Zebrafish Model for Thrombosis and Brain‐Behavior Studies

Hemostasis is a defense mechanism for preventing fluid loss in an injured organism with a circulatory system. In mammals, it begins at the site of the injury, with platelet adhesion to components of the subendothelial matrix activating a series of platelet signaling events that ultimately form a primary hemostatic plug. This process is followed by coagulation cascade activation, leading to fibrin formation and reinforcement of the plug. Thrombosis, an intravascular form of fibrin formation, is unpredictable and often associated with severe pain. Despite decades of research, many hemostatic and thrombotic factors remain unknown. To address this, we introduced zebrafish as a genetic model for studying thrombosis and pain. The piggyback knockdown method is used to knock down genes related to hemostasis and other biochemical or physiological pathways. Blood collection in zebrafish is important in characterizing hemostasis mutants such as hemophilia, in assaying blood coagulation, and in counting thrombocytes in diseases such as thrombocytopenia. FACS analysis is used to study thrombocyte development. Thrombocyte aggregation by flow cytometry and a plate tilt method is used in the functional evaluation of thrombocytes in hemostasis. Defects in coagulation can be studied by kinetic blood coagulation assays and bleeding assays. Laser thrombosis is a global assay that measures blood clotting, thrombocyte function, and endothelial function, including stasis. This technique is useful when screening for genes affecting hemostasis in a genome-wide manner. As pain is sensed by the PAR2 receptor, the trypsin aversion assay detects this pathway and can be used to identify genes related to pain pathways. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Gene silencing by piggyback knockdown Basic Protocol 2: Blood collection from adult zebrafish Basic Protocol 3: Quantification of thrombocytes by FACS analysis Basic Protocol 4: Thrombocyte aggregation assay by flow cytometry Alternate Protocol 1: Whole-blood aggregation assay by the plate tilt method Basic Protocol 5: Kinetic blood coagulation assays Support Protocol: Preparation of zebrafish thromboplastin Basic Protocol 6: Laser thrombosis assay Basic Protocol 7: Chemically induced gill bleeding assay Alternate Protocol 2: Mechanically induced bleeding assay Basic Protocol 8: Trypsin aversion assay in zebrafish larvae.