JAK2 V617F mutation dramatically enhanced the binding of AGK to JAK2 and greatly facilitated JAK2/Stat3 signaling in megakaryocytes/platelets in response to thrombopoietin. We also found that the JAK2 JAK homology 2 domain peptide YGVCF617CGDENI enhanced the binding of AGK to JAK2 and that cell-permeable peptides containing YGVCF617CGDENI sequences accelerated proplatelet formation. Therefore, our study reveals critical roles of AGK in megakaryocyte differentiation and platelet biogenesis and suggests that targeting the interaction between AGK and JAK2 may be a novel strategy for the treatment of thrombocytopenia or thrombocythemia.

Increased levels of the megakaryocyte and platelet expressed cysteine proteases stefin A and cystatin A prevent thrombosis.

Increased platelet activity occurs in type 2 diabetes mellitus (T2DM) and such platelet dysregulation likely originates from altered megakaryopoiesis. We initiated identification of dysregulated pathways in megakaryocytes in the setting of T2DM. We evaluated through transcriptomic analysis, differential gene expressions in megakaryocytes from leptin receptor-deficient mice (db/db), exhibiting features of human T2DM, and control mice (db/+). Functional gene analysis revealed an upregulation of transcripts related to calcium signaling, coagulation cascade and platelet receptors in diabetic mouse megakaryocytes. We also evidenced an upregulation (7- to 9.7-fold) of genes encoding stefin A (StfA), the human ortholog of Cystatin A (CSTA), inhibitor of cathepsin B, H and L. StfA/CSTA was present in megakaryocytes and platelets and its expression increased during obesity and diabetes in rats and humans. StfA/CSTA was primarily localized at platelet membranes and granules and was released upon agonist stimulation and clot formation through a metalloprotease-dependent mechanism. StfA/CSTA did not affect platelet aggregation, but reduced platelet accumulation on immobilized collagen from flowing whole blood (1200 s-1). In-vivo, upon laser-induced vascular injury, platelet recruitment and thrombus formation were markedly reduced in StfA1-overexpressing mice without affecting bleeding time. The presence of CA-074Me, a cathepsin B specific inhibitor significantly reduced thrombus formation in-vitro and in-vivo in human and mouse, respectively. Our study identifies StfA/CSTA as a key contributor of platelet-dependent thrombus formation in both rodents and humans.

JAK2V617F Megakaryocytes Promote Hematopoietic Stem/Progenitor Cell Expansion in Mice Through Thrombopoietin/MPL Signaling.

The myeloproliferative neoplasms (MPNs) are stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired kinase mutation JAK2V617F plays a central role in these disorders. The mechanisms responsible for HSPC expansion in MPNs are not fully understood, limiting the effectiveness of current treatments. One hallmark feature of the marrow in patients with MPNs is megakaryocyte (MK) hyperplasia. Previously, we reported that JAK2V617F-bearing MKs cause a murine myeloproliferative syndrome with HSPC expansion. Here we show that JAK2V617F MKs promote MPN stem cell function by inducing HSPC quiescence with increased repopulating capacity. In addition, we demonstrate that thrombopoietin and its receptor MPL are critical for the JAK2V617F-bearing MK-induced myeloproliferation, both by directly affecting the quantity and quality of MKs and by altering the MK-endothelial interaction and vascular niche function. Therefore, targeting HSPC niche-forming MKs and/or their interactions within the vascular niche could provide novel, more effective therapeutic strategies in patients with MPNs. Stem Cells 2018;36:1676-1684.

Cyanidin-3-o-ß-Glucoside Induces Megakaryocyte Apoptosis via PI3K/Akt- and MAPKs-Mediated Inhibition of NF-κB Signalling.

Apoptotic-like phase is an essential step in thrombopoiesis from megakaryocytes. Anthocyanins are natural flavonoid pigments that possess a wide range of biological activities, including protection against cardiovascular diseases and induction of tumour cell apoptosis. We investigated the effects and underlying mechanisms of cyanidin-3-o-ß-glucoside (Cy-3-g, the major bioactive compound in anthocyanins) on the apoptosis of human primary megakaryocytes and Meg-01 cell line in vitro. We found that Cy-3-g dose-dependently increased the dissipation of the mitochondrial membrane potential, caspase-9 and caspase-3 activity in megakaryocytes from patients with newly diagnosed acute myeloid leukaemia but not in those from healthy volunteers. In Meg-01 cells, Cy-3-g regulated the distribution of Bak, Bax and Bcl-xL proteins in the mitochondria and cytosol, subsequently increasing cytochrome c release and stimulating caspase-9 and caspase-3 activation and phosphatidylserine exposure. However, Cy-3-g did not exert significant effects on factor-associated suicide (Fas), Fas ligand, caspase-8 or Bid expression. Cy-3-g inhibited nuclear factor kappa B (NF-κB) p65 activation by down-regulating inhibitor of NF-κB kinase (IKK)α and IKKß expression, followed by the inhibition of inhibitor of NF-κB (IκB)α phosphorylation and degradation and subsequent inhibition of the translocation of the p65 sub-unit into the nucleus, and finally stimulating caspase-3 activation and phosphatidylserine exposure. The inhibitory effect of Cy-3-g on NF-κB activation was mediated by the activation of extracellular signal-regulated kinases (Erk1/2) and p38 mitogen-activated protein kinase (MAPK) and the inhibition of phosphoinositide 3-kinase (PI3K)/Akt signalling. U0126 (Erk1/2 inhibitor), SB203580 (p38 MAPK inhibitor) and 740 Y-P (PI3K agonist) significantly reversed Cy-3-g-reduced phosphorylation of p65. Taken together, our data indicate that Cy-3-g induces megakaryocyte apoptosis via the inhibition of NF-κB signalling, which may play important roles in regulating thrombopoiesis.

Kinesin-1 Is a New Actor Involved in Platelet Secretion and Thrombus Stability.

OBJECTIVE: Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in platelets, the underlying mechanisms are not yet fully characterized. APPROACH AND RESULTS: By studying a mouse model (cKO [conditional knockout]Kif5b) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKOKif5b platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKOKif5b platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKOKif5b platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 (Ras-related protein Rab-27) protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein. CONCLUSIONS: Our results indicate that a kinesin-1-dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.

A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis.

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard-Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.

The heterotrimeric G protein Gß1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gßγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gß1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gß1 revealed that Gß1 interacts with the PP1c α, ß, and γ1 isoforms. Purified PP1c bound to recombinant Gß1-GST protein, and PP1c co-immunoprecipitated with Gß1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gß1 complex, which correlated with an association of PP1c with phospholipase C ß3 (PLCß3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLCß3. siRNA-mediated depletion of GNB1 (encoding Gß1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα-/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gßγ. Finally, disruption of PP1c-Gß1 complexes with myristoylated Gß1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gß1 protein enlists PP1c to modulate GPCR signaling in platelets.

Matrix Metalloproteinases and Platelet Function.

Platelets contain and release several matrix metalloproteinases (MMPs) and their tissue inhibitors of matrix metalloproteinases (TIMPs), including MMP-1, -2, -3, -9, and -14 and TIMP-1, -2, and -4. Although devoid of a nucleus, platelets also synthesize TIMP-2 upon activation. Platelet-released MMPs/TIMPs, as well as MMPs generated by other cells within the cardiovascular system, modulate platelet function in health and disease. In particular, a normal hemostatic platelet response to vessel wall injury may be transformed into pathologic thrombus formation by the release from platelets and/or by the local generation of some MMPs. Moreover, platelets may localize the production of leukocyte-derived MMPs to sites of vascular damage, contributing to atherosclerosis development and complications and to arterial aneurysm formation. Finally, the interaction between platelets and tumor cells is strongly influenced by MMPs/TIMPs. All these mechanisms are emerging as important in atherothrombosis, inflammatory disease, and cancer growth and dissemination. Increasing knowledge of these mechanisms may open the way to novel therapeutic approaches.

Non-apoptotic functions of caspases in myeloid cell differentiation.

Subtle caspase activation is associated with the differentiation of several myeloid lineages. A tightly orchestrated dance between caspase-3 activation and the chaperone HSP70 that migrates to the nucleus to protect the master regulator GATA-1 from cleavage transiently occurs in basophilic erythroblasts and may prepare nucleus and organelle expel that occurs at the terminal phase of erythroid differentiation. A spatially restricted activation of caspase-3 occurs in maturing megakaryocytes to promote proplatelet maturation and platelet shedding in the bloodstream. In a situation of acute platelet need, caspase-3 could be activated in response to IL-1α and promote megakaryocyte rupture. In peripheral blood monocytes, colony-stimulating factor-1 provokes the formation of a molecular platform in which caspase-8 is activated, which downregulates nuclear factor-kappa B (NF-κB) activity and activates downstream caspases whose target fragments such as those generated by nucleophosmin (NPM1) cleavage contribute to the generation of resting macrophages. Human monocytes secrete mature IL-1ß in response to lipopolysaccharide through an alternative inflammasome activation that involves caspase-8, a pathway that does not lead to cell death. Finally, active caspase-3 is part of the proteases contained in secretory granules of mast cells. Many questions remain on how these proteases are activated in myeloid cell lineages, which target proteins are cleaved, whereas other are protected from proteolysis, the precise role of cleaved proteins in cell differentiation and functions, and the link between these non-apoptotic functions of caspases and the death of these diverse cell types. Better understanding of these functions may generate therapeutic strategies to control cytopenias or modulate myeloid cell functions in various pathological situations.

[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

[SP600125-induced polyploidization of megakaryocytic leukemia cell lines by ribosomal protein S6 kinase 1 depends on the degree of cell differentiation].

Objective To investigate regulatory role of ribosomal protein S6 kinase 1 (S6K1) in the polyploidization of different megakaryocytic leukemia cell lines at the different differentiation stages. Methods Megakaryocytic leukemia cell lines (Dami, Meg-01 and HEL cells) were induced towards polyploidization by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. The SP600125-inducing process was blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor. The phenotype (CD41a, CD42a and CD42b) and DNA ploidy were detected by flow cytometry. The expression and phosphorylation of S6K1 and related proteins were detected by Western blotting. Results SP600125 induced polyploidization and increased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in Dami, Meg-01 and HEL cells. However, the effect of SP600125 on polyploidization of the three cell lines was different, with the strongest effect on Dami cells and the weakest on Meg-01 cells. Moreover, SP600125 increased the phosphorylation of S6K1 Thr421/Ser424 and decreased the phosphorylation of Thr389 in Dami cells. However, it only increased the phosphorylation of Thr389 in HEL cells and had no effect on the phosphorylation of S6K1 in Meg-01 cells. Interestingly, H-89 only partially blocked the polyploidization of Dami cells, although it decreased the phosphorylation of 4E-BP1 in all SP600125-induced three cell lines. Noticeably, H-89 decreased the phosphorylation of S6K1 Thr421/Ser424 and increased the phosphorylation of Thr389 in Dami cells. However, H-89 had no effect on the phosphorylation of Thr421/Ser424, although it increased the phosphorylation of Thr389 in Meg-01 and HEL cells. Phenotypic analysis showed that the three cell lines were at different levels of differentiation in megakaryocytic lineage, with the highest differentiation in Dami and the lowest in Meg-01 cells. Conclusion SP600125-induced polyploidization of megakaryocytic leukemia cell lines is dependent on the effect of SP600125 on phosphorylation of S6K1 in cell lines at the different differentiation stages.

Virtual screening, optimization, and identification of a novel specific PTP-MEG2 Inhibitor with potential therapy for T2DM.

Megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2) is a tyrosine phosphatase expressed in megakaryocytic cells, and causes insulin sensitization when down regulated. Therefore, specific inhibitors of PTP-MEG2 are potential candidates for novel Type 2 Diabetes (T2DM)therapy. In this study, we discovered PTP-MEG2 inhibitors using high throughput and virtual screening (HTS/VS) and structural optimization in silicon.Eight compound-candidates were identified from the interactions with PTP-MEG2, protein tyrosine phosphatase 1B (PTP1B) and T cell protein tyrosine phosphatase (TCPTP). Results from enzymatic assays show compounds 4a and 4b inhibited PTP-MEG2 activity with an IC50 of 3.2 µM and 4.3 µM, respectively. Further, they showed a 7.5 and 5.5 fold change against PTP1B and TCPTP, respectively. We propose compounds 4a and 4b are PTP-MEG2 inhibitors with potential therapeutic use in T2DM treatment.

Nuclear factor-κB regulates expression of platelet phospholipase C-ß2 (PLCB2).

Phospholipase C (PLC)-ß2 (gene PLCB2) is a critical regulator of platelet responses upon activation. Mechanisms regulating of PLC-ß2 expression in platelets/MKs are unknown. Our studies in a patient with platelet PLC-ß2 deficiency revealed the PLCB2 coding sequence to be normal and decreased platelet PLC-ß2 mRNA, suggesting a defect in transcriptional regulation. PLCB2 5'- upstream region of the patient revealed a heterozygous 13 bp deletion (-1645/-1633 bp) encompassing a consensus sequence for nuclear factor-κB (NF-κB). This was subsequently detected in three of 50 healthy subjects. To understand the mechanisms regulating PLC-ß2, we studied the effect of this variation in the PLCB2. Gel-shift studies using nuclear extracts from human erythroleukaemia (HEL) cells or recombinant p65 showed NF-κB binding to oligonucleotide with NF-κB site; in luciferase reporter studies its deletion reduced PLCB2 promoter activity. PLCB2 expression was decreased by siRNA knockdown of NF-κB p65 subunit and increased by p65 overexpression. By immunoblotting platelet PLC-ß2 in 17 healthy subjects correlated with p65 (r=0.76, p=0.0005). These studies provide the first evidence that NF-κB regulates MK/platelet PLC-ß2 expression. This interaction is important because of the major role of PLC-ß2 in platelet activation and of NF-κB in processes, including inflammation and atherosclerosis, where both are intimately involved.

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans.

Human blood cell counts are tightly maintained within narrow physiologic ranges, largely controlled by cytokine-integrated signaling and transcriptional circuits that regulate multilineage hematopoietic specification. Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin/cellular myeloproliferative leukemia virus liganding is dispensable for definitive thrombopoiesis, establishing that fundamentally important modifier loci remain unelucidated. In this study, platelet transcriptome sequencing and extended thrombocytosis cohort analyses identified a single loss-of-function mutation (BLVRB(S111L)) causally associated with clonal and nonclonal disorders of enhanced platelet production. BLVRB(S111L) encompassed within the substrate/cofactor [α/ß dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXß tetrapyrrole(s) and results in exaggerated reactive oxygen species accumulation as a putative metabolic signal leading to differential hematopoietic lineage commitment and enhanced thrombopoiesis. These data define the first physiologically relevant function of BLVRB and implicate its activity and/or heme-regulated BV tetrapyrrole(s) in a unique redox-regulated bioenergetic pathway governing terminal megakaryocytopoiesis; these observations also define a mechanistically restricted drug target retaining potential for enhancing human platelet counts.

Enhanced Megakaryopoiesis and Platelet Activity in Hypercholesterolemic, B6-Ldlr-/-, Cdkn2a-Deficient Mice.

BACKGROUND: Genome-wide association studies for coronary artery disease/myocardial infarction revealed a 58 kb risk locus on 9p21.3. Refined genetic analyses revealed unique haplotype blocks conferring susceptibility to atherosclerosis per se versus risk for acute complications in the presence of underlying coronary artery disease. The cell proliferation inhibitor locus, CDKN2A, maps just upstream of the myocardial infarction risk block, is at least partly regulated by the noncoding RNA, ANRIL, overlapping the risk block, and has been associated with platelet counts in humans. Thus, we tested the hypothesis that CDKN2A deficiency predisposes to increased platelet production, leading to increased platelet activation in the setting of hypercholesterolemia. METHODS AND RESULTS: Platelet production and activation were measured in B6-Ldlr(-/-)Cdkn2a(+/-) mice and a congenic strain carrying the region of homology with the human 9p21.3/CDKN2A locus. The strains exhibit decreased expression of CDKN2A (both p16(INK4a) and p19(ARF)) but not CDKN2B (p15(INK4b)). Compared with B6-Ldlr(-/-) controls, both Cdkn2a-deficient strains exhibited increased platelet counts and bone marrow megakaryopoiesis. The platelet overproduction phenotype was reversed by treatment with cyclin-dependent kinase 4/6 inhibitor, PD0332991/palbociclib, that mimics the endogenous effect of p16(INK4a). Western diet feeding resulted in increased platelet activation, increased thrombin/antithrombin complex, and decreased bleeding times in Cdkn2a-deficient mice compared with controls. CONCLUSIONS: Together, the data suggest that one or more Cdkn2a transcripts modulate platelet production and activity in the setting of hypercholesterolemia, amenable to pharmaceutical intervention. Enhanced platelet production and activation may predispose to arterial thrombosis, suggesting an explanation, at least in part, for the association of 9p21.3 and myocardial infarction.