Page 268 - Haematologica April 2020

L. Zheng et al.
we demonstrated that there was no detectable a13 activity towards a FRETS-VWF73 substrate in zebrafish plasma (Figure 1D). Together, our results demonstrate that the a13 gene in zebrafish is successfully deleted.
a13-/- zebrafish are in a prothrombotic state
Under unprovoked conditions, plasma levels of VWF antigen and multimer size were increased in a13-/- zebrafish compared with those in the wt controls (Figure 2A-C). When a caudal vein of a zebrafish larva (gata- 1/dsRed and fli-1/eGFP) was exposed to 0.3% FeCl3 (Figure 2D), an oxidative injury to vascular endothelium (the loss of green fluorescence) and an accumulation of erythrocytes (red fluorescence) were observed in a13-/- zebrafish (Figure 2E). The time to complete occlusion of the injured venule in a13-/- zebrafish was significantly shorter (mean ± SEM, 2.1±0.3 min) than that in the wt con-
trols (13.6±1.8 min) (Figure 2F) (P<0.001).
To confirm the incorporation of thrombocytes into the
growing thrombus, we performed a similar experiment in a different transgenic zebrafish line, in which gata- 1/dsRed and cd41/eGFP were expressed to label erythro- cytes (red) and thrombocytes (green), respectively. Confocal image analysis demonstrated the incorporation of thrombocytes and erythrocytes into the occlusive thrombus in the caudal vein of zebrafish larva after the oxidative injury (Figure 2G). Additionally, when anticoag- ulated whole blood was perfused over a fibrillar collagen- coated surface under arterial shear (15 dyne/cm2), the sur- face coverage of a13-/- thrombocytes was dramatically increased compared with that of wt thrombocytes (Figure 2H) despite the similar initial rates of thrombocyte accu- mulation (Figure 2I). Supporting this, the length of throm- bocyte-decorated VWF strings (mean±SEM) following perfusion of whole blood of a13-/- zebrafish (87.0±1.1 μm) was significantly longer than that following perfusion of whole blood of wt zebrafish (49.5±1.2 μm) (P<0.01) (Figure 2J). Together, these results demonstrate functional conservation of the a13/vwf axis in zebrafish hemostasis; and that the deletion of a13 in zebrafish results in a pro- thrombotic phenotype.
a13-/- zebrafish develop a spontaneous but mild feature of thrombotic thrombocytopenic purpura
Using total cell counts coupled with flow cytometry, we were able to quantify total, immature, and mature throm- bocytes in whole blood accurately. Under fluorescent microscope, rare mature (green) and immature (orange) thrombocytes on the background of a large number of erythrocytes could be identified (Figure 3A), as previously described.39 The mean blood cell counts in wt and a13-/- adult zebrafish were 3.2 x1012/L and 3.0 x1012/L, respec- tively (P>0.05). Total thrombocytes (mean ± SEM) in wt zebrafish account for ~1% of total blood cells (i.e., 33.9±1.2 x109/L) while total thrombocytes in a13-/- zebrafish account for ~0.7% of total blood cells (i.e. 22.4±0.8 x109/L) (P<0.0001) (Figure 3B). There was a sim- ilar degree of reduction (~30%) in both immature (mean ± SEM, 16.5±0.6 x109/L) (Figure 3C) and mature (5.9±1.7 x109/L) (Figure 3D) thrombocytes in the a13-/- group com- pared with those in the wt controls (24.3±1.1 x109/L and 8.5±0.4 x109/L, respectively) (P<0.0001). The ratio of immature to mature thrombocytes (~74:26) was similar in both the a13-/- and wt groups, suggesting no thrombocyte maturation defect in a13-/- zebrafish. There was no differ-
ence in the number of total, immature, and mature throm- bocytes observed between wt and a13+/- zebrafish (data not shown). The percentages of thrombocytes were also simi- lar in different transgenic zebrafish lines (gata1-dsRed/fli- 1-eGFP vs. cd41-mCherry) (Online Supplementary Figure S3), which further confirmed the accuracy of thrombocyte counts based on eGFP/dsRed expression under the fli- 1/gata-1 promotor.
Microscopic examination of the peripheral blood smears revealed the presence of fragmented erythrocytes in a13-/- (Figure 3E, right), but not in wt (Figure 3E, left) and a13+/- zebrafish (not shown). Quantitative analysis of more than ten blood smears from each group demonstrated a significant increase in the number of fragmented erythro- cytes per high power field in a13-/- zebrafish (mean ± SEM, 3.9±2.3) compared to wt controls (0.4±0.5) (P<0.005) (Figure 3F). Together, our results demonstrate for the first time that a13-/- zebrafish could develop a spontaneous but mild phenotype of TTP.
Lysine-rich histone induces a more severe and persistent thrombotic thrombocytopenic purpura phenotype in a13-/- zebrafish than in wt ones
The plasma levels of histone/DNA complexes are signif- icantly elevated in patients with acute thrombotic microangiopathy, including immune-mediated TTP.29,40 When zebrafish were challenged with a single dose of lysine-rich histone, a significant drop in the number of total, immature, and mature thrombocytes within 24 to 48 h was observed in both wt and a13-/- zebrafish. As shown, in wt zebrafish the number of total (Figure 4A) and imma- ture thrombocytes (Figure 4B) recovered within 48 h, while mature thrombocyte counts recovered 72 h after the histone challenge (Figure 4C). In a13-/- zebrafish, however, thrombocytopenia persisted after the histone challenge for the entire duration of observation (14 days) (Figure 4D). This was not the result of a decrease in immature thrombocytes (Figure 4E) but rather a reduction in mature thrombocytes (Figure 4F). At the end of 14 days after his- tone challenge, there were statistically significant differ- ences in total (Figure 4G), immature (Figure 4H), and mature (Figure 4I) thrombocyte counts between wt and a13-/- zebrafish. Histone challenge also resulted in a marked reduction of erythrocyte counts in a13-/- zebrafish but not wt ones (Online Supplementary Figure S4). Together, these results indicate that an inflammatory mediator such as histone may trigger acute TTP in individuals with severe A13 deficiency.
Kaplan-Meier survival analysis demonstrated a higher mortality rate (~30%) in a13-/- zebrafish than in wt zebrafish (~10%) following the histone challenge (P=0.0002) (Figure 5A). No spontaneous death occurred in either wt or a13-/- zebrafish over a 6-month period of observation without additional stress. To determine whether neutrophil activation and death or other tissue injuries occurred in zebrafish that were challenged with histones, we determined plasma levels of histone/DNA complexes in zebrafish prior to (D0) and 7 days (D7) after histone challenge which allowed for the exogenous his- tone to be cleared. We showed that while there was no statistically significant difference in the plasma levels of histone/DNA complexes between wt and a13-/- zebrafish prior to histone challenge, there were significantly higher levels of these complexes in a13-/- than wt zebrafish 7 days after histone challenge when thrombocytopenia persisted
1110
haematologica | 2020; 105(4)