Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Human red cell differentiation requires the action of erythropoietin on committed progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in hypoplastic anemia. To address the basis for iron regulation of erythropoiesis, we established primary hematopoietic cultures with transferrin saturation levels that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identified as a critical regulatory element the aconitases, multifunctional iron-sulfur cluster proteins that metabolize citrate to isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or megakaryocytic progenitors. An active site aconitase inhibitor, fluorocitrate, blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid iron restriction response in vitro and reversed anemia progression in irondeprived mice. The mechanism for aconitase regulation of erythropoiesis most probably involves both production of metabolic intermediates and modulation of erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase C␣/␤, or possibly protein kinase C␦, whose activities were regulated by iron, isocitrate, and erythropoietin. IntroductionRed cell production results from erythropoietin (Epo)-driven survival, proliferation, and maturation of committed bone marrow progenitor cells. This process critically depends on cellular uptake of adequate bioavailable iron, provided in the form of diferric transferrin. Compromise in iron uptake or intracellular trafficking results in iron-restricted erythropoiesis, characterized by diminished marrow responsiveness to Epo. 1 Epo acts during an early phase of erythroid development, between late (erythroid burst-forming unit) and early pronormoblast stages, before the initiation of hemoglobin synthesis. [1][2][3] Therefore, iron restriction serves as a checkpoint to restrain Epo-driven progenitor expansion in the face of limited iron stores. It has been documented in zebrafish, in which defects in intracellular iron utilization block early erythroid differentiation, 4 and in mammals, in which dietary iron deficiency impairs the transition from erythroid colony-forming unit to pronormoblast. 5 In the clinical setting, iron-restricted erythropoiesis underlies many of the anemias that are refractory to Epo treatment (eg, anemias of chronic renal disease and inflammation). 1 The lineage-and stage-selective nature of the erythroid iron restriction response has suggested involvement of a specialized signaling pathway distinct from the iron depletion response that occurs in a wide variety of cell types treated with chelators. Supporting this notion, chelator-induced iron depletion in MCF-7 cells causes cell-cycle arrest in G 1 phase followed by apoptosis, 6 neither of which is seen in iron-restricted erythropoiesis in vivo. 2,6,7 Thus, to identify mechanisms applicable to ...

The transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclindependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy. IntroductionMegakaryocytes and erythroblasts develop from bipotential megakaryocyte-erythroid progenitors (MEPs) under the influence of multiple critical transcription factors. Some of these factors, such as GATA-1, GATA-2, FOG1, and SCL, promote both lineages, whereas others, such as RUNX1 and EKLF, promote only megakaryocyte or erythroid development, respectively. 1,2 Many of these key factors serve dual functions in development, activating lineage-appropriate genes while simultaneously repressing lineageinappropriate genes. 2 Numerous in vitro and in vivo studies have emphasized the centrality of GATA-1 in megakaryocyte development. Virtually all megakaryocytic promoters contain functionally important GATA binding sites. 3 Mice with diminished GATA-1 in megakaryocytes display thrombocytopenia and aberrant megakaryocytic maturation. 4 In particular, the GATA-1-deficient megakaryocytes show defects in growth regulation, polyploidization, proplatelet formation, granule biogenesis, and surface antigen expression. [5][6][7] Interestingly, these mice also display MEP abnormalities, implicating GATA-1 in development of a properly primed bipotent progenitor. 8,9 In humans, hereditary and acquired GATA-1 mutations have both been associated with defective megakaryopoiesis. Familial X-linked thrombocytopenia results from missense mutations affecting the N-terminal zinc finger of GATA-1, a domain involved in recruitment of the cofactor FOG1. [10][11][12][13][14] Acquired mutations of GATA-1 occur in the setting of the 2 Down syndrome (DS)-associated megakaryoblastic proliferative disorders, transient myeloproliferative disorder (DS-TMD) and...

Iron deficiency causes resistance in erythroid progenitors against proliferative but not survival signals of erythropoietin. Khalil et al. link this response to the down-regulation of Scribble, an orchestrator of receptor trafficking and signaling. With iron deprivation, transferrin receptor 2 drives Scribble degradation, reconfiguring erythropoietin receptor function.

The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.