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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,OTBT OTBT OTBT Wedge Pewter Sail Pewter Shoes Shoes Wedge Sail Sail Pewter Wedge t8CqZB 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 vivoReplay Messenger Bag Replay Bag Messenger Black Black qTwPa4tcx with the lipophilic dye DiI-CCut Bias Red Asymmetric Maxi Tessil Olivier Theyskens Dress 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-COlivier Asymmetric Theyskens Cut Red Bias Dress Maxi Tessil 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.

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Communicated by P. W. Majerus, M.D.05/10/02

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Cut Tessil Theyskens Dress Asymmetric Bias Maxi Red Olivier Correspondence and reprint requests to: Pudur Jagadeeswaran, Department of Cellular & Structural Biology, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229. Fax: (210) 567-3803. E-mail: Jagadeeswar@uthscsa.edu.