Identification of genomic loci regulating platelet plasminogen activator inhibitor-1 in mice.

Background: Plasminogen activator inhibitor-1 (PAI-1, Serpine1) is an important circulating fibrinolysis inhibitor. PAI-1 exists in 2 pools, packaged within platelet α-granules and freely circulating in plasma. Elevated plasma PAI-1 levels are associated with cardiovascular disease.

Genetic analysis of human RNA binding motif protein 48 (RBM48) reveals an essential role in U12-type intron splicing.

U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for the evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals.

Intratumoural haematopoietic stem and progenitor cell differentiation into M2 macrophages facilitates the regrowth of solid tumours after radiation therapy.

Background: Bone-marrow-derived haematopoietic stem and progenitor cells (HSPCs) are a prominent part of the highly complex tumour microenvironment (TME) where they localise within tumours and maintain haematopoietic potency. Understanding the role HSPCs play in tumour growth and response to radiation therapy (RT) may lead to improved patient treatments and outcomes.

Methods: We used a mouse model of non-small cell lung carcinoma where tumours were exposed to RT regimens alone or in combination with GW2580, a pharmacological inhibitor of colony stimulating factor (CSF)-1 receptor.

Deficiency of plasminogen activator inhibitor-2 results in accelerated tumor growth.

Background: Upregulation of the plasminogen activation system, including urokinase plasminogen activator (uPA), has been observed in many malignancies, suggesting that co-opting the PA system is a common method by which tumor cells accomplish extracellular matrix proteolysis. PAI-2, a serine protease inhibitor, produced from the SERPINB2 gene, inhibits circulating and extracellular matrix-tethered uPA. Decreased SERPINB2 expression has been associated with increased tumor invasiveness and metastasis for several types of cancer.

Tissue factor pathway inhibitor is required for cerebrovascular development in mice.

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.

A coagulation defect arising from heterozygous premature termination of tissue factor.

Tissue factor (TF) is the primary initiator of blood coagulation in vivo and the only blood coagulation factor for which a human genetic defect has not been described. As there are no routine clinical assays that capture the contribution of endogenous TF to coagulation initiation, the extent to which reduced TF activity contributes to unexplained bleeding is unknown. Using whole genome sequencing, we identified a heterozygous frameshift variant (p.

Assessing Blood Clotting and Coagulation Factors in Mice.

The mammalian blood coagulation system was designed to restrict blood loss due to injury as well as keep the blood fluid within the blood vessels of the organism. Blood coagulation activity in inbred mouse strains varies widely among strains, suggesting that many genomic variants affect hemostasis. Some of these molecules have been discovered and characterized; however, many are still unknown.

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse.

Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice ('rescues') using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci.

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

Factor V Leiden ( ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized -ethyl--nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for ( ) and haploinsufficient for tissue factor pathway inhibitor ( ). deficiency enhanced the survival of mice, demonstrating that lethality is genetically suppressible.

Spontaneous 8bp Deletion in Nbeal2 Recapitulates the Gray Platelet Syndrome in Mice.

During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp) deletion causing a frameshift in exon 27 of the Nbeal2 gene was identified. Though initially considered as a plausible thrombosis modifier, this Nbeal2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules.

Spontaneous Irs1 passenger mutation linked to a gene-targeted SerpinB2 allele.

In characterizing mice with targeted disruption of the SerpinB2 gene, we observed animals that were small at birth with delayed growth and decreased life expectancy. Although this phenotype cosegregated with homozygosity for the inactive SerpinB2 allele, analysis of homozygous SerpinB2-deficient mice derived from two additional independent embryonic stem (ES) cell clones exhibited no growth abnormalities. Examination of additional progeny from the original SerpinB2-deficient line revealed recombination between the small phenotype (smla) and the SerpinB2 locus.

The endothelial-specific regulatory mutation, Mvwf1, is a common mouse founder allele.

Mvwf1 is a cis-regulatory mutation previously identified in the RIIIS/J mouse strain that causes a unique tissue-specific switch in the expression of an N-acetylgalactosaminyltransferase, B4GALNT2, from intestinal epithelium to vascular endothelium. Vascular B4galnt2 expression results in aberrant glycosylation of von Willebrand Factor (VWF) and accelerated VWF clearance from plasma. We now report that 13 inbred mouse strains share the Mvwf1 tissue-specific switch and low VWF phenotype, including five wild-derived strains.

Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice.

The signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)), likely functions in multiple signaling pathways. Here, we report the characterization of a mouse mutant lacking Vac14, a regulator of PI(3,5)P(2) synthesis. The mutant mice exhibit massive neurodegeneration, particularly in the midbrain and in peripheral sensory neurons.

Plasminogen activator inhibitor-1 in vascular thrombosis.

Thrombotic complications of vascular disease constitute the leading cause of morbidity and mortality in much of the developed world. Current drug therapies available to treat the thrombotic component of arterial and venous vascular complications remain limited. Novel safe and effective treatment strategies to reduce formation of occlusive thrombosis will likely have a major impact on reducing the economic burden of vascular disease on the healthcare system.

Heme oxygenase-1 deficiency accelerates formation of arterial thrombosis through oxidative damage to the endothelium, which is rescued by inhaled carbon monoxide.

Heme oxygenase (HO)-1 (encoded by Hmox1) catalyzes the oxidative degradation of heme to biliverdin and carbon monoxide. HO-1 is induced during inflammation and oxidative stress to protect tissues from oxidative damage. Because intravascular thrombosis forms at sites of tissue inflammation, we hypothesized that HO-1 protects against arterial thrombosis during oxidant stress.

Murine models of vascular thrombosis (Eitzman series).

Thrombotic complications of vascular disease are the leading cause of morbidity and mortality in most industrialized countries. Despite this, safe and effective drugs targeting these complications are limited, especially in the chronic setting. This is because of the complexity of thrombosis in both arteries and veins, which is becoming increasingly evident as numerous factors are now known to affect the fate of a forming thrombus.

Fetal gene defects precipitate platelet-mediated pregnancy failure in factor V Leiden mothers.

We describe a mouse model of fetal loss in factor V Leiden (FvL) mothers in which fetal loss is triggered when the maternal prothrombotic state coincides with fetal gene defects that reduce activation of the protein C anticoagulant pathway within the placenta. Fetal loss is caused by disruption of placental morphogenesis at the stage of labyrinth layer formation and occurs in the absence of overt placental thrombosis, infarction, or perfusion defects. Platelet depletion or elimination of protease-activated receptor 4 (Par4) from the mother allows normal placentation and prevents fetal loss.

Signaling pathways and genes that inhibit pathogen-induced macrophage apoptosis--CREB and NF-kappaB as key regulators.

Certain microbes evade host innate immunity by killing activated macrophages with the help of virulence factors that target prosurvival pathways. For instance, infection of macrophages with the TLR4-activating bacterium Bacillus anthracis triggers an apoptotic response due to inhibition of p38 MAP kinase activation by the bacterial-produced lethal toxin. Other pathogens induce macrophage apoptosis by preventing activation of NF-kappaB, which depends on IkappaB kinase beta (IKKbeta).

Homozygosity for factor V Leiden leads to enhanced thrombosis and atherosclerosis in mice.

Background: Activated protein C resistance due to factor V Leiden (FVL) is a common genetic risk factor for venous thrombosis in humans. Although the impact of FVL on the development of venous thrombosis is well established, its effect on arterial thrombosis and atherosclerosis is controversial.

Methods And Results: To determine the effect of the FVL mutation on arterial thrombosis in the mouse, wild-type (Fv+/+), heterozygous FVL (FvQ/+), and homozygous FVL (FvQ/Q) mice underwent photochemical carotid arterial injury to induce occlusive thrombosis.

Lethal perinatal thrombosis in mice resulting from the interaction of tissue factor pathway inhibitor deficiency and factor V Leiden.

Background: Factor V Leiden (FVL) is a common genetic risk factor for thrombosis in humans. The incomplete penetrance of FVL suggests important contributions from other genetic or environmental modifying factors. Variation in the expression of tissue factor pathway inhibitor (TFPI) has also been proposed as a risk factor for venous thrombosis and has been shown to enhance the prothrombotic effect of FVL in vitro.

Effect of leptin on arterial thrombosis following vascular injury in mice.

Context: Complications of atherosclerosis are the leading cause of morbidity and mortality in industrialized societies. Obesity has emerged as an independent risk factor for complications of atherosclerotic vascular disease. Leptin, a hormone produced by the adipocyte, increases with obesity and appears to modulate energy balance and food intake.

Heparin cofactor II inhibits arterial thrombosis after endothelial injury.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate, heparan sulfate, or heparin. HCII has been proposed to regulate coagulation or to participate in processes such as inflammation, atherosclerosis, and wound repair. To investigate the physiologic function of HCII, about 2 kb of the mouse HCII gene, encoding the N-terminal half of the protein, was deleted by homologous recombination in embryonic stem cells.