Histone-induced thrombotic thrombocytopenic purpura in adamts13-/- zebrafish depends on von Willebrand factor

Thrombotic thrombocytopenic purpura (TTP) is caused by severe deficiency of ADAMTS13 (A13), a plasma metalloprotease that cleaves endothelium-derived von Willebrand factor (VWF). However, severe a13(-/-) deficiency alone is often not sufficient to cause an acute TTP; additional factors may be required to trigger the disease. Using CRISPR/Cas9, we created and characterized several novel zebrafish lines carrying a null mutation in a13(-/-), vwf, and both. We further used these zebrafish lines to test the hypothesis that inflammation that results in neutrophil activation and release of histone/DNA complexes may trigger TTP. As shown, a13(-/-) zebrafish exhibit increased levels of plasma VWF antigen, multimer size, and ability of thrombocytes to adhere to a fibrillar collagen-coated surface under flow. The a13(-/-) zebrafish also show an increased rate of occlusive thrombus formation in the caudal venules after FeCl3 injury. More interestingly, a13(-/-) zebrafish exhibit similar to 30% reduction in the number of total, immature, and mature thrombocytes with increased fragmentation of erythrocytes. Administration of a lysine-rich histone results in more severe and persistent thrombocytopenia and a significantly increased mortality rate in a13(-/-) zebrafish than in wildtype (v) ones. However, both spontaneous and histone-induced TTP in a13(-/-) zebrafish are rescued by the deletion of vwf. These results demonstrate a potentially mechanistic link between inflammation and the onset of TTP in light of severe A13( )deficiency; the novel zebrafish models of TTP may help accelerate our understanding of pathogenic mechanisms and the discoveries of novel therapeutics for TTP and perhaps other arterial thrombotic disorders.