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Keywords:

  • ADAMTS13 protein;
  • animal models;
  • hemolytic anemia;
  • thrombocytopenia;
  • thrombotic thrombocytopenic purpura;
  • von Willebrand factor

Summary

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

Thrombotic thrombocytopenic purpura (TTP) is a puzzling disorder in many ways. The disease is difficult to diagnose as analogous symptoms are also found in other microangiopathic disorders. Although ADAMTS13 deficiency is generally required to develop TTP, only some patients with severe ADAMTS13 deficiency do spontaneously develop this disease. It is therefore assumed that environmental and/or genetic factors are needed to cause acute TTP. Nevertheless, acute TTP-like symptoms have also been observed in patients with moderate or normal levels of ADAMTS13. The development of animal models for TTP has allowed a closer look at the specific need for ADAMTS13 deficiency and the necessity for additional triggers in the pathophysiology of TTP. Mouse models for congenital TTP and a baboon model for acquired TTP have been generated. These animal models have also proven to be extremely valuable in developing new treatment strategies for TTP. In the current review, we discuss current animal models for TTP, what we have learned from them and how they were used to test new treatment strategies.


Thrombotic thrombocytopenic purpura

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

Thrombotic thrombocytopenic purpura (TTP) is a rare but life-threatening disease that belongs to the thrombotic microangiopathic disorders. Diagnosis of TTP is difficult as there is an overlap between the clinical presentations of TTP and other thrombotic microangiopathies. Typical for TTP are (i) thrombocytopenia and hemolytic anemia that cannot be explained by another cause of thrombotic microangiopathy, (ii) presence of von Willebrand factor (VWF)-rich microthrombi in capillaries and arterioles of heart, kidney and brain, and (iii) a fast therapeutic response to plasma exchange or infusion [1].

The pathophysiology of TTP can be largely explained by a deficiency of the VWF-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) [2, 3]. ADAMTS13 is an intriguing multidomain metalloprotease (Fig. 1) with VWF as its only known substrate. Regulation of ADAMTS13 activity is unique as conformational changes in VWF are needed before proteolysis can occur [4-6]. Under normal conditions, ultra-large (UL), hyperreactive VWF multimers are secreted from endothelial cells. Due to shear stress in flowing blood, they unfold and are processed by ADAMTS13 into normally sized, quiescent multimers (Fig. 2A). This process prevents spontaneous interaction between UL-VWF and the glycoprotein (GP) Ib/IX/V receptor on circulating platelets. In the absence of ADAMTS13 activity, UL-VWF multimers can accumulate, leading to formation of disseminated thrombi that are rich in platelets and VWF. These microthrombi can block arterioles and capillaries (Fig. 2B) resulting in organ failure and death when left untreated. Thrombocytopenia observed in TTP is explained by consumption of platelets in this thrombotic process, and microangiopathic hemolytic anemia is assumed to be caused by mechanical rupture of red blood cells in the obstructed microvasculature [7, 8].

image

Figure 1. The multidomain structure of ADAMTS13. The multidomain structure of ADAMTS13 is depicted: the metalloprotease domain (M), disintegrin-like domain (D), thrombospondin type 1 repeats (TSR), cysteine-rich domain (C), spacer domain (S) and ‘complement component Clr/Cls, Uegf, and bone morphogenic protein 1’ (CUB) domains.

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image

Figure 2. ADAMTS13 and its role in the pathophysiology of TTP. (A) UL-VWF (blue) is secreted from endothelial cells, after which it unfolds by shear forces in the flowing blood thereby exposing the ADAMTS13 cleavage site. ADAMTS13 (scissors) proteolysis trims the UL-VWF multimers into smaller multimers that do not spontaneously react with platelets (red) (B). When ADAMTS13 is absent or inactive (e.g. by inhibiting autoantibodies, as depicted), UL-VWF is not cleaved and accumulates. Spontaneous interaction of UL-VWF with platelets results in the formation of microthrombi that block capillaries and arterioles.

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TTP exists in both a congenital and an acquired form. Congenital TTP (also known as Upshaw–Schulman syndrome) is caused by defects in the Adamts13 gene including homozygous or compound heterozygous mutations [8-10]. More than 140 different Adamts13 mutations have been reported including missense mutations, small deletions and insertions, non-sense mutations, and splice-site mutations. Mutations are spread over the entire ADAMTS13 protein and mainly lead to decreased secretion and/or reduced activity of mutant ADAMTS13. Incidence of congenital TTP is low and represents < 5% of all TTP cases [11, 12]. More than 80% of TTP patients have the acquired form of the disease caused by circulating anti-ADAMTS13 autoantibodies [11]. Inhibitory antibodies are present in the majority of the acquired TTP patients although non-inhibitory antibodies, probably causing increased ADAMTS13 clearance, have also been reported [13, 14]. Almost each acquired TTP patient has developed inhibitory antibodies against the spacer domain (Fig. 1A), but the majority of these patients also have additional antibodies against other domains of ADAMTS13 [15-17].

Current TTP treatment is based on either infusion of plasma to provide active ADAMTS13 in congenital TTP patients or plasma exchange to remove anti-ADAMTS13 autoantibodies and restore ADAMTS13 activity in acquired TTP patients. Immunosuppressants are also administered to acquired TTP patients to reduce or suppress anti-ADAMTS13 autoantibody formation. Introduction of plasma exchange using fresh frozen plasma for the treatment of TTP reduced the mortality rate from over 90% to below 30% [1, 18].

The complex etiology of TTP

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

Whereas it is commonly accepted that absence of ADAMTS13 activity (< 5%) is an important factor in TTP development, many congenital TTP patients are not diagnosed until early adulthood, with some individuals remaining free of TTP symptoms even through their third decade of life [19]. This led to the idea that a second hit is needed as a trigger to precipitate acute TTP episodes. Various triggering conditions have been described in TTP patients, including pregnancy, infection, heavy alcohol intake, trauma, myocardial infarction, and surgical procedures. A reasonable common feature might be endothelial activation during which UL-VWF is released from the endothelial Weibel-Palade bodies [20]. Besides the environmental modifiers, there is one report on a genetic modifier (a mutation in complement factor H gene) that influenced the pattern of clinical presentation of congenital TTP in different family members [21]. In addition, normal or only slightly reduced ADAMTS13 levels have been found in some patients diagnosed with TTP. Whether the latter is due to misdiagnosis of TTP or lack of uniformity in ADAMTS13 activity assays is still unclear. To better understand the pathophysiologic process of TTP, several efforts have been made to develop animal models of this disease (Table 1).

Table 1. Overview of ADAMTS13-deficient mice and animal models for TTP and their TTP-like symptoms
AnimalStrain/speciesForm of TTPMethodTriggerEarly symptomsof disease: (thrombocytopenia, hemolytic anemia, increase in LDH,VWF-rich microthrombi)End-stage disease: (severe organ failure, death)Reference
MouseC57BL/6J and 129X1/SvJCongenitalDisruption of Adamts13 gene (exons 1–6)NoNoNo [22]
Mouse129/SvCongenitalDisruption of Adamts13 gene (exons 3–6)NoNoNo [23]
MouseC57BL/6J and 129X1/SvJ and CASA/RkCongenitalDisruption of Adamts13 gene (exons 1–6)NoNot overtSome isolated cases [22]
MouseC57BL/6J and 129X1/SvJ and CASA/RkCongenitalDisruption of Adamts13 gene (exons 1–6)Shiga toxinOnly in a subset of miceOnly in a subset of mice [22, 44]
MouseC57BL/6J and 129X1/SvJCongenitalDisruption of Adamts13 gene (exons 1–6)Recombinant human VWFYesNo [46]
BaboonPapio ursinusAcquiredInjection of inhibiting anti-ADAMTS13 monoclonal antibodyNoYesNo [47]

Mouse models of TTP

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

ADAMTS13 deficiency is not sufficient to develop TTP in mice

Given that ADAMTS13 deficiency is the main factor in development of TTP, the generation of ADAMTS13 knockout mice was a logical evolution. Two groups have generated ADAMTS13-deficient mice via gene targeting, and both groups came to the same remarkable conclusion that ADAMTS13-deficient mice did not spontaneously develop TTP.

Motto et al. [22] were the first to report the generation of ADAMTS13-deficient mice. In a mixed-strain C57BL/6J and 129X1/SvJ genetic background, the murine Adamts13 gene was disrupted, by replacing exons 1–6 with a neomycin resistance cassette. This resulted in a complete loss of ADAMTS13 activity in Adamts13−/− mice. However, none of the Adamts13−/− animals showed any evidence of TTP. Indeed, despite the total lack of ADAMTS13 activity, peripheral blood analysis of Adamts13−/− mice did not show any signs of thrombocytopenia or schistocytosis (fragmented red blood cells), nor did histopathologic examination of kidney, brain, and heart reveal the presence of VWF-rich thrombi typically seen in affected organs from humans with TTP.

Also, Banno et al. [23] generated ADAMTS13-deficient mice. Here, the Adamts13 gene was disrupted, by eliminating exons 3–6 to produce Adamts13−/− mice on a pure 129/Sv genetic background. The resulting Adamts13−/− mice showed complete absence of ADAMTS13 enzymatic activity. Similar to the observations of Motto et al., no differences were observed between Adamts13−/− and Adamts13+/+ animals. Platelet counts and plasma levels of haptoglobin (reflecting hemolysis) were normal, and peripheral blood smears did not show erythrocyte fragmentation. These two studies clearly demonstrated that complete deficiency of ADAMTS13 is not sufficient to produce the typical symptoms of TTP in these strains of mice. Of note, whereas pregnancy is a known potential trigger for TTP in humans [24], both strains of ADAMTS13-deficient mice had uncomplicated pregnancies and produced viable offspring that exhibited normal development and survival.

One obvious factor that could influence TTP development is the quantity and quality of VWF. As it was previously reported that mice on a CASA/Rk genetic background have 5–10 fold higher VWF levels than mice on a mixed C57BL/6J-129X1/SvJ background [25], Motto et al. [22] backcrossed the CASA/Rk genetic background into the C57BL/6J-129X1/SvJ Adamts13−/− mice. Introduction of the Adamts13 gene disruption into the CASA/Rk genetic background resulted in ADAMTS13-deficient mice that exhibited lower average platelet counts and decreased survival. Although spontaneous TTP symptoms were only sporadically observed in Adamts13−/− mice on the CASA/Rk genetic background, a trend of higher frequency was observed when compared with mice on a mixed C57BL/6J-129X1/SvJ background.

ADAMTS13-deficient mice exhibit a prothrombotic phenotype

Despite the lack of spontaneous TTP in ADAMTS13-deficient mice on a 129/Sv or a mixed C57BL/6J and 129X1/SvJ genetic background, the generation of ADAMTS13 knockout mice revealed a clear prothrombotic phenotype when ADAMTS13 is absent. Ablation of the Adamts13 gene was reported to result in a slight increase in VWF levels [23, 26]. ADAMTS13 deficiency led to an increase in the longevity of VWF strings that are released by and stay anchored to activated endothelial cells [6, 27, 28], thereby prolonging VWF-mediated platelet–endothelial interactions [22, 29, 30]. In flow chambers, thrombus formation on immobilized collagen was significantly elevated in blood from ADAMTS13-deficient mice compared with wild-type mice [23]. Shida et al. [31] demonstrated that ADAMTS13 down-regulated thrombus growth by cleaving VWF in the growing thrombus in a shear-rate-dependent manner. Upon injection of a mixture of collagen and epinephrine, ADAMTS13-deficient mice developed more severe thrombocytopenia than wild-type mice (without developing TTP), also indicative of a prothrombotic state in ADAMTS13-deficient mice [23]. When tested in in vivo thrombosis models, ADAMTS13-deficient mice showed accelerated arterial thrombus formation leading to faster occlusion of injured blood vessels [30, 32-34]. The antithrombotic effect of ADAMTS13 seems to partly depend on the C-terminal domains of ADAMTS13 because a C-terminally truncated ADAMTS13 variant (lacking the 2 last TSR and the 2 CUB domains) that is present in certain mouse strains was less capable of delaying thrombus formation than full-length ADAMTS13 [32, 35]. Notably, ADAMTS13 deficiency did not affect bleeding when assessed in a tail clipping bleeding model [23]. Next to the prothrombotic phenotype, ADAMTS13-deficient mice also exhibit a pro-inflammatory state, evidenced by increased leukocyte rolling and adhesion in (stimulated) venules and enhanced extravasation of neutrophils in thioglycollate-induced peritonitis [26]. Together, these data strongly support an anti-inflammatory and antithrombotic effect of ADAMTS13, which was recently further confirmed in several clinically relevant mouse models of stroke [36-39], myocardial infarction [40, 41], and atherosclerosis [42, 43].

Additional triggers induce TTP in mice

Given the suggested need for a second hit to precipitate TTP in certain patients, triggers have also been tested in Adamts13−/− mice to see whether these can recapitulate the clinical TTP symptoms seen in humans. So far, two have been shown to successfully induce TTP-like symptoms in mice: bacterial Shiga toxin and high-dose recombinant (UL-)VWF. The use of bacterial Shiga toxin was originally described by Motto et al. [22]. Shiga toxins are bacterial toxins mainly produced by Shigella dysenteriae and certain strains of Escherichia coli. Infection with these strains is associated with the development of acute hemolytic uremic syndrome (HUS), a thrombotic microangiopathy that shows close clinical resemblance with TTP. Intravenous administration of Shiga toxin triggered TTP in ADAMTS13-deficient mice on CASA/Rk background [22]. Of the thirteen Adamts13−/− mice injected with Shiga toxin, twelve developed thrombocytopenia (> 50% decrease in platelet count). Five mice developed severe anemia, and six mice had died within 10 days after toxin administration. Blood smears and tissue histology confirmed microangiopathic hemolytic anemia (schistocytes and fragmented red blood cells) and the presence of VWF-rich and fibrin-poor hyaline thrombi in the microvasculature of brain, heart, and kidney. Recently, it was shown that the pentameric B subunits of Shiga toxin are sufficient to induce UL-VWF secretion from endothelial cells via the Gb3 receptor present in lipid rafts [44]. Similar to the complete toxin, infusion of the B subunits also provoked TTP-like symptoms in ADAMTS13-deficient mice on CASA/Rk background [44]. Thus, infusion of Shiga toxin in Adamts13−/− mice gives rise to symptoms that closely resemble human TTP, at least in a subset of treated animals. The requirement for VWF in this model was experimentally confirmed by showing that CASA/Rk mice deficient in both ADAMTS13 and VWF were not susceptible to Shiga toxin–induced TTP [45].

Strikingly, Shiga toxin administration to Adamts13−/− mice on mixed C57BL/6J-129X1/SvJ genetic background does not result in ADAMTS13-specific TTP-like symptoms [22]. The enhanced susceptibility to Shiga toxin–induced TTP of CASA/Rk Adamts13−/− mice compared with C57BL/6J-129X1/SvJ Adamts13−/− mice could not be attributed to the higher VWF levels because no correlation was found between elevated VWF plasma levels and mortality or thrombocytopenia [22, 45]. Thus, other factors than high VWF levels, such as genetic and/or environmental factors, appear to determine the response of Adamts13−/− mice to Shiga toxin.

However, a recent study by Schiviz et al. [46] showed that administration of recombinant human VWF containing UL-VWF multimers to C57BL/6J-129X1/SvJ Adamts13−/− mice did induce TTP in all treated mice. Indeed, intravenous administration of 2000 U kg−1 recombinant human VWF to ADAMTS13-deficient mice led to rapid severe thrombocytopenia in all animals. In parallel, hematocrit decreased, lactate dehydrogenase (LDH, a marker for tissue damage and hemolysis) increased, and schistocytes became present in blood smears. None of the mice died, suggesting that no end-stage disease was obtained. Necropsy revealed the heart as the most sensitive target organ with rapid onset of extensive platelet aggregation in the ventricles and myocardial necrosis. Lesions in brain and kidney, typically seen in human TTP, were not observed in this model.

Baboon model of TTP

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

Given the closer resemblance of non-human primates to man, we were interested in developing a baboon model of TTP. To this end, a series of monoclonal antibodies was generated against human recombinant ADAMTS13 [47, 48]. Of 33 selected anti-ADAMTS13 antibodies, we identified one, named 3H9 that fully inhibited the activity of human ADAMTS13 in vitro. We could locate the epitope of 3H9 to the active site containing metalloprotease domain of the enzyme and, importantly, 3H9 cross-reacted with baboon ADAMTS13. With the aim of developing an acquired model of TTP in baboons, we administered this inhibitory antibody to healthy baboons and indeed observed that ADAMTS13 activity fell below detection limit (< 5%). Control animals received a non-inhibitory antibody (5C11), the epitope of which is located in the second TSR domain (Fig. 1). Interestingly, the clinical outcome of ADAMTS13 deficiency in our baboon model of acquired TTP did not corroborate the findings in Adamts13−/− mice. Indeed, mere 3H9-mediated inhibition of ADAMTS13 function in baboons was sufficient to immediately provoke TTP symptoms in all treated animals [47]. Administration of control antibody (5C11) had no effect [47]. Inhibition of ADAMTS13 function resulted in severe thrombocytopenia (Fig. 3A) and hemolytic anemia as was evidenced by the decrease in hemoglobin and haptoglobin levels and increase in schistocyte counts (Fig. 3B). Serum LDH levels were also increased. Immunohistochemical staining of different tissues revealed the presence of microthrombi rich in VWF and platelets but poor in fibrin in kidney, heart, spleen, and brain but not in lung (Fig. 3C). Whereas these results show that inhibition of ADAMTS13 function in the absence of additional triggers is sufficient to cause TTP in baboons, none of the animals showed severe organ failure or died during the study. No signs of renal failure were detected, and only one baboon had signs of myocardial ischemia. Hence, this model represents early-stage TTP, and a second hit could be needed to induce end-stage disease with organ failure and death in these baboons [47].

image

Figure 3. ADAMTS13 deficiency alone is sufficient to induce TTP in baboons. (A) The inhibitory antibody 3H9 was administered intravenously to baboons in two boluses (of 600 μg kg−1, indicated by arrows) over the course of two days with a 96-hour follow-up regimen. Platelet counts showed severe thrombocytopenia in animals treated with 3H9 (●, n = 6) but were not affected in animals treated with control antibody (■, n = 5). (B) Peripheral blood smears prepared 48 and 72 h after injection of antibody revealed the gradual appearance of schistocytes (closed arrowheads) and reticulocytes (open arrowheads) in animals treated with 3H9 (right panels) but not in animals treated with control antibody (left panels). (C) Histopathologic analysis confirms disseminated platelet- and VWF-rich aggregates (indicated by arrows) in tissue sections of kidney, heart, brain, and spleen but not in lung. All panels are original magnification x400. This research was originally published in Blood. Feys, et al. [47] © The American Society of Hematology.

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Development of novel therapies for TTP

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

Plasma exchange and plasma infusion are the treatment of choice in patients presenting with TTP. However, plasma exchange remains cumbersome, expensive and increases the risk for minor venipuncture effects but also for major complications including non-fatal cardiac arrest, systemic infection, catheter obstruction, hypotension, venous thrombosis, and even death [1, 18]. Apart from studying the pathogenesis of TTP, establishment of animal models for TTP also allowed to test new treatment strategies for the prevention and treatment of this disease. Administration of recombinant ADAMTS13 or inhibitors of the VWF–GPIb/IX/V interaction have shown promising results and also gene therapy for TTP has been explored.

Recombinant ADAMTS13

Schiviz et al. used their VWF-induced TTP model in mice to test the effect of recombinant human ADAMTS13 on the development of TTP-like symptoms. Upon prophylactic administration of recombinant human ADAMTS13 prior to challenge with recombinant human VWF, none of the ADAMTS13-deficient animals showed clinical, hematologic or pathological signs of TTP [46]. Therapeutic administration of recombinant human ADAMTS13 up to 3 h after challenge with recombinant human VWF reduced the incidence and severity of TTP findings in a treatment interval-dependent manner. Thus, as expected, these data provide compelling evidence that recombinant human ADAMTS13 exerts a protective effect in this mouse model of TTP.

Inhibitors of the VWF–GPIb interaction

As UL-VWF can spontaneously interact with platelet GPIb to form obstructing microthrombi in TTP, inhibition of this interaction has gained increasing attention as a new and attractive approach to prevent or treat TTP. Many such inhibitors have already been developed as novel antithrombotic agents [39, 49, 50]. We used the humanized anti-human VWF monoclonal antibody GBR600 to assess whether it could prevent TTP in our baboon model [51, 52]. In the control group, repeated administration of 3H9 again induced TTP in all treated animals (Fig. 4A). Interestingly, when GBR600 was administered simultaneously with the TTP-provoking antibody 3H9, baboons did not develop severe thrombocytopenia (Fig. 4B) nor hemolytic anemia [52]. One baboon treated with GBR600 did develop moderate thrombocytopenia, but this was linked to an unexplained inefficient inhibition of the GPIb–VWF interaction by GBR600. This relationship between platelet count and degree of VWF inhibition underscored the specificity of this treatment strategy. Together, these data show that blocking the VWF–GPIb interaction can effectively prevent TTP-like symptoms in baboons. From a clinical point of view, it is particularly interesting to develop a treatment strategy that allows reversing ongoing TTP when patients arrive in the hospital. We therefore tested whether administration of GBR600 could reverse already existing TTP symptoms in baboons. When severe 3H9-induced thrombocytopenia occurred, we administered GBR600 to block binding of VWF to GPIb. As soon as GBR600 was injected, platelet counts started to increase to reach again normal values 4 days after injection of GBR600 (Fig. 4C). Recovery of hemolytic anemia was much slower and only started by the end of the study (7 days after injection of GBR600), which can be explained by the slower turnover of red blood cells. Injection of GBR600 did not induce any obvious hemorrhagic events.

image

Figure 4. Blocking the VWF–GPIb interaction prevents and treats TTP symptoms in baboons. All baboons in the control (A), prevention (B), and treatment (C) group received repeated injections of the inhibitory anti-ADAMTS13 antibody 3H9 (black arrow heads). Severe thrombocytopenia was observed in the control group, which did not receive GBR600 (A). Treatment of baboons in the prevention group with GBR600 (white arrow heads) for 5 days prevented 3H9-induced thrombocytopenia (B). Baboons in the treatment group received daily injections of GBR600 (white arrow heads) from day 4 onward. Severe thrombocytopenia was observed at day 3 and 4 but quickly normalized when GBR600 was injected. Data are mean ± SEM, n = 3 in each group (C). This research was originally published in Blood. Feys et al. [52] © The American Society of Hematology.

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The effectiveness of this treatment strategy was independently confirmed by Callewaert et al. who used the nanobody ALX-0681, another inhibitor of the VWF–GPIb interaction in our baboon model of acquired TTP [53]. In the same study setup, they showed that 3H9-induced thrombocytopenia, schistocytosis, and intravascular hemolysis were completely prevented upon prophylactic treatment with ALX-0681. In the therapeutic part of their study, administration of ALX-0681 after TTP onset also convincingly and rapidly reversed platelet counts. Similar to our results, recovery of hemolytic anemia was also much slower and was not complete by the end of the study. Interestingly, Callewaert et al. scored the number of occluded vessels in the kidneys, heart, spleen, brain, and lungs but saw no difference between animals that received ALX-0681 or control. These data suggest that ALX-0681 did not dissolve microthrombi already present in the vessels of affected organs but most probably prevented new aggregate formation, which leads to restoration of platelet counts. Again, no bleeding was detected via brain CT scans and postmortem analysis of organs. The efficacy and safety of this nanobody as adjunctive treatment to plasma exchange in patients with acquired TTP is now assessed in the TITAN trial, which is a Phase II, single-blind, randomized, placebo-controlled trial [54].

Gene therapy

Another novel therapy that is being explored using ADAMTS13-deficient mice is gene therapy, in which ADAMTS13 expression is restored via gene-based approaches. Several strategies have been reported to result in sustained expression of transgene-encoded ADAMTS13, including in utero lentiviral Adamts13 gene transfer [34], autologous transplantation of hematopoietic progenitor cells transduced ex vivo with lentiviral Adamts13 vectors [33] and systemic administration of adenovirus encoding ADAMTS13 [55]. However, none of these approaches have been tested in the context of TTP in these mice to assess their potential protective effect on this disease.

Lessons from TTP animal models

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

TTP is a puzzling disorder in many ways. The disease is difficult to diagnose as analogous symptoms are also found in other microangiopathic disorders, and the exact cause of acute TTP episodes remains unclear. Hence, animal models of TTP are of great value for unraveling the exact pathophysiologic mechanisms underlying TTP. From the congenital mouse model of TTP, we learned that an additional trigger, Shiga toxin, was needed to create TTP symptoms in a meaningful number of mice. This confirmed the situation in some TTP patients who could live for years with severe ADAMTS13 deficiency without acute TTP episodes until a certain trigger like pregnancy or infection induced the acute state of the disease. However, this mouse model cannot explain that some patients with severe ADAMST13 deficiency do appear to spontaneously develop acute TTP. An intriguing part of this mouse model is the observation that only a subset of mice developed TTP symptoms upon injection of Shiga toxin. Unraveling what exactly triggered TTP in these mice will be of great value. As endothelial activation and concomitant UL-VWF release are thought to be major contributors to acute TTP, a careful analysis of UL-VWF levels in mice after treatment with Shiga toxin could establish whether, for example, a certain threshold of UL-VWF levels must be exceeded to induce TTP in mice.

Inducing TTP by injecting large amounts of recombinant human UL-VWF resulted in a valuable TTP model in Adamst13−/− mice. However, this model makes it harder to investigate potential triggers that induce UL-VWF release thereby causing acute TTP.

With the acquired baboon model of TTP, we showed that ADAMTS13 deficiency as such is capable of inducing TTP symptoms. Whether additional triggers will induce end-stage disease is currently not known and subject of ongoing research. The baboon model is in accordance with the clinical situation of some patients, where TTP can present in a mild form (or even unnoticed) until additional triggers like pregnancy accelerate the disease [56, 57].

Animal models of TTP also proved very useful in testing novel treatment strategies for this disease. A promising candidate is recombinant human ADAMTS13, which has been developed to provide an alternative for the demanding procedure of plasma infusion or exchange. As expected, recombinant human ADAMTS13 was effective in treating congenital TTP in mice. Although clinical symptoms were clearly improved, the effect on histopathologic changes was less clear. Differences in numbers of microthrombi were not scored but could give valuable information on the recently described antithrombolytic activity of ADAMTS13 [58] in the context of TTP. Using recombinant ADAMTS13 in acquired, TTP is less straightforward as anti-ADAMTS13 antibodies might inhibit infused recombinant ADAMTS13. Interestingly, however, an in vitro study showed that reasonable doses of recombinant ADAMTS13 could overcome neutralizing inhibitors and restore ADAMTS13 activity, supporting the use of recombinant ADAMST13 for the treatment of acquired TTP [59].

Another promising treatment strategy is to block interactions of platelets with UL-VWF. Inhibiting microthrombi formation would prevent occlusion of microvessels and rupture of red blood cells thereby stabilizing platelet counts and preventing hemolytic anemia. However, TTP patients are thrombocytopenic, and thus, additional inhibition of VWF (or platelet) function might increase their bleeding risk. Two independent studies, however, confirmed that blocking VWF function is a very effective and safe therapy to prevent and treat TTP in baboons without inducing bleeding problems. Although blocking of VWF-GPIb reduces thrombocytopenia and hemolytic anemia, it does not replace the defective ADAMTS13 enzyme nor does it remove inhibitory ADAMTS13 antibodies. Hence, the most effective treatment strategy for TTP might result from a combined administration of a VWF blocker and plasma exchange or recombinant ADAMTS13 therapy, thereby targeting both the formation of new microthrombi and the deficiency of ADAMTS13 activity. Whether this combined therapy could result in reduced numbers of plasma exchange still needs to be established. A clinical study with the VWF-GPIb inhibiting aptamer ARC1779 did show an increase in platelet counts and no major bleeding complications but was not powerful enough to demonstrate a reduced requirement for plasma exchange in patients with congenital TTP [60]. Whereas blocking VWF activity was effective in our model of early-stage-acquired TTP, it remains to be determined whether this treatment strategy would also ameliorate symptoms of end-stage disease.

In the future, it will also be interesting to investigate whether N-acetylcysteine (NAC, an FDA approved drug for the treatment of congestive and obstructive lung disease or acetaminophen intoxication), could be used to treat TTP in animal models. Recently, it has been shown that NAC reduces disulfide bonds that link the VWF monomers and hence also reduces the size of the UL-VWF multimers [61]. Injection of NAC in Adamts13−/− mice resulted in rapid resolution of thrombi growing at the activated endothelium [61]. Hence, it is anticipated that also NAC might prevent microthrombi formation in TTP.

Conclusion

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

Animal models for TTP are valuable tools not only to shed more light onto the pathogenesis of TTP but also to develop new treatment strategies for this devastating disorder. It is important to stress that environmental, genetic and physiologic differences between mice, baboons, and humans can hamper straightforward interpretation of data obtained in different species. Future use of TTP animal models will nonetheless be very valuable to further unravel the type of and specific need for triggers in the different stages of TTP and to further develop novel therapies. It will be exciting to follow these new developments, which without doubt will improve diagnosis and treatment of TTP patients.

Acknowledgements

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References

We thank Louis Deforche (IWT 111507) for help with preparing the figures. Publication and part of the work described here were supported by Fonds voor Wetenschappelijk Onderzoek Vlaanderen (grant G.0607.09) and GOA/2011/03.

References

  1. Top of page
  2. Summary
  3. Thrombotic thrombocytopenic purpura
  4. The complex etiology of TTP
  5. Mouse models of TTP
  6. Baboon model of TTP
  7. Development of novel therapies for TTP
  8. Lessons from TTP animal models
  9. Conclusion
  10. Acknowledgements
  11. Disclosure of Conflict of Interest
  12. References
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