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ABSTRACT

Zebrafish thrombocytes, the nucleated equivalents of mammalian platelets, have been characterized morphologically, but knowledge about their developmental synthesis and biochemistry is limited. Given the increasing use of zebrafish as a genetic model to study hemostasis, it is important to isolate and study the function of zebrafish thrombocytes. Therefore, the objective of this study was to isolate thrombocytes, study their function in vitro,York Cole Indra Soft Kenneth Sandals Wedge Gold New SwEqqZO and identify the developmental stage at which they enter circulation. To achieve these goals, we developed a method for the selective labeling of thrombocytes and assayed these cells for activation by known mammalian platelet agonists. In both in vitro incubations of whole blood and blood labeled in vivoEtanee Red Etanee Isabel Isabel Marant Marant Sandals wTqx0aq4d with the lipophilic dye DiI-C_{Sandals Sandal Outside 2 Women's 1 Storm Black Superfeet 18}, we found labeling in only a single population of cells. These cells were identified as zebrafish thrombocytes by Wright–Giemsa staining. Using selective DiI-C₁₈ labeling, we showed the formation of thrombocyte aggregates, filopodia, and lipid rafts in response to platelet agonists. Additionally, we showed that aggregates are labeled by binding FITC-conjugated annexin V to exposed phosphatidylserine on the thrombocyte membrane. Using these fluorescent-labeling methods, we developed the first microquantitative assay for thrombocyte aggregation. With this assay, we provided evidence for the presence of an ADP receptor, P2Y₁, in the zebrafish thrombocytes. To study the developmental stage at which thrombocytes appear, we microinjected DiI-C₁₈ into the circulation of zebrafish embryos and identified the presence of DiI-C_{Sandals 1 Outside Black Superfeet Storm Women's Sandal 2 18}-labeled thrombocytes at the 36 h postfertilization stage. These findings will prove helpful in dissecting the functions of thrombocytes in hemostasis and provide further insight into the role of platelets in thrombosis.