ALBERT

Beschreibung: The cessation of bleeding following trauma is a crucial element in vertebrate survival.Factor V (F5) serves as an essential cofactor in the penultimate step of coagulation, the conversion of prothrombin to thrombin. In humans, genetic deficiency of F5 is rare and clinically diverse, with presentations ranging from neonatal intracranial hemorrhage to mild bleeding later in life. This patient variability suggests the presence of modifiers, unlinked genes inherited separately from the F5 locus. Complete loss through gene targeting of mouse F5 results in embryonic and neonatal lethality, interfering with further detailed studies. Zebrafish possess many distinct advantages for the study of coagulation and modifier genes, including high fecundity, optical clarity, external development, as well as extensive homology of the mammalian hemostatic system. Here we report the role of F5 in zebrafish development using genome editing mediated targeted mutagenesis. The f5 locus was identified through a BLAST search of zebrafish genomic sequence, identifying a protein with 48% amino acid identity and 66% similarity to human F5. In situ hybridization revealed expression of f5 mRNA localized to the developing liver at 5 days post fertilization (dpf). CRISPR RNA guided nucleases were designed to target exon 4 of F5 and injected into several hundred wild type zebrafish one cell stage embryos to produce adult fish (F0) with a panel of insertion/deletion mutations in f5. Adult fish (F1) heterozygous for a 49 base pair deletion were identified and incrossed to generate groups of offspring for analysis. This deletion was initiated at amino acid 171, within the A1 domain of F5. Genotyping was performed after phenotypic analysis so that observers were blinded during data collection. Homozygous mutants demonstrated significantly decreased survival compared to their heterozygous and wild type siblings, with die off beginning between 2 and 4 weeks post fertilization and 100% mortality by 7 months of age. Visual observation of development and circulation revealed that homozygous mutant embryos and larvae were indistinguishable from wild type siblings, with no signs of hemorrhage. Since there was no observable bleeding, we used induced occlusive thrombus formation by laser mediated endothelial ablation of the posterior cardinal vein at 3 dpf to assess hemostasis. We found that the ability to produce occlusive thrombi was absent in f5-/- mutants, a bleeding phenotype, while wild type and heterozygous siblings were phenotypically normal. This bleeding phenotype was rescued in 73% of embryos (p=0.0006) at 3 dpf after injection of zebrafish f5 cDNA at the one cell stage. In summary, we have demonstrated strong conservation of zebrafish and mammalian F5, including site of synthesis and requirement for hemostasis. Surprisingly, embryos and larvae, as well as young adults, tolerate what is a severe and lethal defect in mammals, allowing accessibility not easily achieved in murine models. This suggests the possibility of species-specific factors enabling survival in fish. Identification of these factors, combined with small molecule screens, could lead to novel therapeutic modalities for patients with bleeding disorders. Disclosures No relevant conflicts of interest to declare.

Beschreibung: The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines, suggesting that WT1 participates in regulating the proliferation of leukemic cells. However, the role of WT1 in normal hematopoiesis is not well understood. To investigate this question, we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1, although contributing efficiently to other organ systems, make only a minimal contribution to the hematopoietic system in chimeras, indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition, fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blast-forming unit (BFU-E), erythroid colony-forming unit (CFU-E), and colony-forming unit–granulocyte macrophage–erythroid–megakaryocyte (CFU-GEMM). However, transplantation of fetal liver hematopoietic cells lackingWT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1.

Beschreibung: Factor X (F10) deficiency is a rare inherited bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted knockout of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering further studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in development. The zebrafish model is characterized by external development, optical transparency, ability to generate thousands of offspring at low cost, and a highly characterized vasculature. We have used these advantages for more in depth study of the role of the coagulation cascade in developmental regulation of hemostasis and vasculogenesis. We generated a 17 base pair deletion in the zebrafish f10 locus by genome editing with TALENs. Although indistinguishable morphologically from f10+/+ and f10+/- siblings at early stages, f10-/- mutants demonstrated progressive lethality between 1 and 5 months of age. Extensive hemorrhage was identified in multiple tissues starting at 3-4 weeks of age, particularly the brain. Notably, intracranial hemorrhage is a common feature in multiple zebrafish mutants with various vascular defects, including anomalies of endothelial differentiation and specification, apoptosis, and vessel integrity/permeability. Gross inspection of f10 mutant embryos and larvae in the first week of life revealed no apparent defects in circulation or vascular development. Expression of arterial and venous endothelial markers were examined by in situ hybridization at 24 and 72 hours post fertilization, the time period during which axial and intersegmental vessels form, along with early establishment of the vascular network. Markers included ephb2a, cdh5, flk1, flt4, and ephb4, and expression patterns were indistinguishable between mutants and wild type siblings. Acridine orange staining at 5 days post fertilization (dpf) did not detect any dysregulation of apoptosis. Hemoglobin staining found no specific hemorrhage in vehicle or warfarin treated mutants. However, 3 dpf f10-/- mutants did not develop occlusive thrombi in response to laser-mediated venous endothelial injury, indicating that F10 is required for hemostasis. We used quantitative PCR to measure transcription of f10 and downstream coagulation factors in 3 dpf larvae. f10 mRNA was undetectable in homozygous mutants, presumably due to nonsense-mediated decay as a consequence of the TALEN-induced frameshift mutation. fga (fibrinogen alpha) and at3 (antithrombin III) mRNAs were increased by 1.8 and 2.3-fold, respectively (p

Beschreibung: Background: Multiple myeloma disproportionately affects the elderly and is currently an incurable malignancy. New therapies for myeloma, particularly oral therapies, are urgently needed. Objectives: To determine if thalidomide with or without other agents, improves response rate (≥ 50% reduction in monoclonal protein), survival, and/or progression in patients with previously untreated myeloma. To determine the frequency and significance of major adverse events associated with thalidomide in this setting. Methods: A literature search of Medline (1966–June 2006), Embase (1980–June 2006), the Cochrane Library, abstracts from the annual meetings of the American Society of Hematology (1999–2005) and the American Society of Oncology (1999–2006) was completed with a pre-specified search strategy. No language restrictions were applied. Randomized controlled trials of induction thalidomide (any dose, any duration) for adults with previously untreated multiple myeloma were included. Trials of exclusively maintenance therapy were excluded. Two reviewers independently extracted data. The methodological quality of selected trials was assessed and summarized. Weighted data was expressed as relative risk, risk difference, number needed to treat (NNT), and number needed to harm (NNH). A random-effects model was used. Results: Six eligible studies involving almost two thousand patients (N=1875) were identified and meta-analyzed. Two studies were published and four were reported in abstract form only. Five studies reported overall response rate (ORR); the four largest trials reported statistically significant improvements in ORR with the addition of thalidomide to standard therapy. The weighted relative risk of responding to a thalidomide-containing regimen versus control was 1.50 (95% CI 1.21 to 1.86). The NNT to achieve one additional response with thalidomide was 4 (95% CI 2.9 to 8.3). Two trials reported improvements in EFS/PFS. One trial reported an improvement in OS. The risk of VTE, peripheral neuropathy, and constipation was consistently elevated with thalidomide such that for every 50 patients treated with a thalidomide-containing-regimen, one could expect 12 to 13 additional patients to respond, 4 additional patients to develop VTE (NNH 12.5; 95% CI 8.3 to 20), 2 additional patients to develop peripheral neuropathy (NNH 25; 95% CI 16.7 to 50), and 4 additional patients to develop constipation (NNH14; 95% CI 10 to 25). In our analyses, prophylactic anticoagulation appeared to decrease, but not abolish, the risk of VTE with thalidomide. Conclusions: Thalidomide improves response rate and possibly progression free and overall survival in patients with previously untreated myeloma. It also increases the incidence of VTE, neuropathy, constipation and other adverse events. Further studies are required to confirm the survival advantage seen in one study, and to determine the optimum strategy for VTE prophylaxis.

Beschreibung: Plasma von Willebrand factor (VWF) levels are highly variable in the normal human population, and twin studies suggest that two-thirds of this variability is heritable. Only one-third of this genetic component is explained by ABO blood group, with the factors responsible for the majority of the effect still unknown. These putative VWF modifier genes contribute to the wide variability in bleeding severity among patients with von Willebrand disease (VWD), as well as the frequent difficulty in establishing this diagnosis. We previously mapped four loci responsible for variable plasma VWF levels among several inbred mouse strains, which we termed Mvwf1-4 (modifier of Vwf). We have identified the mutant alleles for Mvwf1, a variant in a glycosyltransferase with homology to a human blood group antigen, and Mvwf2, a mutation in the Vwf gene itself. For the current study we chose two inbred strains with 3.5-fold divergent VWF plasma levels, C57BL/6J and WSB/EiJ, neither of which carries the Mvwf1 or Mvwf2 alleles. F1 hybrid mice were backcrossed onto C57BL/6J to generate 200 N2 progeny, followed by determination of plasma VWF levels by ELISA. A dense genome scan of 149 markers, an average of one marker every 10 centimorgans, was performed on genomic DNA from all 200 N2 mice by the Mammalian Genotyping Service at the Marshfield Clinic Research Foundation. Analysis of the data with the R/qtl statistical package identified two major candidate loci with significant evidence for linkage to VWF levels. The first locus (Mvwf5) mapped to a region containing the Vwf gene itself on chromosome 6, with a logarithm of the odds (LOD) score of 12.1. Preliminary studies of Vwf mRNA from F1 mice by primer extension SNP (single nucleotide polymorphism) analysis suggest that Mvwf5 exerts its effect at the level of Vwf transcription or mRNA stability. A second potential modifier (Mvwf6) localized to chromosome 10 with a LOD score of 4.6, and displayed additive effects with Mvwf5. Two additional suggestive loci mapped to murine chromosomes 4 and 5, with LOD scores of 2.4 and 3.4, respectively. The former locus maps to the same region of chromosome 4 as Mvwf3, a candidate modifier previously identified in an F2 intercross between the strains A/J and CASA/RkJ. In summary, we have identified a 2nd natural Vwf gene variant among inbred mice (Mvwf5), a novel VWF regulatory locus on murine chromosome 10 (Mvwf6), and 2 other possible VWF modifiers on chromosomes 4 and 5. Surprisingly, two of six Mvwf loci characterized to date correspond to hypomorphic mutations at the Vwf gene itself and appear to interact with modifier loci on other chromosomes. Similar interactions are likely to explain the extensive variability in plasma VWF levels observed in the general human population as well as among patients with VWD.

Beschreibung: Zebrafish are a powerful vertebrate model system for the study of human disease as they share many molecular pathways with mammals. Of note, nearly all of the mammalian coagulation factors are also highly conserved in fish. As part of a whole genome ENU mutagenesis screen, we identified a mutant zebrafish which displayed intraventricular hemorrhage between 2–3 days post fertilization (dpf), which we named redhead. Using an F2 intercross strategy, we mapped this recessive mutant to a 100 kilobase interval on chromosome 2 and identified a splice site mutation in the gene for an ortholog of the p21-activated kinase Pak2. This IVS9 T+2A mutation renders greater than 90% of transcripts nonfunctional, resulting in a hypomorphic allele. Surveying zebrafish expressed sequence tag databases, we identified two Pak2 orthologs in zebrafish, designated Pak2a and Pak2b, with the redhead mutation in pak2a. Central nervous system (CNS) hemorrhage in redhead embryos was rescued by injection of wild type pak2a mRNA. Morpholino knockdown of pak2a in wild type fish phenocopies the redhead mutant, but with an increase in penetrance and severity, including hydrocephalus and pericardial edema secondary to severe hemorrhage. Injection of either pak2a or pak2b mRNA was able to rescue this phenotype. In addition, pak2b knockdown worsened the bleeding phenotype in redhead embryos, with no effect on wild type fish. These results suggest a partial overlap in function between Pak2a and Pak2b. Confocal microscopy was performed at 2.5 dpf in pak2a knockdown embryos transgenic for Gata1-dsRED and Flk1-eGFP, distinctively labeling erythrocytes and endothelial cells, respectively. The sites and magnitude of hemorrhage were variable between individual embryos, but there were no obvious abnormalities in vessel patterning. In summary, we have identified a critical role for Pak2 in maintaining CNS vessel integrity in zebrafish, without apparent effects on other vascular beds. Further analysis of this signaling pathway in the vessel wall may provide novel insight into the mechanisms underlying vascular heterogeneity in mammals, and the tissue specific function of Pak2 in the CNS vasculature.

Beschreibung: Background Given the widespread use of bendamustine, data on long-term outcomes are essential for patients and clinicians to understand potential risks and benefits of therapy. Despite a long history as treatment for indolent non-Hodgkin lymphoma (iNHL), such information has been limited. We retrospectively reviewed the registration SDX-105-01 and SDX-105-03 trials (bendamustine 120 mg/m2 days 1+2 q21 days) and SDX-105-02 trial (bendamustine 90 mg/m2 days 1+2 plus rituximab 375 mg/m2 day 1 q28 days) to characterize long-term toxicity and efficacy of patients treated with bendamustine. Methods Patient level data was retrospectively collected from patients treated on the SDX-01, 02, and 03 trials. Descriptive statistics were used to summarize patient characteristics and events. The Kaplan-Meier method was used to report time-to-event outcomes. Wilcoxon Rank sum test was used to test the difference between events for continuous variables. Results Out of the total 245 subjects at 45 sites, data were available for 149 subjects (60 men, 89 women; SDX-01 N = 40, SDX-02 N = 43, SDX-03 N = 66) at 21 sites (included based on willingness to participate). The median age was 60 years at the start of bendamustine (range 39-84). The histologies included grade 1-2 follicular lymphoma (FL; N = 73), grade 3 FL (N = 23), SLL (N = 20), marginal zone lymphoma (N = 15), mantle cell lymphoma (N = 9), transformed lymphomas (N = 5), lymphoplasmacytic lymphoma (N = 2), and not reported (N = 2). The average time from diagnosis to study entry was 41 months (range 2-229). The median number of therapies prior to bendamustine was 2.5 (range 1-8). Patients received a median of 6 cycles and a median total dose of bendamustine of 1408 mg (max 5216, min 240). With a median follow up of 8.8 years after study entry, 80 patients had experienced progression. The median PFS was 18.4 months (95% C.I. 11.9-27.8); the 3-year PFS was 37%. During follow up, 93 patients had died at a median time of 22.3 months after the start of bendamustine. The median OS after start of bendamustine was 65.9 months (95% C.I. 38.8-91.8). The causes of death were lymphoma (N = 45), bendamustine toxicity (N = 2), subsequent treatment toxicity (N = 8), MDS/AML (N = 5), other cancer (N = 2), other (N = 6), and unknown (N = 25). A total of 98 patients received a median of 2 therapies following bendamustine (range 1-9), with the first treatment occurring a median of 13.2 months (range 0-111.3) following the final dose of bendamustine. The reported best response to the first subsequent treatment was CR (N = 11), PR (N = 6), SD (N = 21), PD (N = 12), not evaluable (N = 25), and unknown (N = 22) and the median OS of these patients was 51.3 months (95% C.I. 33.4-80.3). Fourteen patients had attempted stem cell collection following bendamustine, 10 of which had stem cells collected successfully. Eight patients had stem cells collected with GCSF alone (N = 7) or GCSF plus chemotherapy (N = 1). Twenty-three patients developed 25 cancers following bendamustine. Six patients developed MDS and 2 more developed AML. The median time to MDS/AML following bendamustine was 24 months (range 10-103) with an annualized incidence rate of 0.52%/year. One of patient had a prior myeloid neoplasm and one had a prior germ cell tumor. In univariate analysis, neither age at lymphoma diagnosis (P=0.438), nor total number of systemic regimens (P=0.443), nor total dose of bendamustine (P=0.291) was associated with MDS/AML. Other cancers included adenocarcinoma (colon N = 2; prostate N = 2; lung N = 2; breast N = 1), non-melanoma skin cancer (N = 6), squamous cell carcinoma (N = 2), hepatocellular carcinoma (N = 1), and bladder cancer (N = 1). None of these occurred in the 12 patients with a history of solid tumor before bendamustine. Conclusions With a median follow up of survivors of 〉 8 years, there was no evidence that bendamustine in the setting of previously treated iNHL was associated with a high rate of long-term bone marrow toxicity. Rates of MDS/AML and failure to collect stem cells were lower than expected. However, roughly half of all patients died within 5 years of starting bendamustine, thereby limiting the long-term follow up. A small but meaningful number of patients achieved durable remissions following bendamustine. These rigorously collected, patient-level, long-term follow up data provide reassurance that bendamustine or bendamustine plus rituximab is associated with efficacy and safety for many patients with relapsed or refractory iNHL. Disclosures Martin: Janssen: Consultancy, Honoraria; Acerta: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bayer: Consultancy. Cheson:AstraZeneca: Consultancy; Ascenta: Research Funding; Spectrum: Consultancy; Astellas: Consultancy; MedImmune: Research Funding; Pharmacyclics: Consultancy, Research Funding; Teva: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Roche/Genentech: Consultancy, Research Funding. Williams:Celgene: Consultancy, Other: Research funding to my institution; Takeda: Consultancy, Other: Research Funding to my institution; Genentech: Other: Research funding to my institution. Bartlett:Gilead: Consultancy, Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Millennium: Research Funding; Colgene: Research Funding; Medimmune: Research Funding; Kite: Research Funding; Insight: Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding. Szer:Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Australia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Smith:celegene, spectrum, genentech: Honoraria. Leonard:Weill Cornell Medical College: Employment; Genentech: Consultancy; Medimmune: Consultancy; AstraZeneca: Consultancy; Spectrum: Consultancy; Boehringer Ingelheim: Consultancy; Vertex: Consultancy; ProNAI: Consultancy; Biotest: Consultancy; Seattle Genetics: Consultancy; Pfizer: Consultancy; Mirati Therapeutics: Consultancy; Gilead: Consultancy; Novartis: Consultancy.

Beschreibung: Abstract 3165 Poster Board III-102 Extensive DNA sequence similarity has previously been demonstrated for most known components of the mammalian hemostatic system and their orthologous zebrafish genes, and vascular occlusion has been observed in zebrafish following laser-induced vascular injury (Jagadeeswaran P, et al. Methods Mol Med 2006).To test potential conservation of function across species, activated bovine thrombin (Sigma-Aldrich), over a range of concentrations was injected into the orbital vascular plexus of zebrafish embryos at 72 hours post fertilization (hpf). The severity of thrombus formation was relative to the thrombin concentration, with thrombosis consistently observed in 72 hpf wild type (WT) embryos at 30-90 ng of thrombin (1-3 nl of a 30mg/ml solution diluted in 0.9% NaCl, with 0.25% phenol red as a tracer). With injection of higher doses (90 ng), complete occlusion of the circulation was generally observed within 30 seconds, with a large thrombus visible in the heart. Results were scored as zero when no thrombosis was observed, grade 1 when thrombosis was seen in the vessels but blood flow was preserved, and grade 2 for total occlusion of the circulation. Thirty of 30 WT embryos with grade 1 thrombosis showed resolution of the thrombus by 5 minutes post injection, consistent with the activity of an endogenous fibrinolytic system in zebrafish, compared to only 22 of 30 among WT embryos scored as grade 2. No thrombosis was observed with injection of BSA (N=30) or thrombin inactivated by incubation at room temperature for 24 hours (N=30). One cell stage zebrafish embryos were injected with antisense morpholino oligonucleotides (MOs) designed to target the mRNAs transcribed from the zebrafish genes for the fibrinogen alpha-chain, von Willebrand Factor (VWF), or ADAMTS13. Each resulting embryo (only embryos without morphologic defects were used), along with a matched non-MO-treated control embryo, was then injected at 72 hpf with 30-60 ng bovine thrombin, and thrombus formation scored blindly by 2 independent observers at 30 seconds and 2 minutes post injection. An ∼70% reduction in mean thrombosis score was observed for embryos treated with the fibrinogen alpha-chain MO (N=29) compared to controls (p

Beschreibung: Venous thrombosis is a well-known complication of sex hormone therapy, with onset typically within weeks to months after initiation. Worldwide, more than 100 million pre-menopausal women use combined oral contraceptives, thus tens to hundreds of thousands develop thrombosis annually, resulting in significant morbidity and mortality. Estrogens are thought to be the major thrombotic risk, although the impact of progestins is less clear. It has been hypothesized that progestin effects may be secondary to mitigation of the estrogen effect. Although it is known that estrogens and progestins can alter expression of coagulation factors, the pathways and mechanisms that connect the two systems, as well as the proteins involved in progression to thrombosis, are poorly understood. Identification of these mediators are central to any comprehensive understanding of hormone-induced pathophysiology, could help ascertain patients at higher risk for thrombosis, and might also pinpoint future therapeutic targets. The zebrafish is a powerful genetic model in which the hemostatic system is nearly entirely conserved with humans. Its external development, ability to generate thousands of offspring at low cost, and optical transparency all make it a powerful tool to study the genetics of coagulation disorders. We previously produced a transgenic line (fabp-fgb-egfp) that generates GFP tagged fibrinogen beta (Fgb), which is incorporated into induced and spontaneous fibrin-rich thrombi. Here we show surprisingly rapid onset of estrogen-induced thrombosis. We exposed 5-6 day old transgenic zebrafish larvae to a range of concentrations of estradiol over 4-24 hours. We discovered green fluorescent deposits co-localizing to the posterior cardinal vein (PCV, orthologous to the inferior vena cava), but not other vessels. This occurred within 4 hours in a dose-dependent fashion. Confocal imaging demonstrated that the fluorescence was intravascular and deposited along the endothelium. Pre-incubation with warfarin and the direct oral anticoagulants rivaroxaban and dabigatran inhibited the fluorescent signal, confirming that the deposits were fibrin thrombi. This phenotype was similar to spontaneous PCV thrombosis observed in zebrafish mutants deficient in antithrombin, which also demonstrate a consumptive coagulopathy with absence of venous occlusion in response to endothelial injury. Therefore we treated 3 dpf larvae with estradiol for 4 hours, followed by laser-mediated endothelial injury and measurement of the time to occlusion (TTO) in the PCV by a blinded observer. Although the median TTO only increased from 12.5 to 15 seconds with treatment, this was highly statistically significant (p=0.0001, Mann Whitney U test, n = 81-98 for each group), consistent with a consumptive coagulopathy. To evaluate the role of progestins, 6 dpf larvae were treated with estradiol and levonorgestrel, desogestrel, or norethisterone, alone or in combination, and quantitatively scored for the presence of thrombosis by a blinded observer. The rates of thrombosis were 72% for estradiol, ranged 15-31% for the progestins, and ranged 66-70% for the combined estrogen/progestin treatments. In summary, our results are nearly identical to loss of antithrombin in zebrafish, although the consumptive coagulopathy is not as potent. These data are consistent with human estrogen-induced venous thromboembolism, but it was completely unexpected and concerning to see the production of thrombi in such a rapid fashion. Our data also suggest that progestins do not mitigate nor potentiate this effect. Future screens using this model are ideally posed to identify the mediators of sex hormone-induced thrombosis, and the results could potentially lead to innovative preventive or therapeutic modalities. Disclosures Shavit: CSL Behring: Consultancy; Octapharma: Consultancy; Shire: Consultancy; Bayer: Consultancy, Research Funding.

Beschreibung: Key Points Juvenile zebrafish tolerate widespread coagulopathy due to complete ablation of antithrombin III, but develop lethal thrombosis as adults. In vivo structure/function analysis of antithrombin III in zebrafish reveals limited roles for heparin-binding and anti-IXa/Xa activity.