Zebrafish: from hematology to hydrology.

Hemostasis is a defense mechanism that protects an organism from bleeding. Abnormal hemostasis results in bleeding disorders and thrombosis. Several factors are known to control hemostasis in mammals. Despite this progress, more factors remain to be identified. Classical genetic approaches have resolved physiological pathways. However, classical genetics could not be applied completely to hemostasis pathways a decade ago. We introduced zebrafish as a model system to study hemostasis and thrombosis using classical genetic methods. First, we established that zebrafish hemostasis is essentially similar to mammalian hemostasis. Secondly, we developed screening tools for isolating hemostatic mutants. These tools enabled us to identify hemostatic mutants, as well as providing a means to study hemostasis by knockdown methods. Continued characterization of the physiology of thrombus formation led to the novel finding of thrombocyte clustering. Recently, we have discovered that fish secrete proteases, which participate in hemostasis and may have direct relevance in humans. Future work to identify most of the players in thrombus formation is underway.

Zebrafish: a genetic model for hemostasis and thrombosis.

Here we review the zebrafish hemostatic system, its relevance to mammalian hemostasis, and its efficacy as a vertebrate genetic model to further the understanding of hemostasis and thrombosis.

Demonstration of the extrinsic coagulation pathway in teleostei: identification of zebrafish coagulation factor VII.

It is not known whether the mammalian mechanism of coagulation initiation is conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VIIa. However, amino acid disparities were found in the epidermal growth factor-2/serine protease regions that are present in the human tissue factor-factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates that the Gla-EGF-EGF-SP domain structure, which is common to coagulation factors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor-factor VIIa interaction.

Developmental expression of vitamin K-dependent gamma-carboxylase activity in zebrafish embryos: effect of warfarin.

Vitamin K-dependent gamma-carboxylation is an essential posttranslational modification required for the functional activity of coagulation proteins such as factors VII, IX, X, and prothrombin. Warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, was used in earlier work on adult zebrafish to provide evidence for the presence of vitamin K-dependent carboxylase in zebrafish. Here we demonstrate the presence of vitamin K-dependent carboxylase activity in zebrafish by directly assaying the microsomal fraction prepared from adult, unfertilized eggs, and embryos from different developmental stages. Gamma-carboxylase activity was detected both before and after fertilization of embryos and the activity levels remained relatively constant from 6 h postfertilization (hpf) through other advanced stages of development. The expression of activity in the early embryos (0-6 hpf) may be due to the presence of maternal protein since the activity was detected even in the unfertilized eggs. Gamma-carboxylase activity in the eggs as well as early embryos suggested that vitamin K-dependent carboxylase is important throughout development. The detection of vitamin K-dependent carboxylase mRNA by RT-PCR and inhibitor studies using warfarin confirmed these activity results. Further, these studies provide a basis for selecting warfarin-resistant zebrafish mutants in order to find genes regulating gamma-carboxylase activity including the yet unidentified vitamin K-epoxide reductase.

Characterization of zebrafish full-length prothrombin cDNA and linkage group mapping.

In this paper, we report the complete cDNA sequence of zebrafish prothrombin. The cDNA sequence predicts that zebrafish prothrombin is synthesized as a pre-proprotein consisting of a Gla domain, two kringle domains, and a two-chain protease domain. Zebrafish prothrombin is structurally very similar to human and other vertebrate prothrombins. Zebrafish and human prothrombin share 53% amino acid identity whereas zebrafish and hagfish prothrombin share 51% identity. Amino acid alignments of various prothrombins identified conservation of many of the functional/structural motifs suggesting that the vertebrate prothrombins may have similar functions. The three-dimensional structure of prothrombin predicted by homology modeling also revealed that the prothrombin fragment 1 and the catalytic domain structures are well conserved except for the insertion of an extra 7-amino-acid loop in the connecting region (CR) between the Gla and kringle I domain of fragment 1. Linkage analysis revealed that the prothrombin gene locus on linkage group 7 in zebrafish is syntenic to the human chromosome 11-prothrombin region suggesting its preservation through evolution. The availability of this cDNA sequence in zebrafish adds to our knowledge of the zebrafish hemostatic system and provides support for the view that similarities between zebrafish and mammalian coagulation exist, thus underscoring the relevance of the zebrafish model for studying human hemostasis.

Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man.

Zebrafish were used as a model to study haemostasis, a vertebrate function of paramount importance. A limitation of the zebrafish model is the difficulty in assaying small amounts of blood to detect coagulation mutants. We report the use of a rapid total coagulation activity (TCA) assay to screen for coagulation defects in individual adult zebrafish. We screened the TCA in 1000 gynogenetic half-tetrad diploids derived from 86 clutches. Each clutch was from a single F1 female offspring of males mutagenized with ethylnitrosourea (ENU). We found 30-50% defective zebrafish among six clutches, consistent with a heritable defect. The assay developed here provided a rapid screen to detect overall coagulation defects. However, because of the limited amounts of plasma, we could not detect defects in specific pathways. Therefore, a novel, ultra-sensitive kinetic method was developed to identify specific pathway defects. To test whether the kinetic assay could be used as a screening tool, 1500 Florida wild-type zebrafish pairs were analysed for naturally occurring coagulation defects. We detected 30 fish with extrinsic pathway defects, but with intact common and intrinsic pathways. We conclude that it is now possible to identify specific coagulation pathway defects in zebrafish.

Identification and characterization of zebrafish thrombocytes.

To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.

Analysis of blood coagulation in the zebrafish.

The zebrafish (Danio rerio) is a unique animal model in which saturation mutagenesis has been used to identify genes involved in vertebrate development. The relevance of the zebrafish as a genetic model for hemostasis depends, in large part, on the degree of similarity between the zebrafish and mammalian systems. The diminutive size of the zebrafish poses technical problems for analysis of coagulation. This study describes methods to obtain citrated whole blood and plasma from the zebrafish, analyze in vitro coagulation in small plasma volumes, obtain uniform dosing of zebrafish with oral anticoagulants, and demonstrate specific factor activities via chromogenic assays. Analysis of the zebrafish system demonstrates the presence of both the intrinsic and extrinsic pathways of coagulation, evidence for prothrombin, factor X, protein C, antithrombin, and heparin cofactor II activity, and a requirement for vitamin K dependent gamma-carboxylation of zebrafish hemostatic proteins. Induction of a morphologically recognizable bleeding phenotype by warfarin treatment is also demonstrated. Characterization of zebrafish coagulation provides evidence that major hemostatic pathways are conserved between zebrafish and man. These similarities indicate that the zebrafish is a relevant genetic model for identification of novel genes involved in hemostasis and thrombosis.

Developmental expression of thrombin in zebrafish embryos: a novel model to study hemostasis.

A partial cDNA encoding zebrafish prothrombin has been cloned and used as a probe to study the temporal expression of prothrombin mRNA during early embryonic development. The results revealed accumulation of prothrombin mRNA in diverse tissues such as the eyes and myotomes in early embryogenesis. We have also examined the enzymatic activity of thrombin in converting fibrinogen to fibrin in individual embryos at different stages of development. We found that the fibrin-forming activity does not temporally correlate with the first presence of thrombin mRNA in the early stages of embryogenesis, but does correlate with the initiation of blood formation. Our ability to observe the fibrin-forming activity in single individual embryo will facilitate studies on identifying recessive mutations affecting blood coagulation, such as the regulatory gene mutations controlling the clotting factor genes. Furthermore, the observation of thrombin activity will also facilitate studies on the blood coagulation pathways in the early embryogenesis in this zebrafish model.

A hemophilia model in zebrafish: analysis of hemostasis.

We have developed artificial hemophilia in zebrafish by treating them with copper and measured their clotting function by a newly developed sensitive clotting time assay. The clotting function can be detected rapidly and reliably in 30 hr larvae and in adult fish by measuring the blood clotting time. We have used this assay to screen wild type zebrafish and identified fish with prolonged clotting time. This verifies the usefulness of this assay in future screening for recessive hemostasis defects generated by chemical and radiation mutagenesis methods.

Effects of hirudin (thrombin specific inhibitor) in zebrafish embryos: a developmental role for thrombin.

To address the role of thrombin in early embryogenesis, hirudin, a thrombin specific inhibitor was microinjected into developing zebrafish embryos to inhibit the temporal activity of thrombin during early embryonic development. We found that the fibrin forming activity is inhibited by the presence of hirudin. We also found that hirudin affects development in zebrafish embryos suggesting thrombin's role in early embryogenesis. This ability to inhibit thrombin activity in developing embryos should facilitate studies on identifying signal transduction pathways affected by thrombin during embryogenesis.

Age-specific regulation of clotting factor IX gene expression in normal and transgenic mice.

Factor IX (FIX), a circulating serine protease that serves as an essential component of the blood coagulation pathway, has been shown to increase with age in humans. We show here that murine FIX mRNA and activity levels also increase with age. Furthermore, one form of hemophilia B, hemophilia B Leyden, which is caused by mutations within the promoter region of the FIX gene, has a distinct age-dependent phenotype. To determine the source of the age-related increases in FIX gene expression, we have analyzed the regulation of the normal FIX gene promoter and FIX Leyden gene promoter with the +13 mutation during aging by generating transgenic mice that contain the -189 to +21 bp promoter segment ligated to a chloramphenicol acetyltransferase reporter gene. We have established that the normal FIX promoter and the Leyden promoter transgenes are expressed in a tissue-specific manner in vivo. The normal FIX promoter transgene does not show any differences in the pattern of expression with age or sex of the organism, whereas the Leyden promoter transgene showed age-dependent male-specific expression. This is the first demonstration of the FIX Leyden phenotype in a transgenic mouse model.

Automation of the Maxam-Gilbert chemical sequencing reactions.

A practical automated method of Maxam-Gilbert chemical sequencing reactions that uses solid-phase chromatography methods to purify DNA following chemical modification and cleavage is described in this report. The automation has primarily been made possible by using specially designed BioPak mini-columns, compatible with the Biomek 1000 automated workstation, which can be utilized in a manner similar to that of standard pipet tips. This automated chromatographic sequencing method produces rapid and reliable data as verified by sequencing a known human factor IX exon VIII gene fragment. The procedure presented in this report is a prototype for a single-fragment reaction and can easily be expanded to perform reactions on as many as 8 fragments at a time. The automation eliminates the tedious and time-consuming steps in the original method and increases the rate of sequence acquisition. This technology makes the Maxam-Gilbert chemical sequencing protocol more accessible, especially in large-scale, automated sequencing projects.

Five novel point mutations: two causing haemophilia B and three causing factor X deficiency.

Factors IX and X are plasma glycoproteins important in the middle phase of the coagulation cascade, and a bleeding disorder of variable severity results from abnormalities in the expression of either gene encoding these proteins. Nearly 380 unique molecular mechanisms cause factor IX deficiency, or haemophilia B, but only a limited number of mutations causing congenital factor X deficiency have been characterized to date. In this study enzymatic amplification has been used to examine the molecular basis for factor IX deficiency in two patients and factor X deficiency in two patients. Genomic DNA was isolated from each patient and synthetic oligonucleotide primers were used in the polymerase chain reaction to amplify each exon, splice junction and polyadenylation site. Amplified DNA was then cloned into pUC18 and sequenced. Five novel point mutations were identified, two occurring in the eighth exon of the factor IX gene and three in the eighth exon of the factor X gene. One of the haemophilia B mutations and one of the factor X mutations altered homologous histidine residues near the serine of the catalytic triad.

Automated column equilibration, washing, sample loading and elution of bench-packed mini-columns.

In this paper we describe an easy and efficient way to pack mini-columns and use them in the Biomek 1000 Automated Laboratory Workstation. These procedures allow for automated column equilibration, column washing, sample loading and elution, and utilization of commercially available components. Various resins can be packed, allowing for automation of a variety of procedures in which minicolumns are used.

Human factor IXLincoln Park: a molecular characterization.

Using genomic DNA prepared from peripheral blood samples of a patient with factor IX deficiency, all eight exons as well as sequences around the splice junctions and putative promoter region of human factor IX DNA have been subjected to polymerase chain reaction (PCR) amplification and sequenced. Comparison of these sequences with normal factor IX gene sequences revealed an insertion in exon VIII that resulted in the alteration of 11 amino acids and the addition of 23 amino acids, all at the carboxy terminal of factor IX. This insertion destroys an Msp I restriction site; carrier detection and antenatal diagnosis in affected kindreds can be performed by testing for the absence of this site. This is the first characterization of a mutation in which insertion in the carboxy terminal region of factor IX causes factor IX deficiency. The genetic change in factor IX in this patient is called Factor IXLincoln Park.

Molecular defect (Gla+14----Lys) and its functional consequences in a hereditary factor X deficiency (factor X "Vorarlberg").

Factor X (FX) "Vorarlberg" is a congenital FX deficiency characterized clinically by a mild bleeding tendency. Homozygous individuals have a FX activity of less than 10% in the extrinsic system and 25% in the intrinsic system. FX antigen is 20%. Using molecular techniques, two point mutations were detected in the coding sequence of the FX Vorarlberg gene: a G----A at base pair 160 in exon II resulting in a change of Gla14 (GAA) to Lys (AAA); a G----A at base pair 424 in exon V resulting in a change from Glu102 (GAG) to Lys (AAG). The mutations abolished a TaqI restriction site in exon II and an MnlI site in exon V. To determine whether these mutations are present on one or on both alleles, restriction analyses of amplified exon II and exon V fragments were performed. Analysis of the pedigree showed that the genotype for the mutation on exon II (homozygous versus heterozygous) correlates with the severity of the phenotypic coagulation defect. We therefore conclude that the mutation in exon II is responsible for the functional defect in FX Vorarlberg. We have also purified the mutant FX protein from patient plasma. Purified FX Vorarlberg is indistinguishable from normal FX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its activity is 15% of normal FX upon activation with factor VIIa/tissue factor, 75% upon activation with factor IXa/factor VIIIa, and 100% upon activation with RVV. Activation at varying Ca2+ concentrations shows that the affinity of FX Vorarlberg for Ca2+ is decreased. Factor Xa Vorarlberg is able to convert prothrombin at a normal rate but also shows decreased affinity for Ca2+ in this interaction. Upon addition of Ca2+, FX Vorarlberg does not undergo the same conformational change as normal FX. Our data show that FX Vorarlberg has a decreased affinity for Ca2+ which impedes a normal conformational change. This leads to a decreased rate of activation by factor VIIa/tissue factor and by factor IXa. The decrease is much more marked for the extrinsic than for the intrinsic pathway.

Allelic association of human dopamine D2 receptor gene in alcoholism.

In a blinded experiment, we report the first allelic association of the dopamine D2 receptor gene in alcoholism. From 70 brain samples of alcoholics and nonalcoholics, DNA was digested with restriction endonucleases and probed with a clone that contained the entire 3' coding exon, the polyadenylation signal, and approximately 16.4 kilobases of noncoding 3' sequence of the human dopamine D2 receptor gene (lambda hD2G1). In the present samples, the presence of A1 allele of the dopamine D2 receptor gene correctly classified 77% of alcoholics, and its absence classified 72% of nonalcoholics. The polymorphic pattern of this receptor gene suggests that a gene that confers susceptibility to at least one form of alcoholism is located on the q22-q23 region of chromosome 11.