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.

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Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use zebrafish to confirm 5 novel human variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of variants. These findings as well as the prolonged survival of mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders.

Bacterial pathogens pose an increasing food safety and bioterrorism concern. Current DNA detection methods utilizing sensitive nanotechnology and biosensors have shown excellent detection, but require expensive and time-consuming polymerase chain reaction (PCR) to amplify DNA targets; thus, a faster, more economical method is still essential. In this proof-of-concept study, we investigated the ability of a gold nanoparticle-DNA (AuNP-DNA) biosensor to detect non-PCR amplified genomic Salmonella enterica serovar Enteritidis (S. enteritidis) DNA, from pure or mixed bacterial culture and spiked liquid matrices. Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry. Our preliminary data indicate that non-PCR amplified genomic DNA can be detected at a concentration as low as 100 ng/mL from bacterial cultures and spiked liquid matrices, similar to reported PCR amplified detection levels. These findings also suggest that AuNP-DNA biosensors are a first step towards a viable detection method of bacterial pathogens, in particular, for resource-limited settings, such as field-based or economically limited conditions. Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

Platelets are a critical component of hemostasis, with disorders of number or function resulting in coagulation disturbances. Insights into these processes have primarily been realized through studies using mammalian models or tissues. Increasingly, zebrafish embryos and larvae have been used to study the protein and cellular components of hemostasis and thrombosis, including the thrombocyte, a nucleated platelet analog. However, investigations of thrombocytes have been somewhat limited due to lack of a robust and simple methodology for quantitation, an important component of platelet studies in mammals. Using video capture, we have devised an assay that produces a rapid, reproducible, and precise measurement of thrombocyte number in zebrafish larvae by counting fluorescently tagged cells. Averaging 1000 frames, we were able to subtract background fluorescence, thus limiting assessment to circulating thrombocytes. This method facilitated rapid assessment of relative thrombocyte counts in a population of 372 zebrafish larvae by a single operator in less than 3 days. This technique requires basic microscopy equipment and rudimentary programming, lends itself to high throughput analysis, and will enhance future studies of thrombopoiesis in the zebrafish.

Bacillus anthracis is a bioterrorism agent classified by the Centers for Disease Control and Prevention (CDC). A highly amplified, nanoparticle-based, biobarcoded electrochemical biosensor for the rapid detection of pagA gene (accession number = M22589) in Bacillus anthracis is reported in this paper. The biosensor system is mainly composed of two nanoparticles: gold nanoparticles (AuNPs) and magnetic microparticles (MMPs). The AuNPs are coated with the 1st target-specific DNA probe (pDNA), which can recognize the target DNA (tDNA), and nanoparticle tracer (NT)-labeled barcode DNA (bDNA) as a signal indicator in a 1:100 probe-to-barcode ratio. The MMPs are coated with the 2nd targetspecific pDNA. After mixing the nanoparticles with the tDNA, the sandwich structure (MMPs-2nd pDNA/tDNA/1st pDNA-AuNPsbDNA-NTs) is formed. A magnetic field is applied to separate the sandwiches from the unreacted materials. Because the AuNPs have a large number of NTs per pDNA binding event, there is substantial amplification. Then, the NTs are dissolved in 1-M nitric acid and the metal ions (Pb 2+ and Cd 2+ ) are detected by square-wave anodic stripping voltammetry on a screen-printed carbon electrode sensor. Using this technique, the detection limit of this biobarcoded DNA sensor is as low as 0.2 ng/mL using cadmium sulfide NTs, or 0.02 ng/mL using lead sulfide NTs. The nanoparticle-based biobarcoded DNA sensor has potential applications in multiplexed detection of bioterrorism threat agents.Index Terms-Biobarcode, DNA sensor, nanoparticle, screenprinted carbon electrode (SPCE).

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<0.05), when compared to wild type siblings. Prothrombin (f2) mRNA transcription was slightly decreased, although the effect was not statistically significant. In summary, we have produced a zebrafish model of human F10 deficiency that exhibits a spontaneous adult lethal bleeding phenotype, although early embryonic/larval survival is unaffected despite an underlying severe hemostatic defect. Further study of this mutant may identify species specific factors enabling this early survival. qPCR analysis in mutant larvae suggests that the level of F10 mRNA or protein indirectly regulates other coagulation factors during development. We have also used this model to evaluate the longstanding hypothesis that coagulation cascade mediated thrombin signaling is required for embryonic vasculogenesis. Thus far we have not found evidence for defects in vessel development or integrity, apoptosis, or endothelial differentiation/specification. Taken together, these data suggest that the effects of F10 loss are restricted to hemostasis, and thus therapies should continue to be focused on this in deficient patients. Further study as to why mutant embryos and larvae are able to tolerate what would likely cause lethal hemorrhage in mammals, as well as small molecule screens using this model, could potentially lead to innovative therapeutic modalities for patients with bleeding disorders. Disclosures Joung: Horizon Discovery: Consultancy; Editas Medicine: Other: Financial interest; Poseida Therapeutics: Other: Financial interest; Hera Testing Laboratories: Other: Financial interest; Transposagen Biopharmaceuticals: Other: Financial interest. Shavit:CSL Behring: Consultancy; Octapharma: Consultancy; Bayer: Consultancy; Baxter: Consultancy.

Thrombosis is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Identification of small molecule inhibitors of thrombosis in an in vivo model would facilitate novel and improved therapeutics for patients. 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 screen for genetic and chemical modifiers of coagulation disorders. We generated a zebrafish model of antithrombin III (AT3) deficiency by targeted mutagenesis using zinc finger nucleases. Homozygous at3 mutants displayed a lethal phenotype due to intracardiac thrombosis between 2 and 7 months of age, yet embryos and larvae appeared grossly normal with no overt evidence of pathologic clotting. Induction of thrombosis at 3-4 days post fertilization (dpf) in homozygous mutant larvae by laser-mediated endothelial ablation resulted in diminished rates of posterior cardinal vein (PCV) occlusion, a bleeding phenotype. To prove functional conservation with mammals, we expressed recombinant zebrafish At3 and demonstrated that it binds human thrombin in vitro. Furthermore, while injection of wild type zebrafish and human cDNAs rescued the laser injury phenotype, zebrafish at3 with a mutation in the putative reactive site failed to do so. We hypothesized that the discrepant larval bleeding and adult thrombotic phenotypes could be accounted for by disseminated intravascular coagulation (DIC). Consistent with this, we observed reduced fibrinogen levels in at3 homozygous mutant plasma, and were able to rescue mutant larvae by injection of human fibrinogen prior to laser injury. To identify the location of consumed fibrinogen, we tagged human fibrinogen with FITC (fluorescein isothiocyanate), followed by infusion into larvae from heterozygous intercrosses at 3 dpf. at3 homozygous mutants displayed extensive PCV fluorescence, which was absent in wild type and heterozygous siblings. Pre-incubation with warfarin completely prevented this phenotype, co-injection of tissue plasminogen activator (TPA) partially prevented fluorescence accumulation, and post-injection of TPA reduced the signal, consistent with our hypothesis of DIC. Our data have uncovered the mechanism underlying the discrepant at3 mutant phenotypes and demonstrated conservation of At3 function in zebrafish. Loss of At3 protein results in DIC in zebrafish larvae secondary to unopposed thrombin activity. Mutants survive, only to succumb to lethal thrombosis as adults. Further study as to why larvae are able to tolerate excessive clot formation, as well as small molecule screens for novel anticoagulants using this model, could potentially lead to innovative therapeutic modalities for affected patients. Disclosures: No relevant conflicts of interest to declare.