Search

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.

Unpublished data from our labs led us to hypothesize that activated protein C (aPC) may initiate an anti-inflammatory signal in endothelial cells by modulating both the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.

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.

Previous studies have shown that platelet-specific factor VIII (FVIII) expression (2bF8) restores hemostasis and induces immune tolerance in hemophilia A (HA) mice even with preexisting inhibitors. Here we investigated for the first time whether platelet FVIII expression can prevent severe spontaneous bleeding in rat HA, a model mimicking the frequent spontaneous bleeding in patients with severe HA. A novel FVIII(−/−) rat model in a Dahl inbred background (Dahl-FVIII(−/−)) with nearly the entire rat FVIII gene inverted was created by using a CRISPR/Cas9 strategy. There was no detectable FVIII in plasma. Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 100% of FVIII(−/−) rats. Sixty-one percent developed anti-FVIII inhibitors after ≥2 doses of recombinant human FVIII infusion. However, when 2bF8 transgene was crossed into the FVIII(−/−) background, none of the resulting 2bF8(tg+)FVIII(−/−) rats (with platelet FVIII levels of 28.26 ± 7.69 mU/10(8) platelets and undetectable plasma FVIII) ever had spontaneous bleeding. When 2bF8(tg) bone marrow (BM) was transplanted into FVIII(−/−) rats, only 1 of 7 recipients had a bruise at the early stage of BM reconstitution, but no other spontaneous bleeding was observed during the study period. To confirm that the bleeding diathesis in FVIII(−/−) rats was ameliorated after platelet FVIII expression, rotational thromboelastometry and whole-blood thrombin generation assay were performed. All parameters in 2bF8(tg) BM transplantation recipients were significantly improved compared with FVIII(−/−) control rats. Of note, neither detectable levels of plasma FVIII nor anti-FVIII inhibitors were detected in 2bF8(tg) BM transplantation recipients. Thus, platelet-specific FVIII expression can efficiently prevent severe spontaneous bleeding in FVIII(−/−) rats with no anti-FVIII antibody development.

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.

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.

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.

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.

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.

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

In this study, we have identified a novel member of the AMPK family, namely Sucrose non-fermenting related kinase (Snrk), that is responsible for maintaining cardiac metabolism in mammals. SNRK is expressed in the heart, and brain, and in cell types such as endothelial cells, smooth muscle cells and cardiomyocytes (CMs). Snrk knockout (KO) mice display enlarged hearts, and die at postnatal day 0. Microarray analysis of embryonic day 17.5 Snrk hearts, and blood profile of neonates display defect in lipid metabolic pathways. SNRK knockdown CMs showed altered phospho-acetyl-coA carboxylase and phospho-AMPK levels similar to global and endothelial conditional KO mouse. Finally, adult cardiac conditional KO mouse displays severe cardiac functional defects and lethality. Our results suggest that Snrk is essential for maintaining cardiac metabolic homeostasis, and shows an autonomous role for SNRK during mammalian development.

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.

The calcium-calmodulin-regulated protein phosphatase calcineurin plays an important regulatory role in muscle differentiation, fiber-type determination, hypertrophy, and muscle regeneration. Because calcineurin functions in numerous processes in muscle, multiple mechanisms are likely necessary to ensure that the activity of this phosphatase is appropriately regulated. Here we demonstrate that the muscle-specific scaffolding protein myospryn modulates calcineurin signaling by inhibiting calcineurin-dependent transcriptional activity in C2C12 myoblasts through direct interaction with the enzyme via its noncanonical tripartite motif (TRIM-like). Consistent with these data, transgenic mice overexpressing both the TRIM-like domain of myospryn and constitutively active calcineurin displayed a severe attenuation in the ability of calcineurin to induce a slow-fiber phenotype. Furthermore, transgenic mice overexpressing the TRIM-like domain of myospryn displayed attenuated muscle regeneration after cardiotoxin-induced muscle injury. These results indicate that myospryn functions as a novel inhibitor of the calcineurin signaling pathway in skeletal muscle.—Kielbasa, O. M., Reynolds, J. G., Wu, C-l., Snyder, C. M., Cho, M. Y., Weiler, H., Kandarian, S., Naya, F. J. Myospryn is a calcineurin-interacting protein that negatively modulates slow-fiber-type transformation and skeletal muscle regeneration.

Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TMPro mice expressing a mutant form of TM with reduced thrombin affinity and TMLeD mice lacking the N-terminal lectin-like domain. Studies of TMPro mice revealed that TM is a powerful determinant of hematogenous metastasis. TMPro mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TMPro mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.

The inflammation-induced activation of the protein C pathway provides negative feedback inhibition of coagulation and exerts coagulation-independent anti-inflammatory and cytoprotective effects. The balance between these activities of aPC modulates the outcome of diverse inflammatory diseases such as encephalitis, diabetes, and sepsis; and is affected by naturally occurring aPC-resistance of coagulation factor V Leiden.

Alloantibodies to platelet-specific antigens are responsible for two clinically-important bleeding disorders: Post-transfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The 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 isoforms harboring select humanized residues within the PSI and EGF1 domain, and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a-specific antibodies. Humanizing the PSI domain of murine GPIIIa 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. Using a series of transgenic mice and recombinant constructs, we reveal 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 by providing cells that distinguish specific alloantibody subpopulations, and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti-HPA-1a reagents. Disclosures Curtis: Ionis Pharmaceuticals: Consultancy.

Von Willebrand Disease (VWD) is caused by either an inherited deficiency of von Willebrand factor (VWF) protein or synthesis of a dysfunctional von Willebrand factor (VWF) that is referred to as a type 2 variant. One of the important functions of VWF is to serve as a carrier protein for FVIII - prolonging FVIII's plasma half-life from We selected 8 different human type 2N mutations, and mutated murine VWF at these positions and expressed the mutant VWF in HEK293T cells in vitro. Binding of each of the 2N mutants of mouse VWF to both murine and human FVIII was tested. The G785E 2N mutation had the most severe defect with negligible binding to both human (rhF8) and mouse FVIII (mF8) and was therefore chosen for further study. Using CRISPR/Cas9 gene editing, we generated two lines of VWF2N mice (termed VWF2N1/2N1 and VWF2N2/2N2) each with a 2N causative 2354G>A (G785E) mutation on the C57BL6 background. Plasma VWF levels were measured by ELISA and FVIII was measured by chromogenic assay. VWF binding to FVIII was determined by FVIII binding to monoclonal captured VWF or by VWF binding to monoclonal captured FVIII. For the remainder of this abstract, we will only discuss the VWF2N1/2N1 mice and refer to them as wt VWF+/+, heterozygous VWF2N/+ or homozygous VWF2N/2N mice. The plasma VWF in VWF+/+, VWD2N/+ or VWF2N/2N mice was 109±31, 100±27, and 115±29 U/dL. The plasma FVIII levels in homozygous VWF2N/2N mice were markedly reduced to 4.9±2.2 U/dL compared to either wt (84±21U/dL) or heterozygous (82±18U/dL) controls. VWF binding to rhF8 was reduced to To assess the clinical phenotype of VWF2N/2N mice, tail bleeding times were determined demonstrating prolongation to 3.1±0.4 hrs in VWF2N/2N mice in contrast to 2.4±0.4 in VWF2N/+ and 2.1±1.0 hrs in wt controls. ROTEM assessment of whole blood clotting time (wbCT) and whole blood thrombin generation assays (wbTGA) were performed. The wbCT was 10.7±1.1 min in VWF2N/2N mice compared to 8.3±0.5 min in VWF2N/+ heterozygotes. In wbTGA, the Lag Time and Peak Time in the VWF2N/2N group were 18.53±5.57 and 34.15±8.37 min, respectively, which were significantly longer than those obtained in the VWF2N/+ group (8.3±0.5 and 17.83±2.33 min, respectively). Peak Thrombin, Endogenous Thrombin Potential, and Thrombin Generation rate in the VWF2N/2N group were significantly lower than those obtained in the VWF2N/+ group. In human type 2N VWD, FVIII synthesis is normal but plasma levels are reduced because of increased FVIII clearance in the absence of VWF binding. Infusion of rhVWF (no FVIII) into the VWF2N/2N mice demonstrated rhVWF peaking by 30 minutes with a rescue of the endogenous mouse FVIII (44±1.7 U/dL) at 4-6 hrs post infusion. This demonstrates improved FVIII survival in the presence of normal VWF. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in Type 2N patients. Plasma levels of VWF are normal in our VWF2N/2N mice. This VWF is incapable of binding FVIII at neutral pH, but is otherwise fully functional. This is a unique model of 2N VWD that can be used to investigate the biological properties of VWF/FVIII association. Disclosures Montgomery: BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

The natural cell type(s) that synthesize and release factor VIII (FVIII) into the circulation are still not known with certainty. In vitro studies indicate that artificial expression of FVIII in endothelial cells produces an intracellular pool of FVIII that can be mobilized together with its carrier protein, von Willebrand factor (VWF), by agonists. Here, we show that expression of human B-domain deleted FVIII (hFVIII) in the vascular endothelium of otherwise FVIII-deficient mice results in costorage of FVIII and VWF in endothelial Weibel-Palade bodies and restores normal levels and activity of FVIII in plasma. Stored FVIII was mobilized into the circulation by subcutaneous administration of epinephrine. Human FVIII activity in plasma was strictly dependent on the presence of VWF. Endothelial-specific expression of hFVIII rescued the bleeding diathesis of hemophilic mice lacking endogenous FVIII. This hemostatic function of endothelial cell–derived hFVIII was suppressed in the presence of anti-FVIII inhibitory antibodies. These results suggest that targeting FVIII expression to endothelial cells may establish a releasable pool of FVIII and normalize plasma FVIII level and activity in hemophilia A, but does not prevent the inhibitory effect of anti-FVIII antibodies on the hemostatic function of transgene-derived hFVIII as is seen with platelet-derived FVIII expression.

Disseminated intravascular coagulation in sepsis is associated with microvascular thrombosis and organ dysfunction. It was expected that prothrombotic disposition such as factor V Leiden (FVL) mutation would worsen clinical outcome. Astonishingly, clinical trial and animal experimental data indicate that FVL can be associated with improved survival. This study investigated the effect of FVL on the response to endotoxin of the coagulation and fibrinolytic system in humans. Fourteen healthy male subjects without FVL and 15 healthy males with heterozygous FVL received an intravenous bolus dose of endotoxin, 2 ng/kg of body weight. Blood samples were drawn before and 1, 2, 4, 6, and 24 hours after administration of the endotoxin. Injection of endotoxin led to a more pronounced increase in soluble fibrin in patients with FVL than in controls. Patients with FVL displayed a more sustained increase in plasmin-plasmin inhibitor complex after 4, 6, and 24 hours. Patients with FVL mutation also displayed higher levels of D-dimer and fibrinogen-fibrin degradation products in plasma after 24 hours. Patients with FVL generate higher levels of soluble fibrin, which may serve as cofactor in tissue plasminogen activator–induced plasminogen activation, leading to a more sustained activation of fibrinolysis with production of more fibrinogen- and fibrin-degradation products.

The protein C (PC) pathway is an important anticoagulant mechanism that prevents thrombosis during the systemic inflammatory response. Thrombomodulin (TM), an endothelial cell membrane receptor, accelerates the conversion of PC to activated protein C (APC), which leads to the down-regulation of thrombin production and fibrin formation. Induction of acute endotoxemia in young and aged mice with a low dose of bacterial endotoxin lipopolysaccharide (LPS, 2.5 mg/kg) caused a high mortality rate in aged (80%) but not young (0%) mice. After injection with this dose of LPS, fibrin formation was significantly elevated only in aged mice, plasma APC levels were increased only in young mice, and TM expression was profoundly depressed in the aged. The increased thrombosis, suppressed APC level, and decreased TM expression were not observed in young mice receiving a higher dose of LPS (20 mg/kg), which resulted in a mortality rate (78%) equivalent to that seen in aged mice with the low-dose LPS. Mutant mice with reduced TM showed significantly less plasma APC and increased fibrin formation compared with wild-type mice after LPS. These results demonstrate that PC pathway activation is suppressed with aging and is partly responsible for age-associated thrombosis and high mortality during endotoxemia.

Background—Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis.Methods and Results—De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TMPro/ProApoE−/−and FVLQ/QApoE−/−mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-Cβ, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells.Conclusions—Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-Cβ, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis.

Activated protein C (aPC) is the key effector protease of the natural protein C anticoagulant pathway and exerts anticoagulant, as well as anti-inflammatory activity. This dual mode of action has been thought to underlie the therapeutic efficacy of recombinant aPC in the treatment of patients suffering from severe forms of sepsis. The development and characterization of recombinant variants of aPC with altered bioactivity profiles has generated an opportunity to test this concept by dissecting the roles of aPC's anticoagulant and cell-signaling functions in the treatment of sepsis. Animal studies suggest that aPC variants with near-normal signaling function, but with greatly diminished anticoagulant potential may exhibit a substantially improved risk-to-benefit ratio in sepsis therapy.

The loss of Gimap5 (GTPase of the immune-associated protein 5) gene function is the underlying cause of lymphopenia and autoimmune diabetes in the BioBreeding (BB) rat. The in vivo function of murine gimap5 is largely unknown. We show that selective gene ablation of the mouse gimap5 gene impairs the final intrathymic maturation of CD8 and CD4 T cells and compromises the survival of postthymic CD4 and CD8 cells, replicating findings in the BB rat model. In addition, gimap5 deficiency imposes a block of natural killer (NK)- and NKT-cell differentiation. Development of NK/NKT cells is restored on transfer of gimap5−/− bone marrow into a wild-type environment. Mice lacking gimap5 have a median survival of 15 weeks, exhibit chronic hepatic hematopoiesis, and in later stages show pronounced hepatocyte apoptosis, leading to liver failure. This pathology persists in a Rag2-deficient background in the absence of mature B, T, or NK cells and cannot be adoptively transferred by transplanting gimap5−/− bone marrow into wild-type recipients. We conclude that mouse gimap5 is necessary for the survival of peripheral T cells, NK/NKT-cell development, and the maintenance of normal liver function. These functions involve cell-intrinsic as well as cell-extrinsic mechanisms.

To discuss the potential use of recombinant activated protein C (aPC) variants with altered bioactivity in sepsis therapy.Since the initial Protein C Worldwide Evaluation in Severe Sepsis trial demonstrating efficacy of aPC therapy to reduce mortality of severe sepsis, follow-up studies have failed to resolve concerns about the low overall risk-to-benefit ratio of this therapy and suggest that it might only be effective in severely ill patients with the most aggravated forms of coagulopathy. New studies begin to shed light on the potential mechanisms of how aPC therapy may alter sepsis outcome, and how recombinant aPC variants with altered bioactivities may improve the efficacy and safety of this therapy.aPC variants with selectively diminished antithrombotic activity, but normal cytoprotective potential, may allow more efficient dosing without increasing adverse bleeding effects and therefore provide a safer and possibly more efficient alternative to normal aPC. Critical questions about the precise mechanisms by which aPC therapy reduces mortality remain to be resolved in order to identify patients most likely to benefit from it and to reevaluate potential efficacy of aPC therapy in children and patients with less than severe sepsis.

Absence of the blood coagulation inhibitor thrombomodulin (Thbd) from trophoblast cells of the mouse placenta causes a fatal arrest of placental morphogenesis. The pathogenesis of placental failure requires tissue factor, yet is not associated with increased thrombosis and persists in the absence of fibrinogen. Here, we examine the role of alternative targets of coagulation that might contribute to the placental failure and death of Thbd−/− embryos. We demonstrate that genetic deficiency of the protease-activated receptors, Par1 or Par2, in the embryo and trophoblast cells does not prevent the death of Thbd−/− embryos. Similarly, genetic ablation of the complement pathway or of maternal immune cell function does not decrease fetal loss. In contrast, Par4 deficiency of the mother, or the absence of maternal platelets, restores normal development in one-third of Thbd-null embryos. This finding generates new evidence implicating increased procoagulant activity and thrombin generation in the demise of thrombomodulin-null embryos, and suggests that platelets play a more prominent role in placental malfunction associated with the absence of thrombomodulin than fibrin formation. Our findings demonstrate that fetal prothrombotic mutations can cause localized activation of maternal platelets at the feto-maternal interface in a mother with normal hemostatic function.

Summary. Background and Objective: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are endothelial-associated anticoagulant proteins thought to control hemostasis in specific vascular beds. Here, we have examined the consequences of TFPI deficiency in the presence of a compounding procoagulant state caused by reduced TM function. Methods and results: TFPI+/−/TMpro/pro mice are born at less than expected frequency in either TFPI+/−/TMpro/+ or TMpro/pro mothers but are born at near the expected frequency in TMpro/+ mothers. Adult TFPI+/−/TMpro/pro mice have elevated thrombin–antithrombin complex and increased thrombus volume in an electrical injury model of venous thrombosis. In striking contrast to mice with single deficiency of TFPI or TM, TFPI+/−/TMpro/pro mice exhibit augmented fibrin deposition not only in the liver, but also in the cerebral microvasculature. Conclusions: TFPI+/−/TMpro/pro mice exhibit partial intrauterine lethality when carried by mothers with an underlying prothrombotic state, providing the first experimental evidence in an animal model that TFPI-dependent control of hemostasis in the vascular bed of the placenta fulfills a critical role for successful pregnancy outcome. In addition to the placenta, partial TFPI deficiency interacts with decreased TM function in an organ selective manner to produce fibrin deposition in other specific vascular beds, the liver and brain.

Nogo-B receptor (NgBR) was identified as a receptor specific for Nogo-B. Our previous work has shown that Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and intersomitic vessel formation via Akt pathway in zebrafish. Here, we further demonstrated the roles of NgBR in regulating vasculature development in mouse embryo and primitive blood vessel formation in embryoid body culture systems, respectively. Our results showed that NgBR homozygous knockout mice are embryonically lethal at E7.5 or earlier, and Tie2Cre-mediated endothelial cell-specific NgBR knockout (NgBR ecKO) mice die at E11.5 and have severe blood vessel assembly defects in embryo. In addition, mutant embryos exhibit dilation of cerebral blood vessel, resulting in thin-walled endothelial caverns. The similar vascular defects also were detected in Cdh5(PAC)-CreERT2 NgBR inducible ecKO mice. Murine NgBR gene-targeting embryonic stem cells (ESC) were generated by homologous recombination approaches. Homozygous knockout of NgBR in ESC results in cell apoptosis. Heterozygous knockout of NgBR does not affect ESC cell survival, but reduces the formation and branching of primitive blood vessels in embryoid body culture systems. Mechanistically, NgBR knockdown not only decreases both Nogo-B and VEGF-stimulated endothelial cell migration by abolishing Akt phosphorylation, but also decreases the expression of CCM1 and CCM2 proteins. Furthermore, we performed immunofluorescence (IF) staining of NgBR in human cerebral cavernous malformation patient tissue sections. The quantitative analysis results showed that NgBR expression levels in CD31 positive endothelial cells is significantly decreased in patient tissue sections. These results suggest that NgBR may be one of important genes coordinating the cerebral vasculature development.

Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi(-/-)) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi(+/-) mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4(-/-)), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi(-/-) embryos, but >40% of expected Tfpi(-/-):Par4(-/-) offspring survived to adulthood. Adult Tfpi(-/-):Par4(-/-) mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi(-/-):Par4(-/-) mice have platelet and fibrin accumulation similar to Par4(-/-) mice following venous electrolytic injury but were more susceptible than Par4(-/-) mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi(-/-):Par4(-/-) mice were born with short tails. Tfpi(-/-):Par4(-/-) mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation.

Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A. We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain-deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific alphaIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8trans mice, the bleeding phenotype of FVIIInull mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII-immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII.

The full repertoire of proteins that comprise the striated muscle Z-disc and peripheral structures, such as the costamere, have yet to be discovered. Recent studies suggest that this elaborate protein network, which acts as a structural and signaling center for striated muscle, harbors factors that function as mechanosensors to ensure coordinated contractile activity. Mutations in genes whose products reside in this region often result in skeletal and cardio myopathies, demonstrating the importance of this macromolecular complex in muscle structure and function. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the costamere in striated muscle. This gene, called myospryn, was identified by microarray analysis as a transcript down-regulated in MEF2A knock-out mice. MEF2A knock-out mice develop cardiac failure during the perinatal period with mutant hearts exhibiting several cardiac abnormalities including myofibrillar disarray. Myospryn is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy-associated gene 5 (CMYA5). Myospryn is expressed as a single, large transcript of approximately 12 kilobases in adult heart and skeletal muscle with an open reading frame of 3739 amino acids. This protein, belonging to the tripartite motif superfamily of proteins, contains a B-box coiled-coil (BBC), two fibronectin type III (FN3) repeats, and SPRY domains and interacts with the sarcomeric Z-disc protein, alpha-actinin-2. Our findings demonstrate that myospryn functions directly downstream of MEF2A at the costamere in striated muscle potentially playing a role in myofibrillogenesis.

Objective: The high prevalence of the factor V Leiden mutation in certain populations has prompted speculation that the mutation may have been subject to positive selection during evolution, either by providing a survival benefit or by directly enhancing reproductive performance. We investigated the hypothesis that heterozygous factor V Leiden carrier status might protect against the lethal consequences of severe inflammatory disease. Data Source: Two mouse models (thrombomodulin-deficient TMPro mice and factor V Leiden mice), in which the endogenous protein C anticoagulant pathway is disrupted either at the level of protein C activation (TMPro mice) or at the level of factor V proteolysis by activated protein C (factor V Leiden mice), were employed. The mutant mouse strains were subjected to lethal doses of bacterial lipopolysaccharide. The effects of these two mutations on coagulation activation and inflammatory cytokine elaboration were observed and compared with those in wild-type mice. Data Summary: As has already been shown, heterozygous factor V Leiden carrier status improves the survival of mice subjected to endotoxemia induced by bacterial lipopolysaccharide. The survival of homozygous factor V Leiden mice did not differ from that of normal mice. The survival benefit derived from heterozygous factor V Leiden carrier status was only evident at doses of lipopolysaccharide producing death in approximately 50% of wild-type animals. At higher (LD 90 ) or lower (LD 10 ) doses of lipopolysaccharide, the survival of heterozygous factor V Leiden mice did not differ from that of wild-type mice. Concomitant administration of an LD 90 dose of lipopolysaccharide and therapeutic heparin abolished the relative survival advantage of heterozygous factor V Leiden mice. Analysis of systemic coagulation and cytokine variables failed to provide conclusive evidence for altered coagulation activation or inflammatory cytokine production as the basis for the survival advantage associated with heterozygous factor V Leiden carrier status. Conclusions: The improved survival of mice heterozygous for the factor V Leiden mutation complements results from the analysis of the factor V Leiden subgroup of patients enrolled in the PROWESS trial. Such convergent findings in two different species strongly suggest that the factor V Leiden mutation is indeed a potent modifier of the response to severe inflammatory disease. The striking magnitude of the factor V Leiden survival benefit in the initial PROWESS population, and in mice, suggests that the as-yet unknown mechanism conferring this benefit is a rather potent endogenous modifier of the pathogenic pathways engaged in sepsis. Delineation of this pathway will be important for understanding the therapeutic mechanisms, or absence thereof, of agents designed to act at the interface of coagulation and inflammation.

Elevated plasma levels of fibrinogen are associated with the presence of cardiovascular disease, but it is controversial whether elevated fibrinogen causally imparts an increased risk, and as such is a true modifier of cardiovascular disease, or is merely associated with disease. By investigating a transgenic mouse model of hyperfibrinogenemia, we show that elevated plasma fibrinogen concentration (1) elicits augmented fibrin deposition in specific organs, (2) interacts with an independent modifier of hemostatic activity to regulate fibrin turnover/deposition, (3) exacerbates neointimal hyperplasia in an experimental model of stasis-induced vascular remodeling, yet (4) may suppress thrombin generation in response to a procoagulant challenge. These findings provide direct experimental evidence that hyperfibrinogenemia is more than a by-product of cardiovascular disease and may function independently or interactively to modulate the severity and/or progression of vascular disease.

Sepsis is associated with systemic inflammation, coagulopathy, and disrupted protein C (PC) pathway function. The effect of prothrombotic polymorphism, factor V Leiden (Arg506Gln; FV Leiden), was examined in a large clinical trial (PROWESS) of severe sepsis and a mouse endotoxemia model. In PROWESS, 4.1% (n = 65) of patients were heterozygous FV Leiden (VL+/–) carriers. The 28-day mortality was lower in VL+/– (13.9%) than in non-FV Leiden (VL–/–; 27.9%) patients (P = .013). The mortality benefit of recombinant human activated PC (rhAPC) treatment was similar in VL+/– (placebo, 15.6%; rhAPC,12.1%) and VL–/– patients (placebo, 31.0%; rhAPC, 24.7%; interaction P = .981). VL+/– status did not appear to influence baseline biomarkers of coagulopathy and inflammation or disease severity, with the exception that vasopressor usage was less in VL+/– patients (46.2% versus 63.0%; P = .009). In a median lethal dose (40 mg/kg) endotoxin mouse model, VL+/– mice had lower mortality than wild-type mice (19% versus 57%; P = .008), whereas the mortality of homozygous (VL+/+) mice was almost identical to that of wild-type mice (65% versus 57%; P = .76). The findings suggest that FV Leiden constitutes a rare example of a balanced gene polymorphism that maintains the FV Leiden mutation in the general gene pool due to a survival advantage of VL+/– in severe sepsis.

Genetic or acquired thrombophilia of the pregnant mother has been associated with the occurrence of gestational vascular disease and recurrent fetal loss and may contribute to the aetiology of pre-eclampsia. This chapter reviews insights into this link between thrombophilia and pregnancy complications that were gained from the study of genetically altered mice. These studies strongly support the notion of a cause-effect relationship between altered function of the thrombomodulin-protein C pathway and adverse pregnancy outcome. Analysis of the mouse models highlights unique aspects of vascular structure and function at the feto-maternal interface, and exposes new biological functions of natural anticoagulant pathways in pregnancy. These roles are unrelated to the maintenance of vascular patency and may be mediated through specific signalling pathways activated by coagulation factors. Abnormal signalling by placental trophoblasts at the feto-maternal interface is suggested as a hitherto unrecognized mechanism that may underlie adverse pregnancy outcome associated with haemostatic disorders.

The thrombin mutant W215A/E217A (WE thrombin) has greatly reduced procoagulant activity, but it activates protein C in the presence of thrombomodulin and inhibits binding of platelet glycoprotein Ib to von Willebrand factor and collagen under flow conditions. Both thrombomodulin-dependent protein C activation and inhibition of platelet adhesion could contribute to the antithrombotic activity of WE thrombin.To assess the role of thrombomodulin, we administered WE thrombin to thrombomodulin-deficient (TM(Pro/Pro)) mice and measured the time to occlusive thrombus formation in the carotid artery after photochemical injury of the endothelium.Doses of WE thrombin ≥10μg/kg prolonged the thrombosis time of wild-type mice (1.6-fold), while doses ≥100μg/kg only slightly prolonged the thrombosis time of TM(Pro/Pro) mice. We conclude that thrombomodulin plays a predominate role in mediating the antithrombotic effect of WE thrombin in the arterial circulation of mice after endothelial injury. Thrombomodulin-independent effects may occur only when high doses of WE thrombin are administered.

Defining regulatory elements governing cell-restricted gene expression can be difficult because cis-elements may reside tens of kilobases away from start site(s) of transcription. Artificial chromosomes, which harbor hundreds of kilobases of genomic DNA, preserve a large sequence landscape containing most, if not all, regulatory elements controlling the expression of a particular gene. Here, we report on the use of a bacterial artificial chromosome (BAC) to begin understanding the in vivo regulation of smooth muscle calponin (SM-Calp). Long and accurate polymerase chain reaction, sequencing, and in silico analyses facilitated the complete sequence annotation of a BAC harboring human SM-Calp (hSM-Calp). RNase protection, in situ hybridization, Western blotting, and immunohistochemistry assays showed the BAC clone faithfully expressed hSM-Calp in both cultured cells and transgenic mice. Moreover, expression of hSM-Calp mirrored that of endogenous mouse SM-Calp suggesting that all cis-regulatory elements governing hSM-Calp expression in vivo were contained within the BAC. These BAC mice represent a new model system in which to systematically assess regulatory elements governing SM-Calp transcription in vivo.

Thrombomodulin (TM) is a vascular endothelial cell (EC) receptor that is a cofactor for thrombin-mediated activation of the anticoagulant protein C. The extracellular NH2-terminal domain of TM has homology to C-type lectins that are involved in immune regulation. Using transgenic mice that lack this structure (TMLeD/LeD), we show that the lectin-like domain of TM interferes with polymorphonuclear leukocyte (PMN) adhesion to ECs by intercellular adhesion molecule 1–dependent and –independent pathways through the suppression of extracellular signal–regulated kinase (ERK)1/2 activation. TMLeD/LeD mice have reduced survival after endotoxin exposure, accumulate more PMNs in their lungs, and develop larger infarcts after myocardial ischemia/reperfusion. The recombinant lectin-like domain of TM suppresses PMN adhesion to ECs, diminishes cytokine-induced increase in nuclear factor κB and activation of ERK1/2, and rescues ECs from serum starvation, findings that may explain why plasma levels of soluble TM are inversely correlated with cardiovascular disease. These data suggest that TM has antiinflammatory properties in addition to its role in coagulation and fibrinolysis.

Summary Background The high allelic frequency of the prothrombotic Leiden polymorphism in human blood coagulation factor V (FV) has been speculated to reflect positive selection during evolution. Heterozygous Leiden carriers enrolled in the placebo arm of the PROWESS sepsis trial and heterozygous Leiden mice challenged with endotoxin both showed reduced mortality, whereas homozygous Leiden mice were not protected from lethal endotoxemia. Follow-up analyses of clinical outcomes and of mouse models of infection with various pathogens remained inconclusive. Objective To establish whether activated protein C resistance of FV Leiden modifies the outcome of bacterial infection in murine sepsis models. Methods Homozygous and heterozygous FV Leiden mice were subjected to gram-positive (S. aureus) or gram-negative (Y. pestis; E. coli) septic peritonitis or polymicrobial, focal septic peritonitis induced by cecal ligation and puncture. The effect of FV Leiden on 7-day survival and bacterial dissemination was assessed. Outcomes were compared with the sepsis survival of mice with genetically impaired hemostasis (hemophilia A, thrombocytopenia, thrombin receptor PAR4 [protease activated receptor 4] deficiency, endothelial protein C receptor [ProcR/EPCR] deficiency). Results Heterozygous, but not homozygous, Leiden mice were protected from lethal infection with highly virulent S. aureus and Y. pestis strains. FV Leiden did not affect the outcome of sepsis induced by cecal ligation and puncture, staphylokinase-deficient S. aureus, Pla-deficient Y. pestis, or E. coli. Thrombocytopenia, deficiency of PAR1 or PAR4 did not affect S. aureus sepsis survival, whereas hemophilia A increased mortality. ProcR deficiency selectively abolished the survival advantage of heterozygous Leiden mice. Conclusions In mice, heterozygous FV Leiden carriers are protected from sepsis mortality after infection with clinically relevant human bacterial pathogens.

Systemic administration of recombinant thrombomodulin (TM) confers radiation protection partly by accelerating hematopoietic recovery. The uniquely potent radioprotector gamma tocotrienol (GT3), in addition to being a strong antioxidant, inhibits the enzyme hydroxy-methyl-glutaryl-coenzyme A reductase (HMGCR) and thereby likely modulates the expression of TM. We hypothesized that the mechanism underlying the exceptional radioprotective properties of GT3 partly depends on the presence of endothelial TM. In vitro studies confirmed that ionizing radiation suppresses endothelial TM (about 40% at 4 hr after 5 Gy γ-irradiation) and that GT3 induces TM expression (about 2 fold at the mRNA level after 5 μM GT3 treatment for 4 hr). In vivo survival studies showed that GT3 was significantly more effective as a radioprotector in TM wild type (TM+/+) mice than in mice with low TM function (TMPro/-). After exposure to 9 Gy TBI, GT3 pre-treatment conferred 85% survival in TM+/+ mice compared to only 50% in TMPro/-. Thus, GT3-mediated radiation lethality protection is partly dependent on endothelial TM. Significant post-TBI recovery of hematopoietic cells, particularly leukocytes, was observed in TM+/+ mice (p = 0.003), but not in TMPro/- mice, despite the fact that GT3 induced higher levels of granulocyte colony stimulating factor (G-CSF) in TMPro/- mice (p = 0.0001). These data demonstrate a critical, G-CSF-independent, role for endothelial TM in GT3-mediated lethality protection and hematopoietic recovery after exposure to TBI and may point to new strategies to enhance the efficacy of current medical countermeasures in radiological/nuclear emergencies.

The cellular source of coagulation factor VIII (FVIII) remains controversial. Like many coagulation proteins, FVIII is produced in the liver, and FVIII synthesis has long been associated with hepatocytes. But extrahepatic synthesis also occurs, and mounting evidence suggests that hepatocytes are not responsible for FVIII production. To determine the tissue that synthesizes FVIII, we developed a Cre/lox-dependent conditional knockout (KO) model in which exons 17 and 18 of the murine factor VIII gene (F8) are flanked by loxP sites, or floxed (F8(F)). In cells expressing Cre-recombinase, the floxed sequence is deleted, resulting in F8(F→KO) gene inactivation. When F8(F) mice were crossed with various tissue-specific Cre strains, we found that hepatocyte-specific F8-KO mice are indistinguishable from controls, whereas efficient endothelial-KO models display a severe hemophilic phenotype with no detectable plasma FVIII activity. A hematopoietic Cre model was more equivocal, so experimental bone marrow transplantation was used to examine hematopoietic FVIII synthesis. FVIII(null) mice that received bone marrow transplants from wild-type donors were still devoid of plasma FVIII activity after hematopoietic donor cell engraftment. Our results indicate that endothelial cells are the predominant, and possibly exclusive, source of plasma FVIII.