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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. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome, predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA binding motif protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5′ end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron-containing genes have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of minor intron-containing genes disrupted in both species. Mutations in the majority of these orthologous minor intron-containing genes have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.

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

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. RT-PCR, western blotting and immunohistochemistry were used to quantify expression levels of factors that affect HSPC differentiation. DsRed(+) HSPC intratumoural activity was tracked using flow cytometry and confocal microscopy. RESULTS: We demonstrated that CSF-1 is enhanced in the TME following exposure to RT. CSF-1 signaling induced intratumoural HSPC differentiation into M2 polarised tumour-associated macrophages (TAMs), aiding in post-RT tumour survival and regrowth. In contrast, hyperfractionated/pulsed radiation therapy (PRT) and GW2580 ablated this process resulting in improved tumour killing and mouse survival. CONCLUSIONS: Tumours coopt intratumoural HSPC fate determination via CSF-1 signaling to overcome the effects of RT. Thus, limiting intratumoural HSPC activity represents an attractive strategy for improving the clinical treatment of solid tumours.

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. PAI-2 deficiency has not been reported in humans and PAI-2 deficient (SerpinB2(−/−)) mice exhibit no apparent abnormalities. OBJECTIVES: We investigated the role of PAI-2 deficiency on tumor growth and metastasis. METHODS: To explore the long-term impact of PAI-2 deficiency, a cohort of SerpinB2(−/−) mice were aged to >18 months, with spontaneous malignancies observed in 4/9 animals, all of apparently vascular origin. To further investigate the role of PAI-2 deficiency in malignancy, SerpinB2(−/−) and wild type control mice were injected with either B16 melanoma or Lewis lung carcinoma tumor cells, with markedly accelerated tumor growth observed in SerpinB2(−/−) mice for both cell lines. To determine the relative contributions of PAI-2 from hematopoietic or non-hematopoietically derived sources, bone marrow transplants between wildtype C57BL/6J and SerpinB2(−/−) mice were performed. RESULTS AND CONCLUSIONS: Our results suggest that PAI-2 deficiency increases susceptibility to spontaneous tumorigenesis in the mouse, and demonstrate that SerpinB2 expression derived from a non-hematopoietic compartment is a key host factor in the regulation of tumor growth in both the B16 melanoma and Lewis Lung carcinoma models.

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 evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome (MDS), predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA Binding Motif Protein48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5’ end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron containing genes (MIGs) have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of MIGs disrupted in both species. Mutations in the majority of these orthologous MIGs have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.

Humans carrying the factor V Leiden (FVL) variant have a fivefold increased risk for venous thrombosis. However, incidence of deep vein thrombosis (DVT) is proportionally greater than that of pulmonary embolism (PE) in these individuals. This is known as the FVL paradox. We hypothesized that the rate of initial DVT development is similar in FVL and noncarriers, but thrombi in FVL carriers are more stable and develop into a clinically significant DVT more often than in noncarriers. To test this, we induced thrombi in the femoral vein of wild-type (WT), heterozygous (F5(L/+)), and FVL homozygous (F5(L/L)) mice. Using intravital microscopy, thrombus size and embolization were visualized and emboli in the lungs were quantified. Compared with WT, femoral vein thrombi in F5(L/+) and F5(L/L) mice were larger and embolized less. Total and large embolic events, the percentage of thrombus that embolized, and PE burden were significantly decreased in F5(L/L) mice. This suggests that in noncarriers (reflected by WT), a minor injury initially resulting in a small DVT tends to remain small and asymptomatic because of the embolization of the otherwise growing thrombus. Alternatively, the same insult in people with FVL (reflected by F5(L/L)) leads to thrombus growth as a result of less embolization, and thus symptomatic DVT development.

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.Ser117HisfsTer10) in F3, the gene encoding TF, causing premature termination of TF ("TFshort") in a woman with unexplained bleeding. Routine hematological laboratory evaluation of the proposita was normal. CRISPR-edited human induced pluripotent stem cells recapitulating the variant were differentiated into vascular smooth muscle and endothelial cells that demonstrated haploinsufficiency of TF. The variant F3 transcript is eliminated by nonsense-mediated decay. Neither overexpression nor addition of exogenous recombinant TFshort inhibited factor Xa or thrombin generation, excluding a dominant negative mechanism. F3+/- mice provide an animal model of TF haploinsufficiency and exhibited prolonged bleeding times, impaired thrombus formation, and reduced survival following major injury. Heterozygous TF deficiency is present in at least 1 in 25,000 individuals and could limit coagulation initiation in undiagnosed individuals with abnormal bleeding but a normal routine laboratory evaluation.

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. Genetically modified mouse technologies are providing a plethora of new mouse models for investigating the regulation of blood coagulation. Here we provide a protocol for the tail bleeding time as a primary assessment of in vivo blood coagulation, as well as in vitro methods such as the prothrombin time, activated partial thromboplastin time, and thrombin generation assay. We also provide protocols for the assessment of the activities of specific known factors involved in blood coagulation. © 2019 by John Wiley & Sons, Inc.

Background: Pregnancy is a major risk factor for venous thromboembolism (VTE) and is the leading cause of maternal death during pregnancy and the immediate post-partum period. Although plasma levels of clotting factors and fibrinolysis inhibitors are known to increase during pregnancy peaking during the third trimester as parturition nears, little is known about whether platelets are affected in response to pregnancy. Platelets are derived from megakaryocytes and have a circulating half-life of 5-10 days. They are essential for blood coagulation, delivering procoagulant molecules directly to sites of vascular injury. Platelets contain a spliceosome that processes pre-mRNA, providing them with the necessary machinery to respond to external stimuli. Thus, platelets can be affected by diseases such as cancer, which can alter platelet mRNA profiles and function. The plasticity of platelets in these contexts suggests that they could also change in response to pregnancy as an adaptation to preventing blood loss during parturition. However, little is known about the transcriptional and/or functional changes that could occur in platelets during the course of pregnancy. Aims: We aimed to analyze mouse platelet function at early, middle, late gestation, and postpartum timepoints in order to identify pregnancy related functional changes. Methods: C57BL/6J female mice were mated with C57BL/6J male mice to generate pregnant mice. Pregnancy was confirmed by the presence of a vaginal plug, which was used to estimate embryonic day 0.5. Pregnant mice were randomly assigned to one of four groups based on gestational stages corresponding to human trimesters and postpartum. The stages are early (days 7-9), middle (days 11-14), late (days 16-20), and postpartum (days 22-24). The maximum volume of mouse whole blood was drawn into a 1:9 volume of buffered sodium citrate via cardiac puncture. A 20ul aliquot was used for complete blood counts using the Advia 2120 with settings optimized for C57BL/6 mouse blood. The remaining blood was reserved for platelet aggregation studies using the Roche Multiplate Aggregometer. ADP and type 1 collagen were used as the aggregating agents with the operator blinded to the gestational groups. Studies were initiated at the same time of day in order to control for the circadian rhythm and the gestational age. Results: The maximum blood volume we were able to draw from each mouse was significantly increased for all groups (early, middle and late gestational stages and postpartum) compared to the non-pregnant control group (p ≤ 0.03, n ≥ 9). Platelet counts (per microliter of blood) were statistically unchanged among the control and the three gestational groups. However, postpartum platelet counts were significantly increased relative to all groups (control, early, middle and late gestational stages) (p ≤ 0.02, n ≥ 9). There was a more vigorous platelet aggregation response in the late gestational group compared to the non-pregnant control and early gestational groups (areas under the curve with ADP (p ≤ 0.004, n ≥ 7) and collagen (p ≤ 0.028, n ≥ 7). Conclusions and future directions: Our results demonstrate significant changes in blood volume and a dramatic increase in postpartum platelet counts. Our results are consistent with those observed in pregnant humans, indicating that platelet counts have a similar response to pregnancy in both humans and mice. Our results demonstrating a late gestational increase in platelet reactivity suggest that pregnancy induced platelet alterations could play a role in the increased risk of VTE during pregnancy and the immediate post-partum period. We are currently performing additional analyses including RNA sequencing, to determine the mechanisms responsible for the phenotypic changes that we have observed here. The results of our studies could lead to enhanced treatment strategies for pregnancy associated VTE. Disclosures No relevant conflicts of interest to declare.

Background Factor V Leiden (FVL) is an inherited autosomal dominant condition that is identified in 20%-50% of patients with venous thromboembolic diseases and 5-8% of the population in Canada. People with FVL are often called hypercoagulable, and have a five-fold increase in the risk of venous thrombosis. There is a low prevalence of FVL among patients with fatal pulmonary embolism (PE) and a higher incidence of deep vein thrombosis (DVT) than PE in patients with FVL. This has been referred to as the FVL paradox in thrombosis. We wished to test whether FVL and normal patients are at the same risk of initial thrombus formation, expect FVL patients present with symptomatic DVT while normal patients do not present, because the small asymptomatic DVT in normal patients embolizes to become an asymptomatic PE. Aim Therefore, to explain why patients with FVL are more likely to present with a DVT rather than a PE we will study DVT stability in FVL mice using a modification of our previously published mouse model of venous thrombus stability. Methods Platelets were fluorescently labeled using CD41 fab fragments conjugated to an Alexa Fluor 488. DVT was induced using a ferric chloride saturated filter paper on the femoral vein of female mice. We chose conditions such that only a small thrombus developed in wild type (WT) mice, and then compared them to thrombi in FVL heterozygous (F5L/+) and FVL homozygous (F5L/L) mice. Intravital videomicroscopy recorded embolic events leaving the thrombus and the thrombus sizes every 10 minutes for 2 hours. Lungs were harvested sectioned and stained for presence of PE. Results Although thrombus size remained small in WT mice, it significantly increased over time in F5L/+ and F5L/L mice. The number of total and large embolic events, and the percent of thrombus that embolized was significantly decreased in F5L/+ and F5L/L mice compared to WT mice. Previously we have correlated increased large embolic events with increased PE burden. FVL mice had significantly reduced PE burden compared to WT mice. Mice with F5L had higher F1.2 levels than in WT, consistent with increased thrombin generation. Conclusion This suggests that in noncarriers (reflected by WT), a minor insult initially resulting in a small DVT tends to remain small and asymptomatic, due to the embolization. Alternatively, the same insult in people with FVL (reflected by F5L/L) leads to thrombus growth due to less embolization and thus development of symptomatic DVT, but no PE.

Factor V Leiden (F5L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/- ). F8 deficiency enhanced the survival of F5L/LTfpi+/- mice, demonstrating that F5L/LTfpi+/- lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+Tfpi+/- females were crossed to generate 6,729 progeny, with 98 F5L/LTfpi+/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/- ) suppressed F5L/LTfpi+/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/LTfpi+/- lethality (P = 1.7 × 10-6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

Decreased fibrinolytic activity is strongly associated with cardiovascular disease. Plasminogen activator inhibitor-1, (PAI-1, Serpine1) is a major circulating fibrinolysis inhibitor, >80% of which is found in platelets. Platelet PAI-1 (pPAI-1) is produced by the megakaryocytes and packaged directly into platelets, thus it is distinct from plasma PAI-1. Plasma PAI-1 levels have been extensively studied and consistently linked with cardiovascular disease. However, variation in pPAI-1, either in the presence or absence of cardiovascular disease, is unknown. To study PAI-1 expression, we surveyed ten mouse strains for pPAI-1 antigen. In particular, the LEWES/EiJ mouse strain had significantly increased pPAI-1 (and Serpine1 mRNA), with an average concentration of 3.1 pg/μg total platelet protein compared to C57BL/6J (B6), which had an average pPAI-1 concentration of 0.4 pg/μg total protein (q=0.0018). Outcrossing LEWES/EiJ x C57BL/6J produced F1 mice with average pPAI-1 levels of 1.6 pg/μg total protein (q=0.0018), suggesting a semidominantly-inherited Serpine1 regulatory effect. To identify pPAI-1 regulatory regions, we produced 48 F2 mice, measured pPAI-1 levels and genotyped 12 (with 1.0 pg/μg total protein) using the Mouse Universal Genotyping Array (MegaMUGA). QTL analysis revealed several candidate regions including a major significant 14.4 megabase locus (128.2-142.6 megabases) on Chromosome 5 (LOD score = 4.81). The Serpine1 gene resides within this candidate interval, strongly suggesting that a Serpine1 cis-eQTL is responsible for pPAI-1 expression differences between LEWES/EiJ and C57BL/6J. Identifying pPAI-1 expression control elements offers insights into platelet-specific gene expression and identifies a putative therapeutic target for modulating hemostasis.

Factor V Leiden (FVL) is an incompletely penetrant thrombosis susceptibility variant common in humans. This incomplete penetrance suggests there are modifiers of FVL (F5L) that alter the susceptibility to a thrombotic event. To identify modifiers, we performed a sensitized ethylnitrosourea (ENU) mutagenesis screen in mice to induce mutations suppressing the F5L/LTfpi+/- perinatal lethal thrombotic phenotype. Although we identified thrombosuppressor mutations in two of our 22 independent MF5L mouse lines thus far, the others are still unknown. Our mouse line MF5L16 has produced a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/LTfpi+/- mice. We performed the Prothrombin Time (PT) assay on plasma from a subset of these mice and observed a significantly longer PT in MF5L16F5L/LTfpi+/- mice compared to F5+/LTfpi+/- and Tfpi+/- control mice (q Disclosures No relevant conflicts of interest to declare.

Venous thromboembolism (VTE) is a prevalent human disease that exhibits significant heritability. Factor V Leiden (F5L) is the most common known VTE risk factor, but modifier genes significantly influence VTE development. We recently identified a p.R258G missense mutation in the Actr2 gene (Actr2+/G, ARP2 protein) as a genetic suppressor of lethal thrombosis in mice homozygous for F5L (F5L/L) and hemizygous for tissue factor pathway inhibitor (Tfpi+/-). However, the antithrombotic mechanism of Actr2+/G is unknown. We used genetic and genomic techniques to investigate the phenotypic effects of the Actr2G variant on cells and mice. We previously analyzed progeny from Actr2+/G x Actr2+/G (N7 backcross generations to C57BL6/J) to investigate the effects of Actr2G on mouse survival. The progeny deviated from Mendelian frequencies, as only ~12% were Actr2G/G mice (N=303; p We next studied the mechanism(s) of Actr2G thrombosis suppression by gene expression studies. RT-qPCR for multiple coagulation genes was performed in liver samples from Actr2+/+ and Actr2+/G mice. Serpine2 mRNA (Protease Nexin-1 (PN-1)) was significantly increased in Actr2+/G mice (N=3; p To further investigate the effects of the Actr2G mutation, we isolated mouse embryonic fibroblasts (MEFs) from Actr2+/+ and Actr2G/G mice. The Actr2G/G MEFs grew poorly in culture, and displayed a significant reduction in cell surface area 20 minutes after plating on fibronectin when compared to Actr2+/+ (q=0.0003). Further analysis revealed F-actin aggregation in the root of cellular protrusions of Actr2G/G MEFs. These results indicate cytoskeletal remodeling defects in Actr2G/G cells. In summary, our studies indicate that mutations affecting ARP2 function can have profound effects on mouse and cell survival, as well as pronounced effects on specific physiological processes like blood coagulation and the transcriptional regulation of coagulation related genes. Disclosures No relevant conflicts of interest to declare.

Factor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized ENU mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous forF5L(F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/−).F8deficiency enhanced survival ofF5L/LTfpi+/−mice, demonstrating thatF5L/LTfpi+/−lethality is genetically suppressible. ENU-mutagenizedF5L/Lmales andF5L/+Tfpi+/−females were crossed to generate 6,729 progeny, with 98F5L/LTfpi+/−offspring surviving until weaning. Sixteen lines exhibited transmission of a putative thrombosuppressor to subsequent generations, with these lines referred to asMF5L(Modifier ofFactor5 Leiden) 1-16. Linkage analysis inMF5L6identified a chromosome 3 locus containing the tissue factor gene (F3). Though no ENU-inducedF3mutation was identified, haploinsufficiency forF3(F3+/−) suppressedF5L/LTfpi+/−lethality. Whole exome sequencing inMF5L12identified anActr2gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression ofF5L/LTfpi+/−lethality (p=1.7x10−6), suggesting thatActr2p.R258Gis thrombosuppressive. CRISPR/Cas9 experiments to generate an independentActr2knockin/knockout demonstrated thatActr2haploinsufficiency is lethal, supporting a hypomorphic or gain of function mechanism of action forActr2p.R258G. Our findings identifyF8and theTfpi/F3axis as key regulators in determining thrombosis balance in the setting ofF5Land also suggest a novel role forActr2in this process.Significance StatementVenous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate blood clots. Inheritance of specific genetic variants, such as the Factor V Leiden polymorphism, increases VTE susceptibility. However, only ~10% of people inheriting Factor V Leiden develop VTE, suggesting the involvement of other genes that are currently unknown. By inducing random genetic mutations into mice with a genetic predisposition to VTE, we identified two genomic regions that reduce VTE susceptibility. The first includes the gene for blood coagulation Factor 3 and its role was confirmed by analyzing mice with an independent mutation in this gene. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood clotting risk.

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. Mice homozygous for the Nbeal2 8 bp deletion (Nbeal2gps/gps) exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10-7). Nbeal2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted Nbeal2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains.

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 locus responsible for smla was mapped to a 2.78-Mb interval approximately 30 Mb proximal to SerpinB2 , bounded by markers D1Mit382 and D1Mit216. Sequencing of Irs1 identified a nonsense mutation at serine 57 (S57X), resulting in complete loss of IRS1 protein expression. Analysis of ES cell DNA suggests that the S57X Irs1 mutation arose spontaneously in an ES cell subclone during cell culture. Although the smla phenotype is similar to previously reported Irs1 alleles, mice exhibited decreased survival, in contrast to the enhanced longevity reported for IRS1 deficiency generated by gene targeting. This discrepancy could result from differences in strain background, unintended indirect effects of the gene targeting, or the minimal genetic interference of the S57X mutation compared with the conventionally targeted Irs1 -KO allele. Spontaneous mutations arising during ES cell culture may be a frequent but underappreciated occurrence. When linked to a targeted allele, such mutations could lead to incorrect assignment of phenotype and may account for a subset of markedly discordant results from experiments independently targeting the same gene.

Most inherited hemostatic disorders exhibit incomplete penetrance and variable expressivity, which can be because of genetic or environmental interactions. This wide phenotypic variability for a given disease can be partly explained by modifier gene interactions. Modifier gene interactions have been described for VWD, TTP and venous thrombosis associated with the factor V Leiden mutation. We have exploited advances in mouse genetics in an effort to identify novel genetic loci that may serve as candidate genetic modifiers for bleeding and thrombosis in humans. We have identified several loci affecting plasma VWF levels and have identified and characterized mouse models of ADAMTS13 deficiency and Factor V Leiden that could be useful for identifying novel genes contributing to thrombosis risk in humans.

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. Genomic sequencing identified a highly conserved 97-kb Mvwf1 haplotype block shared by these strains that encompasses a 30-kb region of high nucleotide sequence divergence from C57BL6/J flanking B4galnt2 exon 1. The analysis of a series of bacterial artificial chromosome (BAC) transgenes containing B4galnt2 derived from the RIIIS/J or C57BL6/J inbred mouse strains demonstrates that the corresponding sequences are sufficient to confer the vessel (RIIIS/J) or intestine (C57BL6/J)-specific expression patterns. Taken together, our data suggest that the region responsible for the Mvwf1 regulatory switch lies within an approximately 30-kb genomic interval upstream of the B4galnt2 gene. The observation that Mvwf1 is present in multiple wild-derived strains suggests that this locus may be retained in wild mouse populations due to positive selection. Similar selective pressures could contribute to the high prevalence of von Willebrand disease in humans. Electronic supplementary material The online version of this article (doi:10.1007/s00335-007-9079-4) contains supplementary material, which is available to authorized users.

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. Cell bodies of affected neurons are vacuolated, and apparently empty spaces are present in areas where neurons should be present. Similar vacuoles are found in cultured neurons and fibroblasts. Selective membrane trafficking pathways, especially endosome-to-TGN retrograde trafficking, are defective. This report, along with a recent report on a mouse with a null mutation in Fig4 , presents the unexpected finding that the housekeeping lipid, PI(3,5)P 2 , is critical for the survival of neural cells.

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. To fully characterize thrombus formation in these settings, in vivo models are necessary to study the various components and intricate interactions that are involved. Genetic manipulations in mice are greatly facilitating the dissection of relevant pro- and antithrombotic influences. Standardized models for the study of thrombosis in mice as well as evolving techniques that allow imaging of molecular events during thrombus formation are now available. This review will highlight some of the recent developments in the field of thrombosis using mouse models and how these studies are expanding our knowledge of thrombotic disease.

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. Enhancing endogenous fibrinolysis by targeting plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of circulating plasminogen activators, has been shown to be effective in markedly attenuating the formation of arterial and venous occlusive thrombosis in animal models. In addition, animal and human studies of PAI-1 deficiency indicate that spontaneous bleeding complications associated with even complete PAI-1 deficiency would be rare. Patients most likely to benefit from PAI-1 inhibition would be those at high risk for vascular events where PAI-1 is elevated, such as is observed in obesity, diabetes and the metabolic syndrome. Since obesity and metabolic syndrome are now epidemic, and will likely have a major adverse impact on vascular thrombotic events, it may be time to test the clinical effectiveness of PAI-1 inhibition in a patient population at high risk for vascular thrombosis.

ISTH07A1_P-W-473: Contact View [P-W-473] AN ENU MUTAGENESIS SCREEN FOR DOMINANT GENETIC MODIFIERS OF THROMBOSIS IN THE FACTOR 5 LEIDEN MOUSE R.J. Westrick, S.L. Manning, M.E. Winn, G.M. Stotz, E. Sanford, D. Ginsburg. Human Genetics; Howard Hughes Medical Institute; Internal Medicine; Life Sciences Institute, University of Michigan, Ann Arbor, United States Introduction: Factor V Leiden, (F5Q) is the most common inherited risk factor for venous thrombosis. However, a relatively small percentage of people carrying F5Q will develop venous thrombosis in their lifetimes. This variability results from complex interactions between the environment and unknown genetic factors (modifier genes). We previously identified a synthetically lethal interaction between mice inheriting two copies of F5Q (F5Q/Q) and partial Tfpi (Tfpi+/-) deficiency (F5Q/Q Tfpi+/-). Complete Tfpi deficiency in mice is embryonic lethal, whereas heterozygosity is compatible with normal survival. However, all mice inheriting F5Q/Q and Tfpi+/die of thrombosis shortly after birth. Methods: The F5Q/Q Tfpi+/lethal interaction was utilized as the basis for a sensitized ENU mutagenesis screen designed to uncover novel dominant suppressor mutations that alter the hemostatic balance. Male F5Q/Q mice were ENU mutagenized and bred to F5Q/+ Tfpi+/double heterozygous females. Surviving offspring were analyzed to identify rescued mice with the F5Q/Q Tfpi+/genotype. Results: Analysis of 5600 G1 offspring to date has identified 64 mice that survived to weaning. Of the 12 F5Q/Q Tfpi+/mutants tested for heritability, five produced additional F5Q/Q Tfpi+/progeny, demonstrating that the associated thrombosis suppressor is heritable. The latter progeny have been genotyped with a panel of markers spanning the genome. For one of these mutant lines, this analysis has mapped the corresponding suppressor mutation to a small region on Chromosome 9, with fine mapping to identify the corresponding gene underway. Conclusions: We have demonstrated the existence of novel dominant mutations that improve hemostatic balance leading to survival of F5Q/Q Tfpi+/mice. Characterization of the genes may provide novel insight into the regulation of hemostasis, as well as identifying potential candidates for thrombosis modifiers in humans. Westrick RJ, Manning SL, Winn ME, Stotz GM, Sanford E, Ginsburg D. AN ENU MUTAGENESIS SCREEN FOR DOMINANT GENETIC MODIFIERS OF THROMBOSIS IN THE FACTOR 5 LEIDEN MOUSE. J Thromb Haemost 2007; 5 Supplement 2: P-W-473 Date: Wednesday, July 11, 2007 Session Info: Poster: Thrombophilia and familial thrombosis Room: Exhibition Area file:///E|/working/RAJAN/Wednesday/P-W-473.htm [1/22/2014 3:39:03 PM]

The carboxylation of glutamic acid residues to γ-carboxyglutamic acid (Gla) by the vitamin K–dependent γ-glutamyl carboxylase (γ-carboxylase) is an essential posttranslational modification required for the biological activity of a number of proteins, including proteins involved in blood coagulation and its regulation. Heterozygous mice carrying a null mutation at the γ-carboxylase (Ggcx) gene exhibit normal development and survival with no evidence of hemorrhage and normal functional activity of the vitamin K–dependent clotting factors IX, X, and prothrombin. Analysis of a Ggcx+/− intercross revealed a partial developmental block with only 50% of expected Ggcx−/− offspring surviving to term, with the latter animals dying uniformly at birth of massive intra-abdominal hemorrhage. This phenotype closely resembles the partial midembryonic loss and postnatal hemorrhage previously reported for both prothrombin- and factor V (F5)–deficient mice. These data exclude the existence of a redundant carboxylase pathway and suggest that functionally critical substrates for γ-carboxylation, at least in the developing embryo and neonate, are primarily restricted to components of the blood coagulation cascade.

Atherosclerotic plaque rupture and subsequent arterial plaque-associated thrombus formation (atherothrombosis) leads to myocardial infarctions and ischemic strokes. Although hyperlipidemia and abnormalities in hemostatic balance contribute to atherothrombosis, the sequence of events and mechanisms are largely unclear and remain of interest. Platelets are thought to play a major role in driving atherothrombosis by localizing to lesions, facilitating inflammatory cell infiltration into lesions, and locally delivering prothrombotic and proinflammatory molecules. Mechanisms underlying platelet dysregulation in atherosclerosis may be caused by exogenous factors, like diet. In fact, Fuller et al investigated the genetic effects of combined scavenger receptor class B, type 1, and Ldlr −/− (double knockout) deficiency on atherogenesis and the development of occlusive coronary artery atherothrombosis in response to 4 different dietary challenges.1 They observed that double knockout mice had increased spontaneous coronary artery disease and decreased survival in response to diets containing high fat and high cholesterol (22% fat, 0.15% cholesterol), high cholesterol (2% cholesterol), and diets high in fat, cholesterol, and choline (ie, the Paigen diet).1,2 Diet-induced mortality in the double knockout mice was likely attributed to abundant platelet accumulation surrounding atherosclerotic plaques as assessed by CD41 staining. Inflammatory markers and plaque-associated monocytes also correlated with increased plaque-associated platelets. These studies suggest that diet not only contributes to the development of atherosclerosis, but also the degree of platelet accumulation within plaques. Overproduction of platelets is a major risk factor for cardiovascular disease, yet the mechanisms that govern excessive platelet production in hyperlipidemic conditions are widely uncharted. Recent findings indicated the lack of ATP-binding cassette transporters (eg, ABCG4 and ABCB6) leads to increased platelet production and atherosclerosis in hypercholesterolemic mice.3,4 Specifically, the lack of ABCB6 in the bone marrow leads to unrestrained platelet production, enhanced proinflammatory platelet activity, and accelerated atherosclerosis in Ldlr …

Factor V Leiden (FVL) is the most common genetic risk factor for venous thromboembolism (VTE). FVL is incompletely penetrant as only 10% of FVL heterozygotes develop VTE. Previously, nearly uniform perinatal lethality was observed in C57BL/6J (B6) mice hemizygous for tissue factor pathway inhibitor (Tfpi+/-) on a homozygous FVL background (FVQ/Q). However, inbred mouse strains exhibit significant genetic variation and can exert a profound influence on phenotype. We tested the lethality of FVQ/Q Tfpi+/- on the DBA/2J inbred strain background by crossing FVQ/+ Tfpi+/- to DBA/2J followed by a double heterozygous (FVQ/+ Tfpi+/-) x FVQ/Q cross. Surprisingly, FVQ/Q Tfpi+/- mice on the DBA/2J genetic background are born at expected Mendelian frequencies (N=62, nexp=15, nobs=15, N.S.) and display no overt thrombosis. This demonstrates the existence of dominant antithrombotic DBA/2J strain-specific thrombosis modifier gene(s). Analysis of progeny from a DBA/2J-B6 F1 FVQ/Q Tfpi+/- x B6 FVQ/Q cross yielded equal proportions of FVQ/Q Tfpi+/- and FVQ/Q Tfpi+/+ (N=130, nexp=43, nobs=63, p

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). To better understand how p38 and NF-kappaB maintain macrophage survival, we searched for target genes whose products prevent TLR4-induced apoptosis and a p38-dependent transcription factor required for their induction. Here we describe key roles for transcription factor CREB, a target for p38 signaling, and the plasminogen activator 2 (PAI-2) gene, a target for CREB, in maintenance of macrophage survival.

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. Methods and Results — To examine the potential in vivo interaction between Tfpi and FvL , we analyzed crosses between mice carrying FvL and a deficiency of TFPI. The Fv Q/Q , Tfpi +/− genotype was nearly completely fatal in the early perinatal period. Increased fibrin deposition was observed in multiple organs from the Fv Q/Q , Tfpi +/− fetuses, suggesting disseminated thrombosis. Conclusions — These observations demonstrate the prothrombotic effect of modest variations in the level of TFPI expression and suggest that TFPI could be an important genetic modifier for the thrombosis associated with FVL in humans.

Elevated expression of plasminogen activator inhibitor-1 (PAI-1) in tumors is associated with a poor prognosis in many cancers. Reduced tumor growth and angiogenesis have also been reported in mice deficient in PAI-1. These results suggest that PAI-1 may be required for efficient angiogenesis and tumor growth. In the present study, we demonstrate that PAI-1 can both enhance and inhibit the growth of M21 human melanoma tumors in nude mice and that this appears to be due to PAI-1 regulation of angiogenesis. Quantitative analysis of angiogenesis in a Matrigel implant assay indicated that in PAI-1 null mice angiogenesis was reduced approximately 60% compared with wild-type mice, while in mice overexpressing PAI-1, angiogenesis was increased nearly 3-fold. Furthermore, addition of PAI-1 to implants in wild-type mice enhanced angiogenesis up to 3-fold at low concentrations but inhibited angiogenesis nearly completely at high concentrations. Together, these data demonstrate that PAI-1 is a potent regulator of angiogenesis and hence of tumor growth and suggest that understanding the mechanism of this activity may lead to the development of important new therapeutic agents for controlling pathologic angiogenesis.

SummaryIncreased fibrin deposition following inflammatory lung injury has been proposed to facilitate the development of pulmonary fibrosis. Therefore, factors predisposing to thrombosis may affect the fibrotic response to injury. Activated protein C (aPC) resistance due to the factor V Leiden mutation (FνL) is a common genetic risk factor for vascular thrombosis. To examine the relationship between aPC resistance and the development of pulmonary fibrosis, lung inflammation was induced by bleomycin in mice carrying the FvL mutation. Three weeks following the instillation of 0.0375 U of bleomycin, the lungs of mice homozygous and heterozygous for FvL contained significantly more hydroxyproline (35 ± 4 and 36 ± 7 ug hydroxyproline/ mg total protein, respectively) than wild-type mice (26 ± 6 ug/mg protein, p

A polymorphism in coagulation factor V, factor V Leiden (FVL), is the major known genetic risk factor for thrombosis in humans. Approximately 10% of mutation carriers experience clinically significant thrombosis in their lifetime. In a small subset of patients, thrombosis is associated with coinheritance of other prothrombotic gene mutations. However, the potential contribution of additional genetic risk factors in the majority of patients remains unknown. To gain insight into the molecular basis for the variable expressivity of FVL, mice were generated carrying the homologous mutation (R504Q [single-letter amino acid codes]) inserted into the endogenous murine Fv gene. Adult heterozygous (FvQ/+) and homozygous (FvQ/Q) mice are viable and fertile and exhibit normal survival. Compared with wild-type mice, adult FvQ/Q mice demonstrate a marked increase in spontaneous tissue fibrin deposition. No differences in fetal development or survival are observed among FvQ/Q,FvQ/+ or control littermates on the C57BL/6J genetic background. In contrast, on a mixed 129Sv-C57BL/6J genetic background,FvQ/Q mice develop disseminated intravascular thrombosis in the perinatal period, resulting in significant mortality shortly after birth. These results may explain the high degree of conservation of the R504/R506 activated protein C cleavage site within FV among mammalian species and suggest an important contribution of other genetic factors to the thrombosis associated with FVL in humans.

A polymorphism in coagulation factor V, factor V Leiden (FVL), is the major known genetic risk factor for thrombosis in humans. Approximately 10% of mutation carriers experience clinically significant thrombosis in their lifetime. In a small subset of patients, thrombosis is associated with coinheritance of other prothrombotic gene mutations. However, the potential contribution of additional genetic risk factors in the majority of patients remains unknown. To gain insight into the molecular basis for the variable expressivity of FVL, mice were generated carrying the homologous mutation (R504Q [single-letter amino acid codes]) inserted into the endogenous murine Fv gene. Adult heterozygous (FvQ/+) and homozygous (FvQ/Q) mice are viable and fertile and exhibit normal survival. Compared with wild-type mice, adult FvQ/Q mice demonstrate a marked increase in spontaneous tissue fibrin deposition. No differences in fetal development or survival are observed among FvQ/Q,FvQ/+ or control littermates on the C57BL/6J genetic background. In contrast, on a mixed 129Sv-C57BL/6J genetic background,FvQ/Q mice develop disseminated intravascular thrombosis in the perinatal period, resulting in significant mortality shortly after birth. These results may explain the high degree of conservation of the R504/R506 activated protein C cleavage site within FV among mammalian species and suggest an important contribution of other genetic factors to the thrombosis associated with FVL in humans.

Abstract —The increased risk of hyperlipidemia on the development of complications of atherosclerosis is well established. Cholesterol-lowering therapies lead to a decrease in the incidence of vascular thrombotic events that is out of proportion to the reduction in plaque size. This suggests that the occurrence of acute thrombosis overlying a disrupted plaque is influenced by changes in lipid levels. The influence of acute hyperlipidemia on the development of thrombosis overlying an atherosclerotic plaque in vivo has not been extensively studied. We used a murine model of vascular injury induced by a photochemical reaction to elicit thrombus formation overlying an atherosclerotic plaque. Fifteen apolipoprotein E–deficient mice were maintained on normal chow until the age of 30 weeks. Five days before the induction of thrombosis, 6 mice were started on a high fat diet, and 9 mice were continued on normal chow. Mice then underwent photochemical injury to the common carotid artery immediately proximal to the carotid bifurcation, where an atherosclerotic plaque is consistently present. Mice maintained on normal chow developed occlusive thrombi, determined by cessation of blood flow, 44±5 minutes (mean±SEM) after photochemical injury, whereas mice fed a high fat chow developed occlusive thrombosis at 27±3 minutes ( P

Abstract —Impaired fibrinolysis has been linked to atherosclerosis in a number of experimental and clinical studies. Plasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of plasminogen activation and has been proposed to promote atherosclerosis by facilitating fibrin deposition within developing lesions. We examined the contribution of PAI-1 to disease progression in 2 established mouse models of atherosclerosis. Mice lacking apolipoprotein E (apoE−/−) and mice lacking the low density lipoprotein receptor (LDLR−/−) were crossbred with transgenic mice overexpressing PAI-1 (resulting in PAI-1 Tg + /apoE−/− and PAI-1 Tg + /LDLR−/−, respectively) or were crossbred with mice completely deficient in PAI-1 gene expression (resulting in PAI-1−/−/apoE−/− and PAI-1−/−/LDLR−/−, respectively). All animals were placed on a western diet (21% fat and 0.15% cholesterol) at 4 weeks of age and analyzed for the extent of atherosclerosis after an additional 6, 15, or 30 weeks. Intimal and medial areas were determined by computer-assisted morphometric analysis of standardized microscopic sections from the base of the aorta. Atherosclerotic lesions were also characterized by histochemical analyses with the use of markers for smooth muscle cells, macrophages, and fibrin deposition. Typical atherosclerotic lesions were observed in all experimental animals, with greater severity at the later time points and generally more extensive lesions in apoE−/− than in comparable LDLR−/− mice. No significant differences in lesion size or histological appearance were observed among PAI-1−/−, PAI-1 Tg + , or PAI-1 wild-type mice at any of the time points on either the apoE−/− or LDLR−/− genetic background. We conclude that genetic modification of PAI-1 expression does not significantly alter the progression of atherosclerosis in either of these well-established mouse models. These results suggest that fibrinolytic balance (as well as the potential contribution of PAI-1 to the regulation of cell migration) plays only a limited role in the pathogenesis of the simple atherosclerotic lesions observed in the mouse.

Occlusive thrombosis depends on the net balance between platelets, coagulation, and fibrinolytic factors. Epidemiologic information suggests that plasminogen activator inhibitor-1 (PAI-1), a central regulator of the fibrinolytic system, plays an important role in determining the overall risk for clinically significant vascular thrombosis. Vitronectin (VN), an abundant plasma and matrix glycoprotein, binds PAI-1 and stabilizes its active conformation. This study assessed the role of PAI-1 and VN expression in the formation of occlusive vascular thrombosis following arterial or venous injury. The common carotid arteries of 17 wild-type (WT) mice and 8 mice deficient in PAI-1 were injured photochemically while blood flow was continuously monitored. WT mice developed occlusive thrombi at 52.0 ± 3.8 minutes (mean ± SEM) following injury; mice deficient in PAI-1 developed occlusive thrombosis at 127 ± 15 minutes (P

Plasminogen activator inhibitor-2 (PAI-2), a member of the serpin gene family, is thought to serve as a primary regulator of plasminogen activation in the extravascular compartment. High levels of PAI-2 are found in keratinocytes, monocytes, and the human trophoblast, the latter suggesting a role in placental maintenance or embryo development. The primarily intracellular distribution of PAI-2 also may indicate a unique regulatory role in a protease-dependent cellular process such as apoptosis. To examine the potential functions of PAI-2in vivo, we generated PAI-2-deficient mice by gene targeting in embryonic stem cells. Homozygous PAI-2-deficient mice exhibited normal development, survival, and fertility and were also indistinguishable from normal controls in response to a bacterial infectious challenge or endotoxin infusion. No differences in monocyte recruitment into the peritoneum were observed after thioglycollate injection. Epidermal wound healing was equivalent among PAI-2 −/− null and control mice. Finally, crossing PAI-2 −/− with PAI-1 −/− mice to generate animals deficient in both plasminogen activator inhibitors failed to uncover an overlap in function between these two related proteins.

We have identified altered lineage-specific expression of an N-acetylgalactosaminyltransferase gene, Galgt2, as the gain-of-function mechanism responsible for the action of the Mvwf locus, a major modifier of plasma von Willebrand factor (VWF) level in RIIIS/J mice. A switch of Galgt2 gene expression from intestinal epithelial cell-specific to a pattern restricted to the vascular endothelial cell bed leads to aberrant posttranslational modification and rapid clearance of VWF from plasma. Transgenic expression of Galgt2 directed to vascular endothelial cells reproduces the low VWF phenotype, confirming this switch in lineage-specific gene expression as the likely molecular mechanism for Mvwf. These findings identify alterations in glycosyltransferase function as a potential general mechanism for the genetic modification of plasma protein levels.

Type 1 von Willebrand disease (VWD), characterized by reduced levels of plasma von Willebrand factor (VWF), is the most common inherited bleeding disorder in humans. Penetrance of VWD is incomplete, and expression of the bleeding phenotype is highly variable. In addition, plasma VWF levels vary widely among normal individuals. To identify genes that influence VWF level, we analyzed a genetic cross between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold difference in plasma VWF level. DNA samples from F 2 progeny demonstrating either extremely high or extremely low plasma VWF levels were pooled and genotyped for 41 markers spanning the autosomal genome. A novel locus accounting for 63% of the total variance in VWF level was mapped to distal mouse chromosome 11, which is distinct from the murine Vwf locus on chromosome 6. We designated this locus Mvwf for “modifier of VWF.” Additional genotyping of as many as 2407 meioses established a high resolution genetic map with gene order Cola1 - Itg3a - Ngfr - Mvwf / Gip - Hoxb9 - Hoxb1 - Cbx · rs2 - Cox5a - Gfap . The Mvwf candidate interval between Ngfr and Hoxb9 is ≈0.5 centimorgan (cM). These results demonstrate that a single dominant gene accounts for the low VWF phenotype of RIIIS/J mice in crosses with several other strains. The pattern of inheritance suggests a gain-of-function mutation in a unique component of VWF biosynthesis or processing. Characterization of the human homologue for Mvwf may have relevance for a subset of type 1 VWD cases and may define an important genetic factor modifying penetrance and expression of mutations at the VWF locus.

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. To investigate the direct function of HO-1 on thrombosis, we used photochemical-induced vascular injury in Hmox1 −/− and Hmox1 +/+ mice. Hmox1 −/− mice developed accelerated, occlusive arterial thrombus compared with Hmox1 +/+ mice, and we detected several mechanisms accounting for this antithrombotic effect. First, endothelial cells in Hmox1 −/− arteries were more susceptible to apoptosis and denudation, leading to platelet-rich microthrombi in the subendothelium. Second, tissue factor, von Willebrand Factor, and reactive oxygen species were significantly elevated in Hmox1 −/− mice, consistent with endothelial cell damage and loss. Third, following transplantation of Hmox1 −/− donor bone marrow into Hmox1 +/+ recipients and subsequent vascular injury, we observed rapid arterial thrombosis compared with Hmox1 +/+ mice receiving Hmox1 +/+ bone marrow. Fourth, inhaled carbon monoxide and biliverdin administration rescued the prothrombotic phenotype in Hmox1 −/− mice. Fifth, using a transcriptional analysis of arterial tissue, we found that HO-1 determined a transcriptional response to injury, with specific effects on cell cycle regulation, coagulation, thrombosis, and redox homeostasis. These data provide direct genetic evidence for a protective role of HO-1 against thrombosis and reactive oxygen species during vascular damage. Induction of HO-1 may be beneficial in the prevention of thrombosis associated with vascular oxidant stress and inflammation.

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. To fully characterize thrombus formation in these settings, in vivo models are necessary to study the various components and intricate interactions that are involved. Genetic manipulations in mice are greatly facilitating the dissection of relevant pro- and antithrombotic influences. Standardized models for the study of thrombosis in mice as well as evolving techniques that allow imaging of molecular events during thrombus formation are now available. This review will highlight some of the recent developments in the field of thrombosis using mouse models and how these studies are expanding our knowledge of thrombotic disease.

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 ( Fv Q /+ ), and homozygous FVL ( Fv Q/Q ) mice underwent photochemical carotid arterial injury to induce occlusive thrombosis. Fv Q/Q mice formed occlusive thromboses 27±3 minutes (n=7) after the onset of injury, which was significantly shorter than that observed for Fv +/+ mice (56±7 minutes, n=9, P Fv Q /+ mice (41±7 minutes, n=5) were intermediate ( P =0.5, compared with Fv +/+ ). To determine the source of FVL relevant to the enhanced vascular thrombosis, bone marrow transplantation experiments were performed between Fv +/+ and Fv Q/Q mice. Fv Q/Q mice transplanted with Fv +/+ bone marrow formed occlusive thromboses at 35±5 minutes (n=7, P Fv +/+ mice), whereas Fv +/+ mice transplanted with Fv Q/Q bone marrow occluded at 59±7 minutes (n=6, P Fv Q/Q mice). To assess the effect of the FVL mutation on the development of atherosclerosis, Fv Q/Q mice were crossed with the atherosclerosis-prone apolipoprotein E (ApoE)–deficient strain ( ApoE −/− ) to generate Fv Q/Q ,ApoE −/− mice. By 52 weeks of age, Fv Q/Q ,ApoE −/− mice (n=8) had developed more aortic atherosclerosis (40±6% lesion area) compared with Fv +/+ ,ApoE −/− mice (15±3% lesion area; n=12, P Conclusions— In conclusion, homozygosity for the FVL mutation in mice leads to enhanced arterial thrombosis and atherosclerosis. The source of the FVL leading to accelerated thrombosis appears to be circulating, non–platelet-derived plasma FVL.

Background —Tissue factor initiates blood coagulation after atherosclerotic plaque disruption. Tissue factor pathway inhibitor (TFPI) inhibits tissue factor activity and may reduce thrombus formation in this setting. We evaluated the effect of heterozygous TFPI deficiency on the development of atherosclerosis and thrombosis in atherosclerosis-prone mice. Methods and Results —Mice with a combined heterozygous TFPI deficiency and homozygous apolipoprotein E deficiency (TFPI +/– /apoE –/– ) were generated by crossbreeding, and they were analyzed for atherosclerosis throughout the vascular tree. Compared with mice with a normal TFPI genotype (TFPI +/+ /apoE –/– ), mice with a TFPI deficiency exhibited a greater atherosclerotic burden involving the carotid and common iliac arteries. Staining for active tissue factor within the plaque revealed more activity in TFPI +/– /apoE –/– mice compared with TFPI +/+ /apoE –/– mice. Consistent with increased plaque tissue factor activity, the time to occlusive thrombosis after photochemical carotid plaque injury was significantly decreased in TFPI +/– /apoE –/– mice. Conclusions —These observations indicate that TFPI protects from atherosclerosis and is an important regulator of the thrombosis that occurs in the setting of atherosclerosis.

Dissolution of the fibrin blood clot is regulated in large part by plasminogen activator inhibitor-1 (PAI-1). Elevated levels of plasma PAI-1 may be an important risk factor for atherosclerotic vascular disease and are associated with premature myocardial infarction. The role of the endogenous plasminogen activation system in limiting thrombus formation following atherosclerotic plaque disruption is unknown. This study found that genetic deficiency for PAI-1, the primary physiologic regulator of tissue-type plasminogen activator (tPA), prolonged the time to occlusive thrombosis following photochemical injury to carotid atherosclerotic plaque in apolipoprotein E-deficient (apoE−/−) mice. However, anatomic analysis revealed a striking difference in the extent of atherosclerosis at the carotid artery bifurcation between apoE−/− mice and mice doubly deficient for apoE and PAI-1 (PAI-1−/−/apoE−/−). Consistent with a previous report, PAI-1+/+/apoE−/−and PAI-1−/−/apoE−/− mice developed similar atherosclerosis in the aortic arch. The marked protection from atherosclerosis progression at the carotid bifurcation conferred by PAI-1 deficiency suggests a critical role for PAI-1 in the pathogenesis of atherosclerosis at sites of turbulent flow, potentially through the inhibition of fibrin clearance. Consistent with this hypothesis, intense fibrinogen/fibrin staining was observed in atherosclerotic lesions at the carotid bifurcation compared to the aortic arch. These observations identify significant differences in the pathogenesis of atherosclerosis at varying sites in the vascular tree and suggest a previously unappreciated role for the plasminogen activation system in atherosclerosis progression at sites of turbulent flow.