Page 20 - Haematologica April 2020

Editorials
in the occurrence of the disease, an insight that was strong- ly supported by the development of animal models of cTTP and iTTP.
Mouse models of thrombotic thrombocytopenic purpura
Several groups have generated ADAMTS13-deficient (ADAMTS13-/-) mice via gene targeting using a mixed- strain C57BL/6J-129X1/SvJ genetic background.17-19 The conclusion from evaluation of these models was that ADAMTS13-/- is not, per se, sufficient to produce the typi- cal symptoms and signs of TTP in these strains of mice which also had normal survival.17 Interestingly, however, when these ADAMTS13-/- mice were backcrossed into the CASA/Rk genetic background resulting in substantially higher plasma VWF levels, the resulting ADAMTS13-/- mice had unusually large VWF multimers, a lower average platelet count and decreased survival, with some mice developing spontaneous TTP manifestations.17 Moreover, when these ADAMTS13-/- mice of C57BL/6J-129X1/SvJ mixed strain that had been backcrossed into the CASA/Rk background were injected with a strong trigger, i.e. shiga- toxin, they developed classic features of acute TTP with severe thrombocytopenia, anemia with schistocytes, increased levels of lactate dehydrogenase (LDH), reflecting both hemolysis and tissue damage, and microthrombi in several organs, especially the heart.17 Using very high doses of intravenous recombinant human (rh) VWF (2000 U/kg of body weight) as a trigger, even the mixed strain C57BL/6J-129X1/SvJ ADAMTS13-/- mice developed acute TTP with severe thrombocytopenia, schistocytic anemia, elevated LDH and myocardial necrosis.19 Interestingly, the prophylactic administration of rhADAMTS13 (200 U/kg) protected the mice from acute TTP and even the therapeu- tic infusion of rhADAMTS13 (320 U/kg), given within 1.5 h after a challenge with rhVWF, could reduce the severity of TTP findings, demonstrating that this model was useful for investigating the efficacy of prophylaxis and treatment of cTTP.19
Thereafter, murine monoclonal antibodies raised in ADAMTS13-/- mice against murine ADAMTS13 were used to generate a mouse model of immune-mediated TTP.20 Injecting a combination of two inhibitory monoclonal antibodies into Adamts13+/+ mice resulted in complete and prolonged inhibition of ADAMTS13 activity for more than 7 days, leading to the appearance of ultralarge VWF multimers in the plasma. In line with the above-men- tioned murine cTTP models, TTP-like symptoms in this murine iTTP model did not occur and could only be induced when rhVWF was injected as an additional trig- ger.20
Baboon model of thrombotic thrombocytopenic purpura
To get closer to man, a non-human primate model of immune-mediated TTP was developed in the baboon (Papio ursinus).21 An inhibitory murine anti-human ADAMTS13 monoclonal antibody was administered intravenously to baboons. In contrast to the mouse mod- els of congenital and immune-mediated TTP discussed above, the monoclonal antibody-mediated complete inhi- bition of ADAMTS13 in the baboon for 4 days resulted in severe thrombocytopenia, microangiopathic hemolytic anemia with increased LDH levels and VWF-rich
microthrombi in most organs without the need for an additional trigger. Nevertheless, none of the animals developed severe organ failure or died during the study.21 Hence, this model represents early-stage iTTP, and a sec- ond hit or prolonged ADAMTS13 inhibition might be needed to induce full-blown TTP with organ failure and death in these baboons.
The baboon iTTP model provided useful data on the feasibility, efficacy and tolerance of new therapeutic strategies for iTTP.22,23 Thus, it could be established that injection of a monoclonal antibody blocking the VWF A1 - glycoprotein Ib interaction was a safe and effective means to prevent and treat the early symptoms of iTTP,22 whereas the therapeutic application of N-acetylcysteine was unable to resolve established TTP manifestations such as thrombocytopenia, hemolytic anemia and organ damage due to failed resolution of microvascular throm- bosis.23
These animal TTP models demonstrated that a com- plete deficiency of ADAMTS13 activity, at least in mice, is not sufficient to produce the typical manifestations of acute TTP which may also depend on the quantity and quality of VWF.24 Several authors have postulated that a “second hit” besides severely deficient ADAMTS13 activ- ity may be needed to bring about acute TTP.25-27 Such sec- ond hits triggering the release of VWF from Weibel-Palade bodies in endothelial cells may include extracellular DNA, histones, e.g. resulting from neutrophil extracellular traps, as well as neutrophil peptides 1-3 (α-defensins) or comple- ment activation products such as sC5b-9 which are gener- ated during infections and inflammation.25-27 Importantly, however, no direct evidence is available to date to support the causative role of these inflammatory mediators in the pathogenesis of TTP. In this regard, more experimental research using additional models is needed to further unravel the pathophysiology and natural course of TTP and to probe new prophylactic and therapeutic interven- tions for this rare and severe disease.
What the ADAMTS13-/- zebrafish reveals about thrombotic thrombocytopenic purpura
The zebrafish (Danio rerio) was reported to be a relevant research tool for studying hemostasis and thrombosis, as it displays a high degree of genetic and functional conser- vation of hemostatic factors including the key functions of VWF, and its nucleated platelets (thrombocytes) seem to have properties comparable to those of human platelets.28 The advantages of this vertebrate model include high fecundity, rapid and external embryonic development, and conservation of virtually all hemostatic factors in the zebrafish genome.
In this issue of Haematologica, Zheng et al.29 report a new model of cTTP in zebrafish with interesting experiments showing the indispensable role of VWF in the occurrence of TTP and the role of a specific histone as an endothelial activator triggering acute severe disease in the context of severe ADAMTS13 deficiency. The authors took advan- tage of a double transgenic zebrafish line (gata-1/dsRed and Fli-1/eGFP), which expresses a red fluorescent protein under the gata-1 promoter in erythrocytes and immature thrombocytes and a green fluorescent protein under the fli-1 promoter in the entire vasculature and thrombocytes.
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