Lysine Histone-Induced Thrombocytopenia in Zebrafish Depends on Endothelial Von Willebrand Factor

Thrombotic thrombocytopenic purpura (TTP), a potentially fatal blood disorder, is caused by severe deficiency of plasma ADAMTS13 activity, primarily resulting from acquired autoantibodies that inhibit ADAMTS13's ability to cleave von Willebrand factor (VWF). However, severe deficiency of plasma ADAMTS13 activity alone is often not sufficient to cause an acute episode of TTP; additional environmental or genetic factors may be required for the development of acute TTP. Here, we report a creation and characterization of a novel zebrafish model of TTP and use the model to identify a potential mechanistic link between an innate immunity and onset of TTP. Using CRISPR/cas9, we generated adamts13^-/-,vwf^-/-, and adamts13^-/-vwf^-/-zebrafish on a double transgenic (Fli1-eGFP/Gata1-dsRed) background in which erythrocytes, thrombocytes, and endothelial cells were uniquely labeled with an endogenously expressed fluorescent protein. Our results demonstrated that adamts13^-/-zebrafish larvae (5-dpf) had an increased rate of developing occlusive thrombi in the caudal venues after FeCl₃ injury; also, adamts13^-/- adult zebrafish (3-4 months) exhibited ~27% reduction of their total or mature thrombocyte counts with a significantly increased number of fragmented erythrocytes; moreover, an intraperitoneal administration of a lysine-rich histone resulted in severe and more persistent thrombocytopenia with an increased mortality in adamts13^-/-zebrafish than their wild-type littermate; finally, both spontaneous and histone-induced thrombocytopenia and thrombotic microangiopathy in adamts13^-/-was eliminated when endothelial vwf was genetically deleted from zebrafish. Together, these results provide experimental evidences to support a mechanistic link of acute infection or inflammation to acute onset of TTP in patients lacking plasma ADAMTS13 activity.