RUNX1-deficient human megakaryocytes demonstrate thrombopoietic and platelet half-life and functional defects.

Heterozygous defects in runt-related transcription factor 1 (RUNX1) are causative of a familial platelet disorder with associated myeloid malignancy (FPDMM). Because RUNX1-deficient animal models do not mimic bleeding disorder or leukemic risk associated with FPDMM, development of a proper model system is critical to understanding the underlying mechanisms of the observed phenotype and to identifying therapeutic interventions. We previously reported an in vitro megakaryopoiesis system comprising human CD34+ hematopoietic stem and progenitor cells that recapitulated the FPDMM quantitative megakaryocyte defect through a decrease in RUNX1 expression via a lentiviral short hairpin RNA strategy. We now show that shRX-megakaryocytes have a marked reduction in agonist responsiveness. We then infused shRX-megakaryocytes into immunocompromised NOD scid gamma (NSG) mice and demonstrated that these megakaryocytes released fewer platelets than megakaryocytes transfected with a nontargeting shRNA, and these platelets had a diminished half-life. The platelets were also poorly responsive to agonists, unable to correct thrombus formation in NSG mice homozygous for a R1326H mutation in von Willebrand Factor (VWFR1326H), which switches the species-binding specificity of the VWF from mouse to human glycoprotein Ibα. A small-molecule inhibitor RepSox, which blocks the transforming growth factor ß1 (TGFß1) pathway and rescued defective megakaryopoiesis in vitro, corrected the thrombopoietic defect, defects in thrombus formation and platelet half-life, and agonist response in NSG/VWFR1326H mice. Thus, this model recapitulates the defects in FPDMM megakaryocytes and platelets, identifies previously unrecognized defects in thrombopoiesis and platelet half-life, and demonstrates for the first time, reversal of RUNX1 deficiency-induced hemostatic defects by a drug.

PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but its role in PAH is unsettled. Here, we report that: (1) PAI-1 is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared to controls; (2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with up-regulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and (3) pharmacological inhibition of uPA in human PAH PASMCs suppresses pro-proliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that down-regulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent up-regulation of mTOR and TGF-b signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.

A novel role for endoplasmic reticulum protein 46 (ERp46) in platelet function and arterial thrombosis in mice.

Although several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has 3 CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, adenosine triphosphate (ATP) release, and αIIbß3 activation. ERp46 protein potentiated αIIbß3 activation, platelet aggregation, and ATP release, whereas inactive ERp46 inhibited these processes. ERp46 knockout mice had prolonged tail-bleeding times and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbß3 activation, platelet aggregation, ATP release, and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the 3 active sites. Platelet aggregation stimulated by an αIIbß3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbß3 by surface plasmon resonance but poorly to platelets lacking αIIbß3 and physically associated with αIIbß3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the ß3 subunit than other PDIs and in contrast to PDI, generated thiols in ß3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in ß3, implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in ß3, implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbß3 activation by targeting disulfide bonds.

Neutrophils promote clearance of nuclear debris following acid-induced lung injury.

Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared with wild type. Size analysis of DNA debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation, they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intratracheal administration of DNase I. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.

RUNX-1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells.

Patients with familial platelet disorder with a predisposition to myeloid malignancy (FPDMM) harbor germline monoallelic mutations in a key hematopoietic transcription factor, RUNX-1. Previous studies of FPDMM have focused on megakaryocyte (Mk) differentiation and platelet production and signaling. However, the effects of RUNX-1 haploinsufficiency on hematopoietic progenitor cells (HPCs) and subsequent megakaryopoiesis remains incomplete. We studied induced pluripotent stem cell (iPSC)-derived HPCs (iHPCs) and Mks (iMks) from both patient-derived lines and a wild-type (WT) line modified to be RUNX-1 haploinsufficient (RUNX-1+/-), each compared with their isogenic WT control. All RUNX-1+/- lines showed decreased iMk yield and depletion of an Mk-biased iHPC subpopulation. To investigate global and local gene expression changes underlying this iHPC shift, single-cell RNA sequencing was performed on sorted FPDMM and control iHPCs. We defined several cell subpopulations in the Mk-biased iHPCs. Analyses of gene sets upregulated in FPDMM iHPCs indicated enrichment for response to stress, regulation of signal transduction, and immune signaling-related gene sets. Immunoblot analyses in FPDMM iMks were consistent with these findings, but also identified augmented baseline c-Jun N-terminal kinase (JNK) phosphorylation, known to be activated by transforming growth factor-ß1 (TGF-ß1) and cellular stressors. These findings were confirmed in adult human CD34+-derived stem and progenitor cells (HSPCs) transduced with lentiviral RUNX1 short hairpin RNA to mimic RUNX-1+/-. In both iHPCs and CD34+-derived HSPCs, targeted inhibitors of JNK and TGF-ß1 pathways corrected the megakaryopoietic defect. We propose that such intervention may correct the thrombocytopenia in patients with FPDMM.

Complement mediates binding and procoagulant effects of ultralarge HIT immune complexes.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex-mediated thrombotic disorders.

Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis.

Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis.

Recognition of PF4-VWF complexes by heparin-induced thrombocytopenia antibodies contributes to thrombus propagation.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT.

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

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.

FcRn augments induction of tissue factor activity by IgG-containing immune complexes.

Thromboembolism complicates disorders caused by immunoglobulin G (IgG)-containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), ß-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.

Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies to platelet factor 4 (PF4)/heparin complexes. PF4 released from platelets binds to surface glycosaminoglycans on hematopoietic and vascular cells that are heterogenous in composition and differ in affinity for PF4. PF4 binds to monocytes with higher affinity than to platelets, and depletion of monocytes exacerbates thrombocytopenia in a murine HIT model. Here we show that the expression of PF4 on platelets and development of thrombocytopenia are modulated by the (re)distribution of PF4 among hematopoietic and endothelial cell surfaces. Binding of PF4 to platelets in whole blood in vitro varies inversely with the white cell count, likely because of the greater affinity of monocytes for PF4. In mice, monocyte depletion increased binding of PF4 to platelets by two- to three-fold. Induction of HIT in mice caused a transient >80-fold increase in binding of HIT antibody to monocytes vs 3.5-fold increase to platelets and rapid transient monocytopenia. Normalization of monocyte counts preceded the return in platelet counts. Exposure of blood to endothelial cells also depletes PF4 from platelet surfaces. These studies demonstrate a dynamic interchange of surface-bound PF4 among hematopoetic and vascular cells that may limit thrombocytopenia at the expense of promoting prothrombotic processes in HIT.

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway.

The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder "phenotype" (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

FLI1 level during megakaryopoiesis affects thrombopoiesis and platelet biology.

Friend leukemia virus integration 1 (FLI1), a critical transcription factor (TF) during megakaryocyte differentiation, is among genes hemizygously deleted in Jacobsen syndrome, resulting in a macrothrombocytopenia termed Paris-Trousseau syndrome (PTSx). Recently, heterozygote human FLI1 mutations have been ascribed to cause thrombocytopenia. We studied induced-pluripotent stem cell (iPSC)-derived megakaryocytes (iMegs) to better understand these clinical disorders, beginning with iPSCs generated from a patient with PTSx and iPSCs from a control line with a targeted heterozygous FLI1 knockout (FLI1+/-). PTSx and FLI1+/- iMegs replicate many of the described megakaryocyte/platelet features, including a decrease in iMeg yield and fewer platelets released per iMeg. Platelets released in vivo from infusion of these iMegs had poor half-lives and functionality. We noted that the closely linked E26 transformation-specific proto-oncogene 1 (ETS1) is overexpressed in these FLI1-deficient iMegs, suggesting FLI1 negatively regulates ETS1 in megakaryopoiesis. Finally, we examined whether FLI1 overexpression would affect megakaryopoiesis and thrombopoiesis. We found increased yield of noninjured, in vitro iMeg yield and increased in vivo yield, half-life, and functionality of released platelets. These studies confirm FLI1 heterozygosity results in pleiotropic defects similar to those noted with other critical megakaryocyte-specific TFs; however, unlike those TFs, FLI1 overexpression improved yield and functionality.

Identifying and enriching platelet-producing human stem cell-derived megakaryocytes using factor V uptake.

Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.

The disulfide isomerase ERp72 supports arterial thrombosis in mice.

Several CGHC motif-containing disulfide isomerases support thrombosis. We here report that endoplasmic reticulum protein 72 (ERp72), with 3 CGHC redox-active sites (ao, a, and a'), supports thrombosis. We generated a new conditional knockout mouse model and found that Tie2-Cre/ERp72fl/fl mice with blood and endothelial cells lacking ERp72 had prolonged tail bleeding times and decreased platelet accumulation in laser-induced cremaster arteriole injury and FeCl3-induced mesenteric arterial injury. Fibrin deposition was decreased in the laser injury model. Both platelet and fibrin accumulation defects were fully rescued by infusion of recombinant ERp72 containing functional a and a' CGHC motifs (ERp72(oo-ss-ss)). Infusion of ERp72 containing inactivated a and a' CGHC motifs (ERp72(ss-oo-oo)) inhibited platelet accumulation and fibrin deposition in wild-type mice. Infusion of ERp72(oo-ss-ss) into ß3-null mice increased fibrin deposition in the absence of platelets. ERp72-null platelets had defective aggregation, JON/A binding, P-selectin expression, and adenosine triphosphate (ATP) secretion. The aggregation and ATP secretion defects were fully rescued by ERp72(oo-ss-ss) but partially rescued by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). Aggregation and ATP secretion of human platelets was potentiated by ERp72(oo-ss-ss) but inhibited by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). These data suggest that both the a and a' active sites are required for platelet function. ERp72 bound poorly to ß3-null mouse platelets, and the addition of ERp72(oo-ss-ss) to human platelets generated thiols in αIIbß3, suggesting a direct interaction of ERp72 with αIIbß3. Defective aggregation of ERp72-null platelets was recovered by ERp72, but not other thiol isomerases. In summary, ERp72 plays a critical role in platelet function and coagulation through the a and a' CGHC motifs.

Pre-clinical development of a cryopreservable megakaryocytic cell product capable of sustained platelet production in mice.

BACKGROUND: Platelet (PLT) transfusions are the most effective treatments for patients with thrombocytopenia. The growing demand for PLT transfusion products is compounded by a limited supply due to dependency on volunteer donors, a short shelf-life, risk of contaminating pathogens, and alloimmunization. This study provides preclinical evidence that a third-party, cryopreservable source of PLT-generating cells has the potential to complement presently available PLT transfusion products. STUDY DESIGN AND METHODS: CD34+ hematopoietic stem/progenitor cells derived from umbilical cord blood (UCB) units were used in a simple and efficient culture system to generate a cell product consisting of megakaryocytes (MKs) at different stages of development. The cultures thus generated were evaluated ex vivo and in vivo before and after cryopreservation. RESULTS: We generated a megakaryocytic cell product that can be cryopreserved without altering its phenotypical and functional capabilities. The infusion of such a product, either fresh or cryopreserved, into immune-deficient mice led to production of functional human PLTs which were observed within a week after infusion and persisted for 8 weeks, orders of magnitude longer than that observed after the infusion of traditional PLT transfusion products. The sustained human PLT engraftment was accompanied by a robust presence of human cells in the bone marrow (BM), spleen, and lungs of recipient mice. CONCLUSION: This is a proof-of-principle study demonstrating the creation of a cryopreservable megakaryocytic cell product which releases functional PLTs in vivo. Clinical development of such a product is currently being pursued for the treatment of thrombocytopenia in patients with hematological malignancies.

CXCL5 regulates chemokine scavenging and pulmonary host defense to bacterial infection.

The chemokine sink hypothesis pertaining to erythrocyte Duffy Antigen Receptor for Chemokines (DARC) during inflammation has received considerable attention, but lacks direct in vivo evidence. Here we demonstrate, using mice with a targeted deletion in CXCL5, that CXCL5 bound erythrocyte DARC and impaired its chemokine scavenging in blood. CXCL5 increased the plasma concentrations of CXCL1 and CXCL2 in part through inhibiting chemokine scavenging, impairing chemokine gradients and desensitizing CXCR2, which led to decreased neutrophil influx to the lung, increased lung bacterial burden and mortality in an Escherichia coli pneumonia model. In contrast, CXCL5 exerted a predominant role in mediating neutrophil influx to the lung during inflammation after LPS inhalation. Platelets and lung resident cells were the sources of homeostatic CXCL5 in blood and inflammatory CXCL5 in the lung respectively. This study presents a paradigm whereby platelets and red cells alter chemokine scavenging and neutrophil-chemokine interaction during inflammation.

Understanding platelet generation from megakaryocytes: implications for in vitro-derived platelets.

Platelets are anucleate cytoplasmic discs derived from megakaryocytes that circulate in the blood and have major roles in hemostasis, thrombosis, inflammation, and vascular biology. Platelet transfusions are required to prevent the potentially life-threatening complications of severe thrombocytopenia seen in a variety of medical settings including cancer therapy, trauma, and sepsis. Platelets used in the clinic are currently donor-derived which is associated with concerns over sufficient availability, quality, and complications due to immunologic and/or infectious issues. To overcome our dependence on donor-derived platelets for transfusion, efforts have been made to generate in vitro-based platelets. Work in this area has advanced our understanding of the complex processes that megakaryocytes must undergo to generate platelets both in vivo and in vitro. This knowledge has also defined the challenges that must be overcome to bring in vitro-based platelet manufacturing to a clinical reality. This review will focus on our understanding of committed megakaryocytes and platelet release in vivo and in vitro, and how this knowledge can guide the development of in vitro-derived platelets for clinical application.