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Objectives Endothelial protein C receptor (EPCR) is highly expressed in synovial tissues of patients with rheumatoid arthritis (RA), but the function of this receptor remains unknown in RA. This study investigated the effect of EPCR on the onset and development of inflammatory arthritis and its underlying mechanisms. Methods Collagen-induced arthritis (CIA) was induced in EPCR gene knockout (KO) and matched wild type (WT) mice. The onset and development of arthritis was monitored clinically and histologically. T cells, dendritic cells (DCs), EPCR and cytokines from EPCRKO and WT mice, RA patients and healthy controls (HC) were detected by flow cytometry and ELISA. Results EPCRKO mice displayed >40% lower arthritis incidence and 50% less disease severity than WT. EPCRKO mice also had significantly less Th1/Th17 cells in synovial tissues with more DCs in circulation. Lymph nodes and synovial CD4 T cells from EPCRKO mice expressed less chemokine receptors CXCR3, CXCR5 and CCR6 than WT. In vitro, EPCRKO spleen cells contained less Th1 and more Th2 and Th17 cells than WT, and, in concordance, blocking EPCR in WT cells stimulated Th2 and Th17 cells. DCs generated from EPCRKO bone marrow were less mature and produced less matrix metalloproteinase-9. Circulating T cells from RA patients expressed higher levels of EPCR than HC cells; blocking EPCR stimulated Th2 and Treg cells in vitro. Conclusion Deficiency of EPCR ameliorates arthritis in CIA via inhibition of the activation and migration of pathogenic Th cells and DCs. Targeting EPCR may constitute a novel strategy for future RA treatment.

BACKGROUND: Rotational thromboelastometry (ROTEM) has been commonly used to assess the viscoelastic properties of the blood clotting process in the clinic for patients with a hemostatic or prothrombotic disorder. OBJECTIVE: To evaluate the capability of ROTEM in assessing hemostatic properties in whole blood from various mouse models with genetic bleeding or clotting disease and the effect of factor VIII (FVIII) therapeutics in FVIII(null) mice. METHODS: Mice with a genetic deficiency in either a coagulation factor or a platelet glycoprotein were used in this study. The properties of platelet- or plasma-FVIII were also assessed. Citrated blood from mice was recalcified and used for ROTEM analysis. RESULTS: We found that blood collected from the vena cava could generate reliable results from ROTEM analysis, but not blood collected from the tail vein, retro-orbital plexus, or submandibular vein. Age and sex did not significantly affect the hemostatic properties determined by ROTEM analysis. Clotting time (CT) and clot formation time (CFT) were significantly prolonged in FVIII(null) (5- and 9-fold, respectively) and FIX(null) (4- and 5.7-fold, respectively) mice compared to wild-type (WT)-C57BL/6J mice. Platelet glycoprotein (GP)IIIa(null) mice had significantly prolonged CFT (8.4-fold) compared to WT-C57BL/6J mice. CT and CFT in factor V (FV) Leiden mice were significantly shortened with an increased α-angle compared to WT-C57BL/6J mice. Using ROTEM analysis, we showed that FVIII expressed in platelets or infused into whole blood restored hemostasis of FVIII(null) mice in a dose-dependent manner. CONCLUSION: ROTEM is a reliable and sensitive assay for assessing therapeutics on hemostatic properties in mouse models with a bleeding or clotting disorder.

Protease-activated receptor 2 (PAR2) signaling controls skin barrier function and inflammation, but the roles of immune cells and PAR2-activating proteases in cutaneous diseases are poorly understood.To dissect PAR2 signaling contributions to skin inflammation with new genetic and pharmacological tools.We found markedly increased numbers of PAR2Myeloid cells and the TF-FXa-PAR2 axis are key mediators and potential therapeutic targets in inflammatory skin diseases.

Activation of blood coagulation and endothelial inflammation are hallmarks of respiratory infections with RNA viruses that contribute significantly to the morbidity and mortality of patients with severe disease. We investigated how signaling by coagulation proteases affects the quality and extent of the response to the TLR3-ligand poly(I:C) in human endothelial cells. Genome-wide RNA profiling documented additive and synergistic effects of thrombin and poly(I:C) on the expression level of many genes. The most significantly active genes exhibiting synergistic induction by costimulation with thrombin and poly(I:C) included the key mediators of 2 critical biological mechanisms known to promote endothelial thromboinflammatory functions: the initiation of blood coagulation by tissue factor and the control of leukocyte trafficking by the endothelial-leukocyte adhesion receptors E-selectin (gene symbol, SELE) and VCAM1, and the cytokines and chemokines CXCL8, IL-6, CXCL2, and CCL20. Mechanistic studies have indicated that synergistic costimulation with thrombin and poly(I:C) requires proteolytic activation of protease-activated receptor 1 (PAR1) by thrombin and transactivation of PAR2 by the PAR1-tethered ligand. Accordingly, a small-molecule PAR2 inhibitor suppressed poly(I:C)/thrombin–induced leukocyte-endothelial adhesion, cytokine production, and endothelial tissue factor expression. In summary, this study describes a positive feedback mechanism by which thrombin sustains and amplifies the prothrombotic and proinflammatory function of endothelial cells exposed to the viral RNA analogue, poly(I:C) via activation of PAR1/2.

Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that lead to the production of thrombin, including prothrombinase (factor Xa [FXa]/FVa), the catalytic complex that directly generates thrombin. Thus, TFPI and FV are directly linked in regulating the procoagulant response. Studies using knockout mice indicate that TFPI and FV are necessary for embryogenesis, but their contributions to vascular development are unclear. We performed extensive histological analyses of Tfpi−/− and Tfpi−/−F5−/− mouse embryos to investigate the importance of the interplay between TFPI and FV in regulating hemostasis and vascular development during embryogenesis. We observed normal tissue development throughout Tfpi−/− embryos, except in the central nervous system (CNS). The CNS displayed stunted brain growth, delayed development of the meninges, and severe vascular pathology characterized by the formation of glomeruloid bodies surrounding areas of cellular death, fibrin deposition, and hemorrhage. Removing FV from Tfpi−/− embryos completely ameliorated their brain pathology, suggesting that TFPI dampens FV-dependent procoagulant activity in a manner that modulates cerebrovascular development. Thus, we have identified a previously unrecognized role for TFPI activity within the CNS. This TFPI activity likely diminishes an effect of excess thrombin activity on signaling pathways that control cerebral vascular development.

Tissue factor (TF) is crucial for embryogenesis, as mice lacking TF are embryonically lethal (E10.5). This lethality may be attributed to defects in vascular development and circulatory failure, suggesting additional roles for TF in embryonic development beyond coagulation. In this study, we characterized the role of one of the TF paralogs (

Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willebrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. We developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167 ± 257 mU/mL), but the VWF was completely incapable of binding FVIII, resulting in 53 ± 23 mU/mL of plasma FVIII levels that were similar to those in VWF-deficient (VWF−/−) mice. When wild-type human or mouse VWF was infused into VWF2N/2N mice, endogenous plasma FVIII was restored, peaking at 4 to 6 hours post-infusion, demonstrating that FVIII expressed in VWF2N mice is viable but short-lived unprotected in plasma due to dysfunctional 2N VWF. The whole blood clotting time and thrombin generation were impaired in VWF2N/2N but not in VWF−/− mice. Bleeding time and blood loss in VWF2N/2N mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF2N/2N mice, but not VWF−/− mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that alternative pathways can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.

Unpublished data from our labs led us to hypothesize that activated protein C (aPC) may initiate an anti-inflammatory signal in endothelial cells by modulating both the integrin αVβ3 and protease-activated receptor 2 (PAR2), which may exist in close proximity on the cellular surface. To test this hypothesis and to probe the possible inflammation-related pathway, we designed and synthesized dual-targeting ligands composed of modified versions of two αVβ3 ligands and two agonists of PAR2. These novel ligands were connected via copper-catalyzed alkyne–azide cycloadditions with polyethylene glycol (PEG) spacers of variable length. Initial in vitro pharmacology with EA.hy926 and HUVEC endothelial cells indicated that these ligands are effective binders of αVβ3 and potent agonists of PAR2. These were also used in preliminary studies investigating their effects on PAR2 signaling in the presence of inflammatory agents, and represent the first examples of ligands targeting both PARs and integrins, though concurrent binding to αVβ3 and PAR2 has not yet been demonstrated.

Background Activation of protease-activated receptor 1 (PAR1) by either thrombin or activated protein C (aPC) differentially regulate the quiescence and bone marrow (BM) retention of hematopoietic stem cells (HSC). Murine HSC co-express THBD, PAR1, and endothelial protein C receptor (EPCR), suggesting that HSC sustain quiescence in a quasi-cell autonomous manner due to the binding of thrombin present in the microenvironment to THBD, activation of EPCR-bound protein C by the thrombin-THBD-complex, and subsequent activation of PAR1 by the aPC-EPCR complex. Objective To determine the role of THBD expression on HSC for sustaining stem cell quiescence and BM retention under homeostatic conditions. Methods Hematopoietic stem cell function was analyzed in mice with constitutive or temporally controlled complete THBD-deficiency by flow cytometry, functional assays, and single cell RNA profiling. Results THBD was expressed in mouse, but not human, HSC, progenitors, and immature B cells. Expression in vascular endothelium was conserved in humans' BM. Mice with constitutive THBD deficiency had a normal peripheral blood profile, altered BM morphology, reduced numbers of progenitors and immature B cells, pronounced extramedullary hematopoiesis, increased HSC frequency, and marginally altered transcriptionally defined HSC stemness. Transplantation experiments indicated near normal engraftment and repopulating ability of THBD-deficient HSC. Transgenic aPC supplementation normalized BM histopathology and HSC abundance, and partially restored transcriptional stemness, but had no effect on B cell progenitors and extramedullary hematopoiesis. Temporally controlled THBD gene ablation in adult mice did not cause the above abnormalities. Conclusion THBD expression on HSPC has minor effects on homeostatic hematopoiesis in mice, and is not conserved in humans.

Laminin-γ1 is required for early embryonic development; however, the need for laminin-γ1 synthesis in adulthood is unknown. A global and inducible mouse model of laminin-γ1 deficiency was generated to address this question. Genetic ablation of the Lamc1 gene in adult mice was rapidly lethal. Despite global Lamc1 gene deletion in tamoxifen-induced mutant mice, there was minimal change in total cardiac, pulmonary, hepatic or renal laminin protein. In contrast, laminin-γ1 was significantly depleted in the small intestines, which showed crypt hyperplasia and dissociation of villous epithelium from adjacent mesenchyme. We conclude that the physiologic requirement for laminin-γ1 synthesis in adult mice is dependent on a tissue-specific basal rate of laminin-γ1 turnover that results in rapid depletion of laminin-γ1 in the intestine.

Background Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species‐specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF. Objectives To develop a VWF knock out (VWF–/–) rat by excision of all 52 exons of the VWF locus. Methods The entire VWF gene was eliminated in Sprague‐Dawley (Crl:SD) rats via CRISPR/Cas9‐mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF–/– rats were infused with FVIII–/– rat platelet poor plasma (PPP) to determine response of plasma FVIII. Results Breeding of VWF ± rats yielded VWF–/– offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF–/– rats. VWF–/– rats bled longer and more than VWF+/– and VWF+/+ rats when challenged. Transfusion of FVIII‐deficient platelet‐poor plasma induced a rapid rise in endogenous FVIII:C in VWF–/– rats. Conclusion This rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.

Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM(−/−)) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM(−/−) cells that resulted in enhanced adhesion of leukocytes to TM(−/−) cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM(−/−) cells. In a flow chamber assay, isolated platelets adhered to TM(−/−) cells, forming characteristic VWF–platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TM(f/f) mice. Reexpression of the TM construct in TM(−/−) cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

Deficiencies in many coagulation factors and protease-activated receptors (PARs) affect embryonic development. We describe a defect in definitive erythropoiesis in PAR2-deficient mice. Embryonic PAR2 deficiency increases embryonic death associated with variably severe anemia in comparison with PAR2-expressing embryos. PAR2-deficient fetal livers display reduced macrophage densities, erythroblastic island areas, and messenger RNA expression levels of markers for erythropoiesis and macrophages. Coagulation factor synthesis in the liver coincides with expanding fetal liver hematopoiesis during midgestation, and embryonic factor VII (FVII) deficiency impairs liver macrophage development. Cleavage-insensitive PAR2-mutant mice recapitulate the hematopoiesis defect of PAR2-deficient embryos, and macrophage-expressed PAR2 directly supports erythroblastic island function and the differentiation of red blood cells in the fetal liver. Conditional deletion of PAR2 in macrophages impairs erythropoiesis, as well as increases inflammatory stress, as evidenced by upregulation of interferon-regulated hepcidin antimicrobial peptide. In contrast, postnatal macrophage PAR2 deficiency does not have any effect on steady-state Kupffer cells, bone marrow macrophage numbers, or erythropoiesis, but erythropoiesis in macrophages from PAR2-deficient mice is impaired following hemolysis. These data identify a novel function for macrophage PAR2 signaling in adapting to rapid increases in blood demand during gestational development and postnatal erythropoiesis under stress conditions.

The tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells such as hematopoietic stem and progenitor cells (HSPCs), megakaryocytes (MKs), and platelets. Individuals expressing somatic activating JAK2 mutations such as JAK2V617F commonly develop myeloproliferative neoplasms (MPNs) associated with serious complications, including venous and arterial thrombosis, a leading cause of mortality. Here, we investigated the role of JAK2 in hemostasis and thrombosis using Jak2fl/flPf4-Cre (Jak2Plt-/-) mice specifically lacking JAK2 within the platelet lineage. Jak2Plt-/- mice developed severe thrombocytosis with a 5-fold increase in circulating platelet number, MK hyperplasia, and splenomegaly. Jak2Plt-/-platelets were of normal size and the expression of major membrane surface glycoproteins was indistinguishable from controls, except for the integrin β3, which was reduced by 20%. Despite the thrombocytosis, Jak2Plt-/- mice had a severe bleeding diathesis, as evidenced by: 1) prolonged tail bleeding time; 2) failure to occlude in a ferric chloride-induced carotid artery injury model; and 3) failure to form stable thrombi in a laser-induced cremaster muscle injury model. Jak2Plt-/- platelets spread poorly on immobilized collagen or on immobilized fibrinogen following GPVI stimulation with the collagen-related peptide (CRP). Jak2Plt-/- platelets had defective α-granule secretion and integrin αIIbβ3 activation following stimulation with CRP, but not thrombin, and showed aggregation defects with low-doses of CRP. Together, the data support a GPVI-specific impairment in platelets lacking JAK2, a notion that was supported by impaired intracellular signaling following GPVI stimulation, as assessed by protein tyrosine phosphorylation. Jak2Plt-/- platelets adhered poorly to type I collagen under arterial shear rates in whole blood. However, JAK2 deletion in platelets did not alter plasma von Willebrand factor (VWF) levels or botrocetin-mediated binding of plasma VWF to GPIbα. Together, the results underline a critical role for JAK2 in platelet GPVI signaling and hemostatic function, which likely contributes to venous and arterial thrombosis observed in patients with MPNs with the activating JAK2V617F mutation. Disclosures No relevant conflicts of interest to declare.

Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a–specific antibodies. Humanizing the PSI domain of muGPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a–specific antibodies; however, humanizing distinct amino acids within the linearly distant but conformationally close EGF1 domain was required to enable binding of others. These results reveal the previously unsuspected complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti–HPA-1a reagents.

Obesity promotes a chronic inflammatory and hypercoagulable state that drives cardiovascular disease, type 2 diabetes, fatty liver disease, and several cancers. Elevated thrombin activity underlies obesity-linked thromboembolic events, but the mechanistic links between the thrombin/fibrin(ogen) axis and obesity-associated pathologies are incompletely understood. In this work, immunohistochemical studies identified extravascular fibrin deposits within white adipose tissue and liver as distinct features of mice fed a high-fat diet (HFD) as well as obese patients. Fibγ390–396A mice carrying a mutant form of fibrinogen incapable of binding leukocyte αMβ2-integrin were protected from HFD-induced weight gain and elevated adiposity. Fibγ390–396A mice had markedly diminished systemic, adipose, and hepatic inflammation with reduced macrophage counts within white adipose tissue, as well as near-complete protection from development of fatty liver disease and glucose dysmetabolism. Homozygous thrombomodulin-mutant ThbdPro mice, which have elevated thrombin procoagulant function, gained more weight and developed exacerbated fatty liver disease when fed a HFD compared with WT mice. In contrast, treatment with dabigatran, a direct thrombin inhibitor, limited HFD-induced obesity development and suppressed progression of sequelae in mice with established obesity. Collectively, these data provide proof of concept that targeting thrombin or fibrin(ogen) may limit pathologies in obese patients.

Unpublished data from our labs led us to hypothesize that activated Protein C (aPC) may initiate an anti-inflammatory signal in endothelial cells by modulating both the integrin αVβ3 and Protease-Activated Receptor 2 (PAR2), which may exist in close proximity on the cellular surface. To test this hypothesis and to probe the possible inflammation-related pathway, we designed and synthesized heterobivalent ligands composed of modified versions of two αVβ3 ligands and two agonists of PAR2. These novel ligands were connected via copper-catalyzed alkyne-azide cycloadditions with polyethylene glycol (PEG) spacers of variable length. Initial in vitro pharmacology with EA.hy926 and HUVEC endothelial cells indicated that these bivalent ligands are effective binders of αVβ3 and potent agonists of PAR2. These bivalent ligands were also used in preliminary studies investigating their effects on PAR2 signaling in the presence of inflammatory agents, and represent the first examples of ligands targeting both PARs and integrins.

Immune evasion in the tumor microenvironment (TME) is a crucial barrier for effective cancer therapy, and plasticity of innate immune cells may contribute to failures of targeted immunotherapies. Here, we show that rivaroxaban, a direct inhibitor of activated coagulation factor X (FX), promotes antitumor immunity by enhancing infiltration of dendritic cells and cytotoxic T cells at the tumor site. Profiling FX expression in the TME identifies monocytes and macrophages as crucial sources of extravascular FX. By generating mice with immune cells lacking the ability to produce FX, we show that myeloid cell-derived FX plays a pivotal role in promoting tumor immune evasion. In mouse models of cancer, we report that the efficacy of rivaroxaban is comparable with anti-programmed cell death ligand 1 (PD-L1) therapy and that rivaroxaban synergizes with anti-PD-L1 in improving antitumor immunity. Mechanistically, we demonstrate that FXa promotes immune evasion by signaling through protease-activated receptor 2 and that rivaroxaban specifically targets this cell-autonomous signaling pathway to reprogram tumor-associated macrophages. Collectively, our results have uncovered the importance of FX produced in the TME as a regulator of immune cell activation and suggest translational potential of direct oral anticoagulants to remove persisting roadblocks for immunotherapy and provide extravascular benefits in other diseases.

Gene therapy offers the potential to cure hemophilia A (HA). We have shown that hematopoietic stem cell (HSC)–based platelet-specific factor VIII (FVIII) (2bF8) gene therapy can produce therapeutic protein and induce antigen-specific immune tolerance in HA mice, even in the presence of inhibitory antibodies. For HSC-based gene therapy, traditional preconditioning using cytotoxic chemotherapy or total body irradiation (TBI) has been required. The potential toxicity associated with TBI or chemotherapy is a deterrent that may prevent patients with HA, a nonmalignant disease, from agreeing to such a protocol. Here, we describe targeted nongenotoxic preconditioning for 2bF8 gene therapy utilizing a hematopoietic cell–specific antibody-drug conjugate (ADC), which consists of saporin conjugated to CD45.2- and CD117-targeting antibodies. We found that a combination of CD45.2- and CD117-targeting ADC preconditioning was effective for engrafting 2bF8-transduced HSCs and was favorable for platelet lineage reconstitution. Two thirds of HA mice that received 2bF8 lentivirus-transduced HSCs under (CD45.2+CD117)-targeting ADC conditioning maintained sustained therapeutic levels of platelet FVIII expression. When CD8-targeting ADC was supplemented, chimerism and platelet FVIII expression were significantly increased, with long-term sustained platelet FVIII expression in all primary and secondary recipients. Importantly, immune tolerance was induced and hemostasis was restored in a tail-bleeding test, and joint bleeding also was effectively prevented in a needle-induced knee joint injury model in HA mice after 2bF8 gene therapy. In summary, we show for the first time efficient engraftment of gene-modified HSCs without genotoxic conditioning. The combined cocktail ADC-mediated hematopoietic cell–targeted nongenotoxic preconditioning that we developed is highly effective and favorable for platelet-specific gene therapy in HA mice.

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications.

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective negative allosteric modulator of PAR1 that inhibits human platelet aggregation. It showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications.

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart, small intestine and dorsal skin were collected for tissue analysis. Female APCHi mice showed the most severe radiation-induced deposition of cardiac collagens but were protected against a radiation-induced loss of microvascular density. Both male and female APCHi mice were protected against a radiation induced upregulation of toll-like receptor 4 in the heart, but this did not translate into a clear protection against immune cell infiltration. In the small intestine, the APCHi genotype had no effect on an increase in the number of myeloperoxidase positive cells (seen mostly in females) or an increase in the expression of T-cell marker CD2 (males). Lastly, both male and female APCHi mice were protected against radiation-induced epidermal thickening and increase in 3-nitrotyrosine positive keratinocytes. In conclusion, prolonged high levels of APC in a transgenic mouse model had little effects on indicators of DEARE in the heart, small intestine and skin, with some differential effects in male compared to female mice.

Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive. SummaryBackground Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin–antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet–leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of platelet-expressed FVIII than are required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection.

Nogo-B receptor (NgBR) was identified as a specific receptor for binding Nogo-B and is essential for the stability of Niemann-Pick type C2 protein (NPC2) and NPC2-dependent cholesterol trafficking. Here, we report that NgBR expression levels decrease in the fatty liver and that NgBR plays previously unrecognized roles in regulating hepatic lipogenesis through NPC2-independent pathways. To further elucidate the pathophysiological role of NgBR in mammals, we generated NgBR liver-specific knockout mice and investigated the roles of NgBR in hepatic lipid homeostasis. The results showed that NgBR knockout in mouse liver did not decrease NPC2 levels or increase NPC2-dependent intracellular cholesterol levels. However, NgBR deficiency still resulted in remarkable cellular lipid accumulation that was associated with increased free fatty acids and triglycerides in hepatocytes in vitro and in mouse livers in vivo. Mechanistically, NgBR deficiency specifically promotes the nuclear translocation of the liver X receptor alpha (LXRα) and increases the expression of LXRα-targeted lipogenic genes. LXRα knockout attenuates the accumulation of free fatty acids and triglycerides caused by NgBR deficiency. In addition, we elucidated the mechanisms by which NgBR bridges the adenosine monophosphate–activated protein kinase alpha signaling pathway with LXRα nuclear translocation and LXRα-mediated lipogenesis. Conclusion: NgBR is a specific negative regulator for LXRα-dependent hepatic lipogenesis. Loss of NgBR may be a potential trigger for inducing hepatic steatosis. (Hepatology 2016;64:1559-1576)

Blood cell production is regulated by peripheral nerve fibers that innervate the bone marrow. However, little is known about the development or maintenance of hematopoietic innervation. Schwann cells (SCs) are the primary axon 'support cells' of the peripheral nervous system (PNS), and abnormal SC development is sufficient to impair peripheral nerve function. SCs are also the primary repair cell for the PNS which makes them an attractive therapeutic target for normalization of drug or malignancy-induced 'hematopoietic neuropathy'. We hypothesized that neural regulation of hematopoiesis is dependent on SC development. To test this hypothesis, we used the Myelin Protein Zero-Cre (MP0-Cre); Lamc1fl/fl mouse line in which laminin-γ1 expression is deleted from SC precursors and their progeny1. Early SC maturation is dependent on autocrine SC precursor-derived molecules such as laminin-γ1. SC differentiation arrests prior to axon sorting and ensheathment in MP0-Cre; Lamc1fl/fl mice, and causes a global peripheral neuropathy that persists throughout the lifetime of the animal. Preliminary hematopoietic analysis of 'steady state' MP0-Cre; Lamc1fl/fl and littermate control mice has shown the following: (1) MP0-Cre; Lamc1fl/fl bone marrow is innervated, and Cre-mediated gene recombination occurs in cells immunophenotypically consistent with SCs throughout the peripheral nervous system, including those in the bone marrow; (2) MP0-Cre; Lamc1fl/fl mice are lymphopenic but not neutropenic; (3) MP0-Cre; Lamc1fl/fl mice have significantly reduced spleen size and cellularity; and (4) MP0-Cre; Lamc1fl/fl bone marrow has an ~50% reduction in Lin-Sca-1+Kit+(LSK) cells (measured as a percentage of the Lin- compartment of the bone marrow). These results are consistent with earlier work by our groups in which we found that global Lamc1 gene deletion in adult mice induced peripheral blood lymphopenia, reduced spleen size, and a niche-dependent reduction of lymphoid progenitor and precursor cells that was secondary to increased lymphoid precursor cell apoptosis and reduced proliferation (UBC-CreERT2; Lamc1fl/fl mouse line). As with the SC-specific laminin-γ1 deficient mice, myelopoiesis was preserved in the UBC-CreERT2; Lamc1fl/fl mice. Based on results from MP0-Cre; Lamc1fl/fl and UBC-CreERT2; Lamc1fl/fl mice, we conclude that early lymphoid but not myeloid development requires laminin-γ1 expression by MP0-Cre-targetted niche cells, i.e. Schwann Cells. Our results are consistent with reports from other labs that hematopoietic sympathetic neuropathy promotes aberrant myeloid expansion at the expense of lymphopoiesis2. Going forward, we will determine whether lymphopoietic development is dependent on global versus laminin-specific SC-derived cues, and whether these signals are transmitted directly between SCs and lymphoid biased HSPCs or indirectly via other components of the hematopoietic niche. We anticipate that this line of investigation will provide molecular insights and pharmacologic targets for prevention and or normalization of the 'hematopoietic neuropathy' induced by diabetes, aging, neurotoxic chemotherapies and myeloid malignancies. REFERENCES: 1 Yu, W. M., Feltri, M. L., Wrabetz, L., Strickland, S. & Chen, Z. L. Schwann cell-specific ablation of laminin gamma1 causes apoptosis and prevents proliferation. J Neurosci25, 4463-4472, doi:10.1523/JNEUROSCI.5032-04.2005 (2005). 2 Maryanovich, M. et al. Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche. Nat Med24, 782-791, doi:10.1038/s41591-018-0030-x (2018). Disclosures No relevant conflicts of interest to declare.

Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as Pl

Essentials Defective binding to collagen IV has been seen in von Willebrand factor (VWF) A1 domain variants. We developed a murine model of defective VWF-collagen IV interactions with VWF variant p.R1399H. p.1399HH homozygous mice had decreased binding to collagen IV and increased bleeding times. p.1399HH homozygous mice had increased time to thrombosis and decreased platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) binding to type IV collagen occurs via the VWF A1 domain, with p.R1399H being the most common VWF variant affecting this interaction. Objectives We generated a murine model of 1399H VWF to investigate its in vivo effects. Methods Mice expressing the murine 1399H variant were generated via gene targeting in embryonic stem cells. VWF antigen and VWF collagen binding were measured with ELISA. Tail bleeding time assays were performed by clipping a 3-mm segment. Ferric chloride-induced thrombosis was measured via ultrasound in the carotid artery. Platelet aggregation in response to collagens I and IV was measured. VWF-dependent platelet adhesion to collagen IV was measured under flow. Results Breeding of heterozygous p.R1399H and homozygous p.1399HH mice was observed to follow normal Mendelian ratios. No spontaneous bleeding was observed for any of the offspring. VWF expression was normal, but VWF binding to collagen IV was decreased in both heterozygous and homozygous offspring. Blood loss following tail resection was increased for p.1399HH mice, and occlusion times following ferric chloride-induced thrombosis were prolonged. Platelet aggregation was unaffected, but platelet adhesion to collagen IV under flow was diminished for p.1399HH mice. Conclusions These results show that a decrease in the ability of 1399H VWF to bind collagen IV under static conditions corresponds to a decrease in binding under flow conditions, an increased bleeding time, and a prolonged time to thrombosis. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagen IV binding.

Coagulation activation accompanied by reduced anticoagulant activity is a key characteristic of patients with idiopathic pulmonary fibrosis (IPF). Although the importance of coagulation activation in IPF is well studied, the potential relevance of endogenous anticoagulant activity in IPF progression remains elusive. We assess the importance of the endogenous anticoagulant protein C pathway on disease progression during bleomycin‐induced pulmonary fibrosis. Wild‐type mice and mice with high endogenous activated protein C APC levels (APC high) were subjected to bleomycin‐induced pulmonary fibrosis. Fibrosis was assesses by hydroxyproline and histochemical analysis. Macrophage recruitment was assessed immunohistochemically. In vitro, macrophage migration was analysed by transwell migration assays. Fourteen days after bleomycin instillation, APC high mice developed pulmonary fibrosis to a similar degree as wild‐type mice. Interestingly, Aschcroft scores as well as lung hydroxyproline levels were significantly lower in APC high mice than in wild‐type mice on day 28. The reduction in fibrosis in APC high mice was accompanied by reduced macrophage numbers in their lungs and subsequent in vitro experiments showed that APC inhibits thrombin‐dependent macrophage migration. Our data suggest that high endogenous APC levels inhibit the progression of bleomycin‐induced pulmonary fibrosis and that APC modifies pulmonary fibrosis by limiting thrombin‐dependent macrophage recruitment.

The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.

Postsurgical peritoneal adhesion bands are the most important causes of intestinal obstruction, pelvic pain, and female infertility. In this study, we used a mouse model of adhesion and compared the protective effect of activated protein C (APC) to that of the Food and Drug Administration-approved antiadhesion agent, sodium hyaluronate/carboxymethylcellulose (Seprafilm) by intraperitoneal administration of either APC or Seprafilm to experimental animals. Pathological adhesion bands were graded on day 7, and peritoneal fluid concentrations of tissue plasminogen activator (tPA), d-dimer, thrombin-antithrombin complex, and cytokines (IL-1β, IL-6, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1) were evaluated. Inflammation scores were also measured based on histologic data obtained from peritoneal tissues. Relative to Seprafilm, intraperitoneal administration of human APC led to significantly higher reduction of postsurgical adhesion bands. Moreover, a markedly lower inflammation score was obtained in the adhesive tissues of the APC-treated group, which correlated with significantly reduced peritoneal concentrations of proinflammatory cytokines and an elevated tPA level. Further studies using variants of human APC with or without protease-activated receptor 1 (PAR1) signaling function and mutant mice deficient for either endothelial protein C receptor (EPCR) or PAR1 revealed that the EPCR-dependent signaling activity of APC is primarily responsible for its protective activity in this model. These results suggest APC has therapeutic potential for preventing postsurgical adhesion bands.

Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd(-/-)) mice to term. Half of the Thbd(-/-) offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd(-/-) animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd(-/-) mice. However, Thbd(-/-) females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy

Platelets are a rich source of many cytokines and chemokines including transforming growth factor β 1 (TGF-β1). TGF-β1 is required to convert conventional CD4+ T (Tconv) cells into induced regulatory T (iTreg) cells that express the transcription factor Foxp3. Whether platelet contents will affect Treg cell properties has not been explored. In this study, we show that unfractionated platelet lysates (pltLys) containing TGF-β1 efficiently induced Foxp3 expression in Tconv cells. The common Treg cell surface phenotype and in vitro suppressive activity of unfractionated pltLys-iTreg cells were similar to those of iTreg cells generated using purified TGF-β1 (purTGFβ-iTreg) cells. However, there were substantial differences in gene expression between pltLys-iTreg and purTGFβ-iTreg cells, especially in granzyme B, interferon γ, and interleukin-2 (a 30.99-, 29.18-, and 17.94-fold difference, respectively) as determined by gene microarray analysis. In line with these gene signatures, we found that pltLys-iTreg cells improved cell recovery after transfer and immune suppressive function compared with purTGFβ-iTreg cells in factor VIII (FVIII)-deficient (F8null, hemophilia A model) mice after recombinant human FVIII (rhF8) infusion. Acute antibody-mediated platelet destruction in F8null mice followed by rhF8 infusion increased the number of Treg cells and suppressed the antibody response to rhF8. Consistent with these data, ex vivo proliferation of F8-specific Treg cells from platelet-depleted animals increased when restimulated with rhF8. Together, our data suggest that pltLys-iTreg cells may have advantages in emerging clinical applications and that platelet contents impact the properties of iTreg cells induced by TGF-β1.

Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)-activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.

HSCs lacking the guanosine nucleotide-binding protein Gimap5, which stabilizes expression of the Mcl-1 Bcl2 family protein, exhibit impaired survival and long-term repopulation capacity., Mice and rats lacking the guanosine nucleotide-binding protein Gimap5 exhibit peripheral T cell lymphopenia, and Gimap5 can bind to Bcl-2. We show that Gimap5-deficient mice showed progressive multilineage failure of bone marrow and hematopoiesis. Compared with wild-type counterparts, Gimap5-deficient mice contained more hematopoietic stem cells (HSCs) but fewer lineage-committed hematopoietic progenitors. The reduction of progenitors and differentiated cells in Gimap5-deficient mice resulted in a loss of HSC quiescence. Gimap5-deficient HSCs and progenitors underwent more apoptosis and exhibited defective long-term repopulation capacity. Absence of Gimap5 disrupted interaction between Mcl-1—which is essential for HSC survival—and HSC70, enhanced Mcl-1 degradation, and compromised mitochondrial integrity in progenitor cells. Thus, Gimap5 is an important stabilizer of mouse hematopoietic progenitor cell survival.