Animal models for thrombotic thrombocytopenic purpura: a narrative review

Introduction

Thrombotic thrombocytopenic purpura (TTP) is caused by a severe deficiency of plasma metalloprotease ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13) (1-5). ADAMTS13 is primarily synthesized in hepatic stellate cells (6-8) and endothelial cells (8-10), it is then released into the blood stream where it cleaves endothelium- and platelet-derived ultra-large (UL) von Willebrand factor (VWF) (11-13). The proteolysis of ULVWF is crucial for normal hemostasis and inhibition of inflammation (13-16). When the ability to cleave ULVWF is compromised, ULVWF multimers accumulate on endothelial surfaces or at the site of vascular injury where they recruit platelets from circulation, thus promoting the formation of occlusive thrombi in small arterioles and capillaries. This leads to systemic tissue ischemia and damage, the pathognomonic feature of TTP (11,17).

TTP is a rare but potentially fatal blood disorder, characterized by severe thrombocytopenia and microangiopathic hemolytic anemia with various degrees of organ dysfunction (18,19). Most TTP cases are caused by immunoglobulin G autoantibodies that bind and inhibit plasma ADAMTS13 (i.e., immune TTP or iTTP) (20-23). Rarely, TTP may also be caused by hereditary deficiency of plasma ADAMTS13 activity resulting from ADAMTS13 mutations (i.e., hereditary TTP or hTTP) (5,24-27). The mortality of TTP was ~90% when left unrecognized or untreated (18,28). Significant progress has been made in the past decades in terms of early diagnosis and therapeutic intervention for TTP, which has dramatically reduced the mortality and morbidity associated with the disease.

Therapeutic plasma exchange (TPE), in conjunction with corticosteroids, caplacizumab, rituximab, or other immunosuppressives, known as “triple therapy”, have been recommended by the International Society of Thrombosis and Haemostasis (ISTH) for all patients with new or relapsed iTTP (29). This combination of therapy has become the standard of care for iTTP (29-34). However, the mechanism underlying the onset, progression, exacerbation and/or relapse of TTP remains poorly understood.

This review will describe several animal models of TTP developed in past decades, which may provide tools for further assessing the potential environmental and/or genetic triggers of TTP and test potential novel therapeutics for TTP. We present this article in accordance with the Narrative Review reporting checklist (available at https://aob.amegroups.com/article/view/10.21037/aob-22-18/rc).

Methods

We performed a literature search through PubMed from 1969 to 2022 using free text: TTP and animal model (Table 1). We found a total of 67 peer-reviewed articles. Of these, 34 articles that did not discuss TTP models were excluded, and this gave us 33 articles for analysis and review. Additional articles were included based on historic reading database.

Results

Hereditary TTP models

In hTTP, mutations in ADAMTS13 result in severe deficiency of plasma ADAMTS13 activity (2,5,24,27,35-38), primarily resulting from defective secretion of the ADAMTS13 protein (39). To model this, Adamts13 in animals was deleted to render ADAMTS13 nonfunctional. Both Adamts13-null mice and zebrafish models were generated with a classic homologous recombination through embryonic stem (ES) cells and CRISPR/cas gene editing approach, respectively. These animals were fully characterized and recapitulated some of the key clinical features of TTP in humans including thrombocytopenia, microangiopathic hemolytic anemia, and microvascular thrombosis in major organ tissue, etc. (Figure 1).

Figure 1 Animal models established for studying TTP. Animal models of TTP can be classified into two major groups: hereditary (non-immune) and acquired (immune-mediated) TTP. Hereditary TTP models include Adamts13^-/- mice in various strains, such as C57BL/6J, B6.129, CASA/Rk, and CAST/Ei, and Adamts13^-/- zebrafish. The first two mouse stains (C57BL/6J and B6.129) have low plasma levels of VWF, while the latter two strains (CASA/Rk and CASA/Ei) have high plasma levels of VWF. In all these mouse strains with severe ADAMTS13 deficiency, spontaneous TTP is lacking, but an acute TTP could be elicited by an exogenous trigger, such as an intravenous administration of Shigatoxin-2, or its subunit B, or rhVWF or rmVWF. However, Adamts13^-/- zebrafish developed a spontaneous thrombocytopenia which was exacerbated by an intraperitoneal administration of histone, resulting in more profound and persistent thrombocytopenia, and an increased mortality rate. Acquired TTP models were created by injection or expression of an inhibitory antibody that inhibits plasma ADAMTS13 activity in various animals including baboons, rats, and mice. In balloons, spontaneous TTP occurred following an injection of a monoclonal inhibitory antibody against human ADAMTS13 even without a trigger; in rats and mice, TTP did not occur unless being challenged with a high dose of rhVWF or rmVWF. TTP, thrombotic thrombocytopenic purpura; VWF, von Willebrand factor; rhVWF, recombinant human VWF; rmVWF, recombinant murine VWF; i.v., intravenous; i.p., intraperitoneal; and r.o., retro-orbital.

Mouse models

The first mouse model of TTP was established by Motto et al. (40) by deletion of several critical exons in the Adamts13 gene. Surprisingly, mice with nonfunctional Adamts13 (i.e., Adamts13^-/-) in C57BL/6J background did not develop signs and symptoms consistent with TTP. However, after being crossed with CASA/Rk mice that have elevated plasma levels of VWF for several generations, the resulting new Adamts13^-/- CASA/Rk mice developed spontaneous thrombocytopenia and showed a significantly decreased survival rate. Moreover, challenging these mice with a bacterial toxin, namely shigatoxin-2, could result in more profound and persistent thrombocytopenia, microangiopathic hemolytic anemia, and widespread VWF-rich thrombi in the small vessels, as well as an increased mortality rate (40). These are the classic features of TTP. These results recapitulate some of the features in human hTTP and suggest that severe ADAMTS13 deficiency alone may not be sufficient for the development of an acute episode of TTP; an additional environmental trigger or other genetic factors may contribute to the onset and progression of TTP resulting from severe ADAMTS13 deficiency.

This mouse model has been widely used by other investigators for characterizing the nature and mechanism of a potential environmental trigger (41), for studying the biological functions of ADAMTS13 for anti-thrombosis and anti-inflammation (12-16,42,43), and for testing potential novel therapeutics (44-47). For instance, Huang et al. (41) utilized the murine model to test which subunit of Shiga toxin is required for triggering TTP and what is the molecular mechanism underlying such a triggering effect. There are two variants of bacterial Shiga toxin: Stx1 and Stx2, each is composed of one catalytically active A subunit and 5 identical B subunits (48). While the A subunit is responsible for cytotoxicity of the toxin, B subunits mediate binding to cell-surface receptors (49). When injected into Adamts13^-/- mice (CAST/Ei mice), the B subunits from either Stx1 or Stx2 appeared to be sufficient to trigger TTP (41), resulting from acute release of endothelial VWF (50,51).

To confirm whether an elevated plasma level of VWF is sufficient to trigger TTP when plasma ADAMTS13 is severely deficient, Schiviz et al. (52) reported the creation of a similar murine model. Again, no spontaneous TTP was observed in the Adamts13^-/- mice. However, a single intravenous bolus of recombinant human VWF (rhVWF) (2,000 units/kg body weight) into Adamts13^-/- mice rapidly induced a profound but transient thrombocytopenia. The platelet count returned to normal within one day of the rhVWF challenge. Despite the transient nature of thrombocytopenia, the presence of fragmentation of red blood cells (or schistocytosis), low hematocrit, and elevated serum lactate dehydrogenase (LDH) levels suggest the development of TTP in these mice following the rhVWF challenge. They further tested the efficacy of recombinant human ADAMTS13 (rhADAMTS13) in preventing or treating TTP in this model. As shown, an administration of rhADAMTS13 prior to or together with the rhVWF challenge protected Adamts13^-/-mice from developing an acute TTP or at least reduced the severity of the disease.

To test the efficacy of adeno-associated virus serotype 8 (AAV8)-mediated gene therapy for hTTP, the Adamts13^-/- mice generated by Dr. Motto (40) were utilized. Jin et al. (45) reported that administration of AAV8-hAAT-mdtcs (a truncated form of ADAMTS13) at doses greater than 2.6×10¹¹ vg/kg body weight resulted in sustained expression of plasma ADAMTS13 activity at therapeutic levels. Expression of this truncated ADAMTS13 variant essentially eliminated the circulating ULVWF multimers, prevented severe thrombocytopenia, and reduced mortality in Adamts13^-/- mice following a challenge with shigatoxin-2 (45). The data demonstrates the efficacy and supports the possibility of using the AAV-mediated gene therapy strategy for treatment of hereditary TTP in humans.

Zebrafish models

Zebrafish have gained their popularity and recognition as an excellent vertebrate animal model to study human blood diseases (53-55). The hemostasis-related genes including vwf and adamts13 in zebrafish are highly conserved with their corresponding orthologs in humans (56-58). Zebrafish thrombocytes, which are functionally equivalent to platelets, contain receptors that respond to human platelet agonists including thrombin, adenosinediphosphate (ADP), and collagen, etc. (59-61).

We reported the first TTP model in zebrafish created by CRISPR/Cas9 (62). The results demonstrated that adamts13^-/- (null) zebrafish were viable, but with a 30% reduction in their total thrombocyte counts (62). An intraperitoneal administration of lysine-rich histone, a molecule of damage-associated molecular patterns (DAMPs) (63-65), into these zebrafish resulted in severe and persistent thrombocytopenia, fragmentation of erythrocytes, formation of VWF-rich microvascular thrombi, and increased mortality, consistent with the TTP phenotype (62). Moreover, a deletion of vwf (vwf^-/-) essentially rescued either spontaneous thrombocytopenia or histone-induced TTP in adamts13^-/- zebrafish (62), further confirming the critical roles of ADAMTS13 and VWF in the pathogenesis of TTP (Figure 1). These zebrafish models will be used to discover the role and mechanism of novel environmental factors or genetic modifiers in the pathogenesis of TTP.

Acquired TTP models

To date, there is no animal of true autoimmune TTP beside humans. In humans, TTP is primarily caused by IgG type autoantibodies against ADAMTS13 (21-23,66-73). Risk factors for the development of such autoantibodies are not clear, although TTP is more commonly seen in young females, particularly of African descent (74,75).

To generate acquired deficiency of ADAMTS13, an inhibitory antibody (monoclonal or polyclonal) against ADAMTS13 was injected into a wild-type animal to inhibit plasma ADAMTS13 activity (Figure 1). Following antibody administration, plasma ADAMTS13 activity and the clinical features of TTP were assessed. Similar to the hTTP model, a certain environmental trigger was used to incite an acute episode of TTP, if animals with acquired deficiency of plasma ADAMTS13 did not develop the signs and laboratory evidence of TTP.

Baboon models

Feys et al. (76) reported the first non-human primate TTP model by repeated injections of an inhibitory monoclonal antibody (mAb) to the wild-type baboon (Papio ursinus). They found that a 4-day functional inhibition of plasma ADAMTS13 activity was sufficient to induce spontaneous TTP in baboons without an additional trigger. The baboons receiving inhibitory mAb presented with a characteristic hematologic picture of TTP, including severe thrombocytopenia, schistocytic hemolytic anemia, and a rapid rise in serum LDH activity; additionally, immunohistochemical studies revealed the presence of disseminated platelet- and VWF-rich thrombi in several major organs including kidneys, heart, brain, and spleen. Interestingly, these baboons did not develop a fatal condition (Figure 1). Nevertheless, these results indicate that there may be an unidentified genetic or environmental factor that confers baboons’ susceptibility to TTP when their plasma ADAMTS13 activity is profoundly inhibited. Additionally, the no-fatality resulting from the baboon model of TTP is consistent with the transient nature of supplemented inhibitory antibodies and some potential mechanisms present in baboons that confer a survival benefit in the case of acute TTP.

Rat models

Tersteeg et al. (77) reported the first rat model of TTP. A polyclonal antibody against ADAMTS13 (650 U/kg) was used to block plasma ADAMTS13 activity in wild-type Sprague-Dawley rats. When challenged with rhVWF (2,000 U/kg), these animals with antibody-mediated inhibition of plasma ADAMTS13 activity displayed severe “TTP-like” signs and symptoms, including thrombocytopenia, hemolytic anemia, and VWF-rich thrombi in the kidneys and brain. Again, a subsequent infusion of rhADAMTS13 (400, 800, or 1,600 U/kg) prevented the full development of “TTP-like” symptoms. The amount of rhADAMTS13 was able to override the injected amount of anti-ADAMTS13 antibodies, thus restoring plasma ADAMTS13 activity and allowing the normal degradation of ULVWF multimers to occur. The rat model may have different uses for investigating the biology of ADAMTS13 and its disease association in vivo.

Mouse models

In addition to the genetic mouse models, Deforche et al. (78) reported a mouse model with acquired ADAMTS13 deficiency. Through extensive screening of their monoclonal antibodies (mAbs) against murine ADAMTS13, they found four mAbs that strongly inhibited murine ADAMTS13 activity in vitro (~68–90% inhibition) using a fluorescent resonance energy transfers (FRETS)-VWF73 based assay. Two inhibitory mAbs (13B4 and 14H7) were injected into wild type mice at 1.25 mg/kg, each resulted in nearly complete inhibition of plasma ADAMTS13 activity (96%±4% inhibition, day 1 post injection). This led to the accumulation of ULVWF in murine plasma. Following a single bolus intravenous injection of these two mAbs, inhibition of murine plasma ADAMTS13 lasted for more than 7 days.

Like genetic deficiency of ADAMTS13 in Adamts13^-/- mice, the mice with acquired inhibition of plasma ADAMTS13 activity did not develop spontaneous TTP either. However, 24 hours following an intravenous infusion of rhVWF (500 U/kg), these mice developed marked thrombocytopenia and elevation of serum LDH, consistent with the clinical features of TTP (78) (Figure 1).

To create a persistent acquired deficiency of plasma ADAMTS13 activity, Ostertag et al. (79) used a hydrodynamic injection of naked DNA encoding human anti-ADAMTS13 scFv4-20 that targets at the spacer domain. Ten days following the hydrodynamic injection of scFv4-20, plasma ADAMTS13 activity was completely absent and ULVWF multimers appeared in plasma and thrombus formation after laser injury in cremaster blood vessel was significantly enhanced. However, no spontaneous thrombocytopenia and hemolysis occurred. When an intravenous injection of Shigatoxin-2 was given to the mice at 50 ng/kg weight, all mice with severe ADAMTS13 deficiency developed severe thrombocytopenia and died within 5 days. Histology analysis revealed the presence of disseminated microvascular thrombosis in several major organs including brain, heart, and kidneys in the deceased mice. These results demonstrate for the first time the ability of a cloned human monoclonal recombinant monovalent antibody fragment against ADAMTS13 to recapitulate the key pathologic features of untreated acquired TTP in vivo, validating the clinical significance of isolated monoclonal antibodies and providing a useful animal model for testing novel targeted therapeutic approaches for TTP.

Conclusions

In summary, the TTP animal models created from zebrafish to non-human primates have helped researchers make important progress in understanding the disease mechanism and developing new therapeutics. While hTTP and iTTP are two distinct forms, in terms of the underlying etiology of severe ADAMTS13 deficiency, most iTTP cases also exhibit low levels of ADAMTS13 antigen resulting from accelerated clearance of ADAMTS13/antibody immune complexes. In this regard, iTTP resembles hTTP (80). Thus, both genetically modified and antibody-mediated animal models may be used to test the role of potential environmental factors such as infection and pregnancy or potential genetic modifiers such as complement factor H mutation (81-83) and ANRKD family proteins (84) in the pathogenesis of TTP. Further utilization of these TTP animal models will allow us to test the efficacy and safety of various targeted therapeutic strategies in addition to the restoration of plasma ADAMTS13 activity.

Prospective

Gene editing using the CRISPR/Cas system has made creation of animal models easier and more rapid than ever. Not only can we create an ADAMTS13 knockout animal mutant, but also generate a point-mutation in ADAMTS13 and other associated genetic modifiers that are found in patients with hTTP. Having a true hTTP model, rather than the ADAMTS13 null model, in conjunction with mutations in other genetic modifiers, will significantly enhance our ability to understand the biology of ADAMTS13 and the pathogenesis of TTP. Finally, we are still in urgent need of a true autoimmune TTP animal model, which may help our understanding of the immunological aspect of the disease, such as the triggers of ADAMTS13 autoantibody production and targeted immune therapies.

Acknowledgments

The authors thank Antonia Bignotti, Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, for proofreading of the manuscript.

Funding: This work was supported in part by grants from National Blood Foundation (NBF 22-09R) (to LZ) and National Institutes of Health (HL144552 and HL157975) (to XLZ).

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Blood for the series “Thrombotic Thrombocytopenic Purpura”. The article has undergone external peer review.

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://aob.amegroups.com/article/view/10.21037/aob-22-18/rc

Peer Review File: Available at https://aob.amegroups.com/article/view/10.21037/aob-22-18/prf

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://aob.amegroups.com/article/view/10.21037/aob-22-18/coif). The series “Thrombotic Thrombocytopenic Purpura” was commissioned by the editorial office without any funding or sponsorship. XLZ is a consultant for Alexion, Sanofi, BioMedica Diagnostics, Argenx, and Takeda, as well as a co-founder of Clotsolution. XLZ served as the unpaid Guest Editor of the series and serves as an unpaid editorial board member of Annals of Blood from March 2022 to February 2024. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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