Page 265 - Haematologica April 2020

Hemostasis
Histone-induced thrombotic thrombocytopenic purpurainadamts13-/- zebrafishdependsonvon Willebrand factor
Liang Zheng,1 Mohammad S. Abdelgawwad,1 Di Zhang,1 Leimeng Xu,1 Shi Wei,2 Wenjing Cao1 and X. Long Zheng1
Divisions of 1Laboratory Medicine and 2Anatomic Pathology, Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL, USA
ABSTRACT
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 fur- ther used these zebrafish lines to test the hypothesis that inflammation that results in neutrophil activation and release of histone/DNA com- plexes may trigger TTP. As shown, a13-/- zebrafish exhibit increased lev- els 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 ~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 persist- ent thrombocytopenia and a significantly increased mortality rate in a13-/- zebrafish than in wildtype (wt) 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.
Introduction
Thrombotic thrombocytopenic purpura (TTP), resulting from severe deficiency of the plasma metalloprotease ADAMTS13 (A13), is characterized by thrombocy- topenia and microangiopathic hemolytic anemia with various degrees of organ dysfunction.1,2 Most patients have immune-mediated TTP, caused by an immunoglobulin G (IgG) autoantibody that inhibits plasma A13 activity;3-7 rarely, TTP may be caused by a hereditary mutation or mutations in A13,8,9 resulting in defective secretion of A13 protein, known as congenital TTP.
A13 is primarily produced in hepatic stellate cells10,11 and released into the blood stream where it cleaves endothelium-derived ultra-large (UL) von Willebrand factor (VWF).12 The proteolysis of ULVWF by A13 is crucial for hemostasis1,13 and inflam- mation.14,15 When the ability to cleave ULVWF is compromised, due to deficiency of plasma A13 activity, ULVWF multimers accumulate on the surface of endothelium or at the site of vascular injury, recruit platelets from circulation, and promote for- mation of occlusive thrombi in small arterioles and capillaries – a pathognomonic feature of TTP.12,16 A13 and VWF are highly conserved from zebrafish to mam- mals,17,18 suggesting the importance of the A13/VWF axis in biology.
In the past two decades, studies have shown that patients with severe deficiency of plasma A13 activity often do not develop an acute TTP episode until a stressful
Ferrata Storti Foundation
Haematologica 2020 Volume 105(4):1107-1119
Correspondence:
X. LONG ZHENG
xzheng@uabmc.edu or longzheng01@gmail.com
Received: September 11, 2019. Accepted: November 21, 2019. Pre-published: November 21, 2019.
doi:10.3324/haematol.2019.237396
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/4/1107
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