Biology subjects

Poncz, M.

Publications and source records attributed to Poncz, M..

15 recordsLinked to original sources

bioRxiv2025

Antiphospholipid syndrome (APS) is a platelet factor 4 (PF4)-centric immunothrombotic disorder

Antiphospholipid syndrome (APS) is an immunothrombotic disorder, frequently attributed to autoantibodies that bind {beta}2-glycoprotein I ({beta}2GPI). A study showed that the platelet-specific chemokine, platelet factor 4 (PF4), binds to {beta}2GPI, enhancing recognition of {beta}2GPI by APS antibodies. APS antibodies induce the release of neutrophil extracellular traps (NETs), webs of decondensed chromatin that bind both PF4 and {beta}2GPI. We propose that PF4 bridges {beta}2GPI to NETs (and other PF4-targeted polyanions), leading to the formation of prothrombotic PF4:{beta}2GPI:NET immunotargets in APS. Dynamic light-scattering studies of isolated IgGs from four patients with triple-positive APS show formation of PF4:{beta}2GPI:NET complexes that bind APS antibodies. NETs released in a microfluidic system bound {beta}2GPI, but only in the presence of PF4, forming a multimolecular APS antigenic target. Whole blood infused through a photochemically-injured, endothelium-lined microfluidic channel formed platelet-, fibrin-, and complement-rich thrombi that bound APS antibody only in the presence of PF4. Thrombi were reduced in size if either ADAMTS13 or DNase1 was infused. In a murine APS model, wildtype and transgenic mice expressing platelet human PF4 {+/-} Fc{gamma}RIIA developed more intense neutrophil rolling along veins, and more extensive thrombus formation following laser injury to cremaster arterioles and venules, whereas mice lacking PF4 did not. Three antigenically distinct anti-hPF4 monoclonal antibodies blocked thrombosis in vitro, and neutrophil rolling and thrombosis in vivo. Our studies provide new insights into the basis of APS that has mechanistic parallels to other known PF4 immunothrombotic disorders and offer potential diagnostic and non-anticoagulant therapeutic strategies for clinical management. Key pointsO_LIPF4 enhances {beta}2GPI binding to NETs and these complexes are central to APS immunothrombosis. C_LIO_LIAnti-PF4 monoclonal antibodies block APS immunothrombosis in microfluidic and murine studies. C_LI

cell biology↗

bioRxiv2025

Platelet factor 4 modulates endothelial cell antimicrobial activity to enhance bacterial clearance and improve sepsis outcomes

Sepsis is a life-threatening condition characterized by dysregulated host responses to infection. Here, we identify platelet factor 4 (PF4) as a key mediator of vascular antimicrobial defense. In vitro, PF4 enhanced endothelial cell internalization of Escherichia coli via interactions with the PF4 receptor CXCR3 and the endothelial glycocalyx, directing bacteria to clathrin-mediated endocytosis and lysosomal degradation. In vivo, PF4 administration improved survival and reduced sepsis severity, bacterial burden, inflammation, and thrombosis in wild-type (WT) and PF4 knockout (PF4-/-) mice challenged with systemic polymicrobial infection. Using intravital microscopy, we observed that infused bacteria were rapidly sequestered in the pulmonary microvasculature. However, PF4-/- mice exhibited impaired bacterial clearance and increased microvascular platelet adhesion and aggregation. In the liver, following Kupffer cell depletion, PF4-/- mice had increased sinusoidal platelet accumulation, larger bacterial aggregates, and elevated hepatic bacterial burden compared to WT controls. Collectively, these findings reveal that PF4 promotes bacterial clearance and restrains immunothrombosis during sepsis in part via endothelial cell uptake and destruction of microbes. By enhancing endothelial antimicrobial function, PF4 represents a significant yet previously underrecognized host defense mechanism that limits bacterial spread and alleviates vascular injury during infection. KEY POINTSO_LIIn vitro, PF4 accelerates bacterial clearance by enhancing endothelial uptake of bacteria and promoting their trafficking to the lysosome. C_LIO_LIIn murine sepsis, PF4 augments pathogen clearance to reduce infection severity, limit organ injury, and improve survival. C_LI

cell biology↗

bioRxiv2024

Platelet integrin αIIbβ3 plays a key role in venous thrombogenesis in a mouse model

AbstractVenous thrombosis (VT) is a common vascular disease associated with reduced survival and a high recurrence rate. Previous studies have shown that the accumulation of platelets and neutrophils at sites of endothelial cell activation is a primary event in VT, but a role for platelet IIb{beta}3 in the initiation of venous thrombosis has not been established. This task has been complicated by the increased bleeding linked to partial agonism of current IIb{beta}3 inhibitory drugs such as tirofiban (Aggrastat(R)). Here, we show that m-tirofiban, an engineered version of tirofiban, is not a partial agonist of IIb{beta}3. This is based on its cryo-EM structure in complex with human full-length IIb{beta}3 and its inability to increase expression of an activation-sensitive epitope on platelet IIb{beta}3. m-tirofiban abolished agonist-induced platelet aggregation ex vivo at concentrations that preserved clot retraction and markedly suppressed the accumulation of platelets, neutrophils, and fibrin on thrombin-activated endothelium in real-time using intravital microscopy in a mouse model of venous thrombogenesis. Unlike tirofiban, however, m-tirofiban did not increase bleeding at the thrombosis-inhibitory dose. These findings establish a key role for IIb{beta}3 in the initiation of VT, provide a guiding principle for designing potentially safer inhibitors for other integrins, and suggest that pure antagonists of IIb{beta}3 like m-tirofiban merit further consideration as potential thromboprophylaxis agents in patients at high-risk for VT and hemorrhage.

pathology↗

bioRxiv2024

Marginated neutrophils in the lungs effectively compete for nanoparticles targeted to the endothelium, serving as a part of the reticuloendothelial system

Nanomedicine has long pursued the goal of targeted delivery to specific organs and cell types but has not achieved this goal with the vast majority of targets. One rare example of success in this pursuit has been the 25+ years of studies targeting the lung endothelium using nanoparticles conjugated to antibodies against endothelial surface molecules. However, here we show that such "endothelial-targeted" nanocarriers also effectively target the lungs numerous marginated neutrophils, which reside in the pulmonary capillaries and patrol for pathogens. We show that marginated neutrophils uptake of many of these "endothelial-targeted" nanocarriers is on par with endothelial uptake. This generalizes across diverse nanomaterials and targeting moieties and was even found with physicochemical lung tropism (i.e., without targeting moieties). Further, we observed this in ex vivo human lungs and in vivo healthy mice, with an increase in marginated neutrophil uptake of nanoparticles caused by local or distant inflammation. These findings have implications for nanomedicine development for lung diseases. These data also suggest that marginated neutrophils, especially in the lungs, should be considered a major part of the reticuloendothelial system (RES), with a special role in clearing nanoparticles that adhere to the lumenal surfaces of blood vessels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/597904v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17e0518org.highwire.dtl.DTLVardef@809eb6org.highwire.dtl.DTLVardef@33d9a7org.highwire.dtl.DTLVardef@169863_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Studies of infused megakaryocytes into mice support a ''catch-and-release'' model of pulmonary-centric thrombopoiesis

Many aspects of thrombopoiesis, the release of platelets from megakaryocytes (Mks), remain under debate, including where this process occurs. Murine lung in situ-microscopy studies suggested that a significant fraction of circulating platelets was released from lung-entrapped, marrow-derived Mks. We now confirm these that endogenous murine (m) Mks are immobilized in the lungs. Intravenously infused in vitro-differentiated, mMks and human (h) Mks are similarly entrapped, followed by shedding of their cytoplasm over [~]30 minutes with a peak number of released platelets occurring 1.5-4 hours later. However, while infused Mks from both species shed large intrapulmonary cytoplasmic fragments that underwent further processing into platelet-sized fragments, the two differed: many mMks escaped from and then recycled back to the lungs, while most hMks were enucleated upon first intrapulmonary passage. Infused immature hMks, inflammatory hMks, umbilical cord-blood-derived hMks and immortalized Mk progenitor cell (imMKCL)-derived hMks were also immobilized in the lung of recipient mice, and released their cytoplasm, but did so to different degrees. Pharmacologically-treated hMks support that membrane stiffness was key to pulmonary thrombopoiesis. Intraarterial infused hMks resulted in few Mks being immobilized in tissues other than the lungs and was accompanied by a blunted and delayed rise in circulating human platelets. These studies demonstrate that the lung entraps and processes both circulating Mks and released large cytoplasmic fragments consistent with a prior lung/heart murine study and support a pulmonary-centric "catch-and-release" model of thrombopoiesis. Thrombopoiesis is a drawn-out process with the majority of cytoplasmic processing of Mks occurring in the pulmonary bed. Key PointsO_LIInfused in vitro-differentiated megakaryocytes synchronously release cytoplasmic fragments selectively in the pulmonary bed. C_LIO_LILarge, released megakaryocyte fragments recycle to the lungs, undergo further fission, terminally forming platelets. C_LI

cell biology↗

bioRxiv2024

G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics

Key PointsO_LIBispecific cis-acting platelet receptor inhibitors (CAPRIs) mediate hetero-clustering of the ITIM receptor G6B with ITAM receptors. C_LIO_LIG6B-GPVI and G6B-CD32A CAPRIs specifically inhibit collagen- and immune complex-induced thrombus formation, respectively. C_LI Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR {gamma}-chain complex or Fc{gamma}RIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies.

cell biology↗

bioRxiv2023

Packaging of supplemented urokinase into naked alpha-granules of in vitro-grown megakaryocytes for targeted therapeutic delivery

Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human platelets in vitro from CD34+ hematopoietic cell-derived megakaryocytes. CD34+-megakaryocytes internalize and store in -granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+-\megakaryocytes was mediated in part via LRP1 and IIb{beta}3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2r{gamma}null (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWFR1326H generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet -granules from in vitro-generated megakaryocytes. Key pointsO_LIUnlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its -granules. C_LIO_LIuPA uptake involves LRP1 and IIb{beta}3 receptors and is functionally available from activated platelets. C_LI

cell biology↗

bioRxiv2023

PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hy-pertension

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

cell biology↗

bioRxiv2023

Neutrophil extracellular trap stabilization by platelet factor 4 reduces thrombogenicity and endothelial cell injury

Neutrophil extracellular traps (NETs) are abundant in sepsis, and proposed NET-directed therapies in sepsis prevent their formation or accelerate degradation. Yet NETs are important for microbial entrapment, as NET digestion liberates pathogens and NET degradation products (NDPs) that deleteriously promote thrombosis and endothelial cell injury. We proposed an alternative strategy of NET-stabilization with the chemokine, platelet factor 4 (PF4, CXCL4), which we have shown enhances NET-mediated microbial entrapment. We now show that NET compaction by PF4 reduces their thrombogenicity. In vitro, we quantified plasma thrombin and fibrin generation by intact or degraded NETs and cell-free (cf) DNA fragments, and found that digested NETs and short DNA fragments were more thrombogenic than intact NETs and high molecular weight genomic DNA, respectively. PF4 reduced the thrombogenicity of digested NETs and DNA by interfering, in part, with contact pathway activation. In endothelial cell culture studies, short DNA fragments promoted von Willebrand factor release and tissue factor expression via a toll-like receptor 9-dependent mechanism. PF4 blocked these effects. Cxcl4-/- mice infused with cfDNA exhibited higher plasma thrombin anti-thrombin (TAT) levels compared to wild-type controls. Following challenge with bacterial lipopolysaccharide, Cxcl4-/- mice had similar elevations in plasma TAT and cfDNA, effects prevented by PF4 infusion. Thus, NET-stabilization by PF4 prevents the release of short fragments of cfDNA, limiting the activation of the contact coagulation pathway and reducing endothelial injury. These results support our hypothesis that NET-stabilization reduces pathologic sequelae in sepsis, an observation of potential clinical benefit. HIGHLIGHTSO_LIIn contrast to intact NETs, degraded NETs and cfDNA are prothrombotic and injure the endothelium. C_LIO_LIPF4 reduces the ability of degraded NETs and cfDNA to promote thrombosis and injure the endothelium. C_LI

cell biology↗

bioRxiv2022

Prevention of thrombocytopenia and thrombosis in heparin-induced thrombocytopenia (HIT) using deglycosylated KKO: A novel therapeutic?

Heparin-induced thrombocytopenia (HIT) is characterized by mild thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human (h) platelet factor 4 (PF4) complexed with various polyanions. While non-heparin anticoagulants and intravenous immunoglobulin (IVIG) are the mainstay of care, bleeding may develop, and risk of new thromboembolic events remain. We had described a mouse IgG{kappa}2b antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4:polyanion complexes. KKO, like HIT IgGs, activates platelets through Fc{gamma}RIIA and induces complement activation. We now asked whether Fc-modified KKO can be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). DGKKO bound to PF4-polyanion complexes, and blocked Fc{gamma}RIIA-dependent activation of PF4 treated platelets by KKO, 5B9 (another HIT-like monoclonal antibody), and isolated IgGs from HIT patients. DGKKO also decreased complement activation and deposition of C3c on platelets. Injection of DGKKO into "HIT mice" lacking mouse PF4, but transgenic for hPF4 and Fc{gamma}RIIA, prevented and reversed thrombocytopenia when injected before or after KKO, 5B9 or HIT IgG, respectively, in a microfluidic system. DGKKO reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis by IgG from a patient with the HIT-related disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT. Key PointsO_LIDeglycosylated (DG) KKO can reverse thrombocytopenia in a HIT murine model. C_LIO_LIDGKKO can prevent/reverse thrombosis in vitro and in a HIT murine model. C_LI

cell biology↗

bioRxiv2022

RUNX1-deficient human megakaryocytes demonstrate thrombopoietic and platelet half-life and functional defects in an in vivo murine model: Therapeutic implications

Heterozygous defects in runt-related transcription factor-1 (RUNX1) are causative of a familial platelet disorder with associated myeloid malignancy (FPDMM). Since RUNX1-deficient animal models do not mimic FPDMMs bleeding disorder or leukemic risk, establishment of a proper model system is critical to understand the underlying mechanisms of the observed phenotype and to identify therapeutic interventions. We previously reported an in vitro-megakaryopoiesis system using human CD34+-hematopoietic stem and progenitor cells that recapitulated the FPDMM quantitative megakaryocyte defect by decreasing RUNX1 expression using a lentiviral short-hairpin RNA (shRNA for RUNX1 or shRX) 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 non-targeting 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 species-binding specificity of the VWF from mouse to human glycoprotein Ib. A small-molecule inhibitor RepSox, which blocks the transforming-growth factor beta pathway, and which rescued defective megakaryopoiesis in vitro, corrected the thrombopoietic defect, platelet half-life and agonist response, and thrombus formation in NSG/VWFR1326H mice. Thus, this model recapitulates the defect in FPDMM megakaryocytes and platelets, identifies previously unrecognized defects in thrombopoiesis and platelet half-life, and demonstrates, for the first time, reversal of RUNX1 deficiencys hemostatic defects by a drug. Key PointsO_LIRUNX1-deficient megakaryocytes exhibit thrombopoietic and platelet defects in NSG/VWFR1326H mice. C_LIO_LIPre-exposure of RUNX1-deficient megakaryocytes to a TGF{beta}1-pathway inhibitor ameliorated both defects, correcting hemostasis. C_LI

cancer biology↗

bioRxiv2021

Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the Fc{gamma}RIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. Cover illustration O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY One-sentence summaryThe Fc{gamma}RIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19

immunology↗

bioRxiv2020

RUNX1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells: Mechanistic studies and drug correction

Patients with familial platelet disorder with a predisposition to myeloid malignancy (FPDMM) harbor germline monoallelic mutations in a key hematopoietic transcription factor RUNX1. Previous studies of FPDMM have focused on megakaryocyte (Mk) differentiation, and platelet production and signaling. However, the effects of RUNX1 haploinsufficiency on hematopoietic progenitor cells (HPCs) and subsequent megakaryopoiesis remains incomplete. To address this issue, we studied induced-pluripotent stem cell (iPSC)-derived HPCs (iHPCs) and Mks (iMks) from both patient-derived lines and a wildtype line modified to be RUNX1 haploinsufficient (RUNX1+/-), each compared to their isogenic wildtype control. All RUNX1+/- lines showed decreased iMk yield and depletion of a 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 FPDMM Mk-biased iHPCs. Analyses of gene sets upregulated in FPDMM iHPCs indicated enrichment for response to stress, regulation of signal transduction and response to cytokine gene sets. Immunoblotting studies 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 {beta}1 and cellular stressors. J-IN8 and RepSox, small drugs targeting these pathways, corrected quantitative defects in FPDMM iHPC production. These findings were confirmed in adult human CD34+-derived stem and progenitor cells transduced with lentiviral RUNX1 short-hairpin (sh) RNA to mimic RUNX1+/-. These mechanistic studies of the defect in megakaryopoiesis in FPDMM suggest druggable pathways for clinical management of thrombocytopenia in affected patients. Key pointsO_LIRUNX1 haploinsufficiency results in a deficiency of megakaryocyte-biased hematopoietic progenitor cells (HPCs). C_LIO_LIRUNX1 haploinsufficiency elevates druggable proinflammatory and TGF{beta}R1-related pathways in HPCs. C_LI

molecular biology↗

bioRxiv2019

Neutrophil extracellular trap stabilization leads to improved outcomes in murine models of sepsis

Sepsis is characterized by multi-organ system dysfunction that occurs due to infection. It is associated with unacceptably high morbidity and mortality and in need of improved therapeutic intervention. 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 likely have a significant NET burden contributing to the multi-organ damage. Therefore, interventions that prevent 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 therefore be of limited therapeutic benefit. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. Platelet factor 4 (PF4, CXCL4) a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to deoxyribonuclease I. A monoclonal antibody, KKO, which binds to PF4-NET complexes, further enhances this resistance. We now show that PF4 increases NET-mediated bacterial capture in vitro, reduces the release of NDPs, and improves outcome in murine models of sepsis. An Fc-modified KKO further enhances deoxyribonuclease resistance, decreases NDP release, and increases survival in these models, supporting a novel NET-targeting approach to improve outcomes in sepsis.

immunology↗

bioRxiv2018

Structure-guided design of a pure orthosteric antagonist of integrin αIIbβ3 that inhibits thrombosis but not clot retraction

Platelet integrin lIb{beta}3 plays a critical role in both hemostasis and thrombosis. Current IIb{beta}3 antagonists are potent anti-thrombotic drugs, but also cause adverse outcomes, which limited their clinical use. Drug-induced serious bleeding, thrombocytopenia and paradoxical thrombosis have been linked to impaired clot retraction and to conformational changes in IIb{beta}3 that promote binding of preformed antibodies, natural ligands or both to IIb{beta}3. We have used structure-guided design to generate the orthosteric inhibitor Hr10 that acts as a pure IIb{beta}3 antagonist, i.e. it does not induce the conformational changes in IIb{beta}3. Hr10 is as effective as the partial agonist drug eptifibatide in blocking platelet aggregation and arteriolar thrombosis in mice. In contrast to eptifibatide, however, Hr10 preserved thrombin-induced clot retraction, suggesting that it may not perturb hemostasis. Our structure-based approach can find general utility in designing pure orthosteric inhibitors for other integrins, in providing vital tools for dissecting structure-activity relationships in IIb{beta}3, and potentially in offering safer alternatives for human therapy.

cell biology↗