Inflammatory platelet production stimulated by tyrosyl-tRNA synthetase mimicking viral infection.

Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRSACT) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRSACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRSACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRSACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP+) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these "inflammatory" MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRSACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.

Mechanisms of anti-GPIbα antibody-induced thrombocytopenia in mice.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by antibody-mediated platelet destruction. Different mechanisms have been suggested to explain accelerated platelet clearance and impaired thrombopoiesis, but the pathophysiology of ITP has yet to be fully delineated. In this study, we tested 2 mouse models of immune-mediated thrombocytopenia using the rat anti-mouse GPIbα monoclonal antibody 5A7, generated in our laboratory. After a single IV administration of high-dose (2 mg/kg) 5A7, opsonized platelets were rapidly cleared from the circulation into the spleen and liver; this was associated with rapid upregulation of thrombopoietin (TPO) messenger RNA. In contrast, subcutaneous administration of low-dose 5A7 (0.08-0.16 mg/kg) every 3 days gradually lowered the platelet count; in this case, opsonized platelets were observed only in the spleen, and TPO levels remained unaltered. Interestingly, in both models, the 5A7 antibody was found on the surface of, as well as internalized to, bone marrow megakaryocytes. Consequently, platelets generated in the chronic phase of repeated subcutaneous 5A7 administration model showed reduced GPIbα membrane expression on their surface. Our findings indicate that evaluation of platelet surface GPIbα relative to platelet size may be a useful marker to support the diagnosis of anti-GPIbα antibody-induced ITP.

Generation of PECAM-1 (CD31) conditional knockout mice.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is an adhesion and signaling receptor that is expressed on endothelial and hematopoietic cells and plays important roles in angiogenesis, vascular permeability, and regulation of cellular responsiveness. To better understanding the tissue specificity of PECAM-1 functions, we generated mice in which PECAM1, the gene encoding PECAM-1, could be conditionally knocked out. A targeting construct was created that contains loxP sites flanking PECAM1 exons 1 and 2 and a neomycin resistance gene flanked by flippase recognition target (FRT) sites that was positioned upstream of the 3' loxP site. The targeting construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo-pregnant females. Resulting chimeric animals were bred with transgenic mice expressing Flippase 1 (FLP1) to remove the FRT-flanked neomycin resistance gene and mice heterozygous for the floxed PECAM1 allele were bred with each other to obtain homozygous PECAM1 flox/flox offspring, which expressed PECAM-1 at normal levels and had no overt phenotype. PECAM1 flox/flox mice were bred with mice expressing Cre recombinase under the control of the SRY-box containing gene 2 (Sox2Cre) promoter to delete the floxed PECAM1 allele in offspring (Sox2Cre;PECAM1 del/WT ), which were crossbred to generate Sox2Cre; PECAM1 del/del offspring. Sox2Cre; PECAM1 del/del mice recapitulated the phenotype of conventional global PECAM-1 knockout mice. PECAM1 flox/flox mice will be useful for studying distinct roles of PECAM-1 in tissue specific contexts and to gain insights into the roles that PECAM-1 plays in blood and vascular cell function.

Tyrosyl-tRNA synthetase stimulates thrombopoietin-independent hematopoiesis accelerating recovery from thrombocytopenia.

New mechanisms behind blood cell formation continue to be uncovered, with therapeutic approaches for hematological diseases being of great interest. Here we report an enzyme in protein synthesis, known for cell-based activities beyond translation, is a factor inducing megakaryocyte-biased hematopoiesis, most likely under stress conditions. We show an activated form of tyrosyl-tRNA synthetase (YRSACT), prepared either by rationally designed mutagenesis or alternative splicing, induces expansion of a previously unrecognized high-ploidy Sca-1+ megakaryocyte population capable of accelerating platelet replenishment after depletion. Moreover, YRSACT targets monocytic cells to induce secretion of transacting cytokines that enhance megakaryocyte expansion stimulating the Toll-like receptor/MyD88 pathway. Platelet replenishment by YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from induced pluripotent stem cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. We suggest megakaryocyte-biased hematopoiesis induced by YRSACT offers new approaches for treating thrombocytopenia, boosting yields from cell-culture production of platelet concentrates for transfusion, and bridging therapy for hematopoietic stem cell transplantation.

Platelet hyperreactivity explains the bleeding abnormality and macrothrombocytopenia in a murine model of sitosterolemia.

Sitosterolemia is a rare, autosomal recessive disease caused by mutations in the adenosine triphosphate-binding cassette transporter genes ABCG5 or ABCG8 that result in accumulation of xenosterols in the body. Clinical manifestations include tendon xanthomas, premature coronary artery disease, hemolytic anemia, macrothrombocytopenia, and bleeding. Although the effect of sterol accumulation on the predisposition for atherosclerosis is evident, how xenosterol accumulation leads to defects in platelet physiology is unknown. Sitosterolemia induced in Abcg5- and Abcg8-deficient mice fed a high plant sterol diet resulted in accumulation of free sterols in platelet plasma membranes, leading to hyperactivatable platelets characterized by constitutive binding of fibrinogen to its αIIbß3 integrin receptor, internalization of the αIIbß3 complex, generation of platelet-derived microparticles, and changes in the quantity and subcellular localization of filamin. The latter was associated with macrothrombocytopenia, shedding of GPIbα, impaired platelet adhesion to von Willebrand factor, and inability to form stable thrombi. Plasma levels of soluble GPIbα were strongly correlated with plasma sitosterol levels in samples from human sitosterolemic patients, implicating a similar mechanism of sterol-induced platelet passivation in the human disease. Intercalation of plant sterols into the plasma membrane therefore results in dysregulation of multiple platelet activation pathways, leading to macrothrombocytopenia and bleeding.

Platelets with a W127X mutation in GPIX express sufficient residual amounts of GPIbα to support adhesion to von Willebrand factor and collagen.

Bernard-Soulier syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib/IX complex. The GPIX W127X mutation is the most common genetic defect in Japanese patients with BSS, which is often misdiagnosed as immune thrombocytopenic purpura, presumably due to residual expression of GPIbα. Neither the mechanism by which this mutation leads to a mild bleeding diathesis, nor whether functional GPIbα is expressed on platelet surfaces is known. We investigated GPIbα expression and function in platelets with a GPIX W127X mutation (GPIXW127X). GPIbα complexed with GPIbß by disulfide bonding was expressed on GPIXW127X platelets and stable CHO-K1 cells lacking GPIX but expressing GPIbα and GPIbß. Expression of GPIbα/ß on GPIXW127X platelets was sufficient to support adhesion to immobilized von Willebrand factor and type III collagen and ristocetin-induced platelet agglutination. A residual amount of functional GPIbα/ß heteromer expressed on GPIXW127X platelets partially compensates for the absence of the GPIb/IX complex. This may account for the mild bleeding phenotype of the BSS variant characterized by a non-sense mutation in GPIX.

Characterization of the light chain-restricted clonal B cells in peripheral blood of HCV-positive patients.

To investigate the association between hepatitis C virus (HCV) and B cell proliferation, we searched for the clonal B cells by flow cytometric analysis of the surface immunoglobulin kappa (kappa):lambda (lambda) light chain ratios of the circulating B (CD19+) cells in 240 HCV-positive patients and 150 negative controls with liver diseases. Clonal B cells with light chain restriction (kappa:lambda ratio >3:1 or <1:2) were analyzed for CD5 expression and the presence of monoclonal immunoglobulin heavy-chain (IGH) gene rearrangements and the t(14;18) chromosomal translocation. Clonal B cells were detected in 7 cases with HCV (2.9%), but was never detected in the controls (p < 0.05). Of the 7 cases, all had monoclonal IGH gene rearrangements and one had the t(14;18) chromosomal translocation. These HCV-related clonal B cells are not uniform in the intensity of CD5 expression and showed no increase in the frequencies of CD5+ population compared with non-clonal B cells. No "chronic lymphocytic leukemia-phenotype" cells were found. The loss of clonality was observed in 2 cases treated with interferon and in one case treated with splenectomy. The longitudinal study is required to determine whether these circulating clonal B cells progress to lymphoproliferative disorders in future or not.

Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells.

Embryonic stem cells (ESCs) could potentially compensate for the lack of blood platelets available for use in transfusions. Here, we describe a new method for generating mouse ESC-derived platelets (ESPs) that can contribute to hemostasis in vivo. Flow cytometric sorting of cells from embryoid bodies on day 6 demonstrated that c-Kit(+) integrin alpha IIb (alpha IIb)(+) cells, but not CD31(+) cells or vascular endothelial cadherin(+) cells, are capable of megakaryopoiesis and the release of platelet-like structures by day 12. alpha IIb beta 3-expressing ESPs exhibited ectodomain shedding of glycoprotein (GP)Ibalpha, GPV, and GPVI, but not alpha IIb beta 3 or GPIb beta. ESPs showed impaired alpha IIb beta 3 activation and integrin-mediated actin reorganization, critical events for normal platelet function. However, the administration of metalloproteinase inhibitors GM6001 or TAPI-1 during differentiation increased the expression of GPIb alpha, improving both thrombogenesis in vitro and posttransfusion recovery in vivo. Thus, the regulation of metalloproteinases in culture could be useful for obtaining high-quality, efficacious ESPs as an alternative platelet source for transfusions.

Periostin and bone marrow fibrosis.

Periostin is a secreted protein that shares structural homology with the insect axon guidance protein fasciclin 1. Periostin is expressed predominantly in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses. We have shown previously that periostin is a novel component of subepithelial fibrosis in bronchial asthma. Here, we investigated the relationship between periostin and bone marrow (BM) fibrosis. Periostin was expressed in the stroma and stromal cells of BM fibrosis specimens and to a great extent its expression levels correlated closely to the grade of fibrosis, as estimated by silver staining. However, in the present study, we found no relationship between plasma periostin levels and the extent of BM fibrosis. We also demonstrated that periostin is secreted by human BM hTERT stromal cells and that its secretion is enhanced by TGF-beta, a cytokine produced by clonal proliferation of megakaryocytes and/or monocytes. These results indicate that periostin is a component of BM fibrosis and that it may play a role in the disease progression.

Characterization of a patient with atypical amegakaryocytic thrombocytopenia.

We report a 6-year-old girl with amegakaryocytic thrombocytopenia, the first case of this rare congenital disorder not to have an MPL gene mutation. Although no mutations were identified in MPL, Mpl protein was absent in the platelets and TPO induced phosphorylation of the Janus tyrosine kinase 2 (Jak2) was not detected. In addition to the defect of Mpl, the patient demonstrated markedly reduced expression of glycoprotein VI (GPVI) in contrast to normal expression of other platelet-specific proteins GPIb alpha, GPIb beta, and GPIIb. To explore the causes for the absence of Mpl, the entire coding region of Jak2 and AML1 were sequenced and no mutations were identified. To our knowledge, this is the first report that describes a case of amegakaryocytic thrombocytopenia that is not caused by a mutation in MPL and demonstrates the severe impairment of GPVI expression on platelets.

Involvement of IL-32 in activation-induced cell death in T cells.

NK cell transcript 4 (NK4), now denoted as IL-32, was originally identified as a transcript whose expression was increased in activated NK cells. It has been very recently demonstrated that NK4 is secreted from several cells upon the stimulation of some inflammatory cytokines such as IL-18, IL-1beta, IFN-gamma and IL-12. Furthermore, NK4 induces production of tumor necrosis factor, macrophage inflammatory protein (MIP)-2 and IL-8 in monocytic cell lines, indicating that this factor would be involved in the inflammatory responses. Based on these findings, NK4 was renamed IL-32. However, the biological activities of IL-32 on other cell types remained undetermined. Furthermore, it was still argued whether IL-32 acts on cells from outside or inside the cells. In this article, we first report that expression of IL-32 was up-regulated in activated T cells and NK cells, and that IL-32beta was the predominantly expressed isoform in activated T cells. IL-32 was specifically expressed in T cells undergoing apoptosis and enforced expression of IL-32-induced apoptosis, whereas its down-regulation rescued the cells from apoptosis in HeLa cells. IL-32 existing in the supernatant would be derived from the cytoplasm of apoptotic cells. These results strongly indicated that IL-32 would be involved in activation-induced cell death in T cells, probably via its intracellular actions. Our present findings expand our understanding of the biological function of IL-32 and argue that IL-32 may act on cells, not only from the outside but also from the inside.

Thrombopoietin initiates demethylation-based transcription of GP6 during megakaryocyte differentiation.

Glycoprotein VI (GPVI) is an essential platelet receptor for collagens that is exclusively expressed in the megakaryocytic lineage. Transcription of the human gene GP6 is driven largely by GATA-binding protein 1 (GATA-1), specificity protein 1 (Sp1), and Friend leukemia integration 1 (Fli-1). In this report, we show that GPVI expression during megakaryocytic differentiation is dependent on cytosine-phosphate-guanosine (CpG) demethylation that can be initiated by thrombopoietin (TPO). Sodium bisulfite genomic sequencing established that a CpG-rich island within the GP6 promoter region is fully methylated at 10 CpG sites in GPVI-nonexpressive cell lines, such as UT-7/EPO and C8161, but completely unmethylated in GPVI-expressive cell lines, including UT-7/TPO and CHRF288-11. To further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated human cord blood cells with TPO. The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+-enriched cells obtained after TPO differentiation. Furthermore, when UT-7/EPO-Mpl cells, which stably express human C-myeloproliferative leukemia virus ligand (c-Mpl), were treated with TPO, demethylation of the GP6 promoter was induced. In every case, demethylation of the GP6 promoter correlated with an increase in mRNA level. Thus, megakaryocyte-specific expression of the GP6 gene is regulated, in part, by CpG demethylation, which can be directly initiated by TPO.

Megakaryocyte proliferation and ploidy regulated by the cytoplasmic tail of glycoprotein Ibalpha.

We have investigated the ability of glycoprotein (GP) Ibalpha, a megakaryocytic gene product, to sequester the signal transduction protein 14-3-3xi and to influence megakaryocytopoiesis. Using a Gp1ba(-/-) mouse colony, we compared the rescued phenotypes produced by a wild-type human GP Ibalpha allele or a similar allele containing a 6-residue cytoplasmic tail truncation that abrogates binding to 14-3-3xi. The observed phenotypes illustrate an involvement for GP Ibalpha in thrombopoietin-mediated events of megakaryocyte proliferation, polyploidization, and the expression of apoptotic markers in maturing megakaryocytes. We developed a hypothesis for the involvement of a GP Ibalpha/14-3-3xi/PI-3 kinase complex in regulating thrombopoietin-mediated responses. An observed increase in thrombopoietin-mediated Akt phosphorylation in the truncated variant supported the hypothesis and led to the development of a model in which the GP Ibalpha cytoplasmic tail sequestered signaling proteins during megakaryocytopoiesis and, as such, became a critical regulator in the temporal sequence of events that led to normal megakaryocyte maturation.

Characterization of the human protein S gene promoter: a possible role of transcription factors Sp1 and HNF3 in liver.

Protein S is a vitamin-K-dependent plasma glycoprotein that serves as a cofactor for activated protein C in the protein C anticoagulation pathway, which regulates blood coagulation by inactivating factors Va and VIIIa. Mechanisms regulating the expression of the protein S gene remain unknown to date. The aim of this study was to characterize the cis-acting DNA elements of the human protein S gene in liver. We found that liver cell lines (HepG2 and PLC) transcribed the human protein S gene to mRNA, whereas non-liver cell lines (HEK293 and HeLa cells) either transcribed the gene weakly or not at all. Isolation and analysis of tissue-specific gene expression in HepG2 and HeLa cells of the 5'-flanking region from -6183 to +294 of the protein S gene indicated that the consensus binding motifs to HNF3 and Sp1 or MAZ transcription factors in the flanking region are essential for protein S gene expression. Exogenous expression of the Sp1 gene augmented the protein S-reporter gene expression in HepG2 or PLC cells but had no effect in HeLa cells. Taken together, we would conclude that transcription factors of HNF3, MAZ, and Sp1 are required for high-level expression of the protein S gene in hepatic cells, but in non-hepatic cells such as HeLa cells, an unknown factor(s) binds to the Sp1 region and disturbs the action of Sp1 and MAZ.

The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion.

Platelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collagen-induced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VInull platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VInull or FcR-gammanull animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VInull platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils.