ADAMTS13
ADAMTS13 is an important enzyme in the blood, responsible to regulate the size of
the VWF multimers. The gene of ADAMTS13 is encoded by 29 exons located on chromosome 9q34.[15 ]
[17 ] This enzyme, consisting of 1,427 amino acids, is mainly synthesized in the hepatic
stellate cells of the liver and results in a circulating plasma concentration of approximately
1 µg/mL with a half-life of 2 to 4 days.[16 ]
[24 ]
[25 ]
[26 ]
ADAMTS13 consists of several domains: a signal peptide (SP), a propeptide (PP), a
metalloprotease domain (M), a disintegrin-like domain (D), a first thrombospondin
type-1 repeat (T1), a cysteine-rich domain (C), a spacer domain (S), seven additional
thrombospondin type-1 repeats (T2–T8), and two CUB domains ([Fig. 2A ]).[18 ] The C-terminal part of ADAMTS13 contains three linker (L) regions located between
T2 and T3 (L1), T4 and T5 (L2), and T8 and CUB1 (L3; [Fig. 2A ]), which introduce a lot of flexibility in the molecule,[27 ] making it difficult to resolve the crystal structure of full-length ADAMTS13. Each
domain of ADAMTS13 has a role to regulate the multimeric size of the VWF multimers
through a molecular zipper mechanism ([Fig. 2B ]) to avoid spontaneous platelet aggregation.[28 ] The proteolysis of VWF by ADAMTS13 is dependent on conformational changes of both
proteins.[27 ]
[29 ]
[30 ]
[31 ]
[32 ]
[33 ] VWF, a multimeric glycoprotein stored in the Weibel–Palade bodies of endothelial
cells or α-granules of platelets, is secreted in a globular form.[34 ]
[35 ] Upon shear stress, VWF multimers unravel thereby exposing their ADAMTS13 binding
and cleavage site Tyr1605-Met1606 (located in the A2 domain of VWF).[29 ]
[31 ] Under normal physiological conditions, ADAMTS13 circulates in a closed conformation
where spacer and T8-CUB2 domains interact.[27 ]
[32 ]
[33 ] An initial interaction between VWF and the T5-CUB2 domains allosterically activates
ADAMTS13, inducing an open ADAMTS13 conformation in which the interaction between
spacer and CUB domains is abrogated.[27 ]
[30 ] VWF substrate recognition by ADAMTS13 is regulated through exosites present in the
spacer domain, cysteine-rich domain, and disintegrin-like domain. Recently, it has
been shown by the group of Prof. Crawley that the exosites in the disintegrin-like,
cysteine-rich, and spacer domains primarily contribute to the substrate recognition
(Km). However, the exosite located in the disintegrin-like domain also has a major
influence on the catalytic efficiency (kcat) of VWF proteolysis.[36 ] Indeed, binding of the disintegrin-like domain to VWF allosterically activates the
otherwise latent metalloprotease domain, giving VWF access to the active-site cleft,
resulting in the proteolysis of VWF.[36 ]
Fig. 2 ADAMTS13 domain organization and molecular zipper mechanism. (A ) ADAMTS13 domain organization. ADAMTS13 consists of several domains: a signal peptide
(SP), a propeptide (PP), a metalloprotease domain (M), a disintegrin-like domain (D),
a first thrombospondin type-1 repeat (T1), a cysteine-rich domain (C), a spacer domain
(S), seven additional thrombospondin type-1 repeats (T2–T8), and 2 CUB domains. The
C-terminal part of ADAMTS13 also contains three linker (L) regions located between:
T2 and T3 (L1); T4 and T5 (L2); T8 and CUB1 (L3). (B ) Molecular zipper mechanism of the proteolysis of von Willebrand factor (VWF) by
ADAMTS13. ADAMTS13 normally circulates in a closed conformation where spacer and T8-CUB2
domains interact (1.). ADAMTS13 will become allosterically activated due to an initial
interaction between D4-CK domains of VWF and its T5-CUB2 domains, thereby inducing
an open ADAMTS13 conformation in which the interaction between spacer and CUB domains
is abrogated (2.). Due to shear stress of the blood, VWF multimers unravel thereby
exposing their ADAMTS13 binding and cleavage sites Tyr1605-Met1606 (located in the
A2 domain of VWF). Exosites present in the spacer domain (3.), cysteine-rich domain
(4.), and disintegrin-like domain (5.) regulate VWF substrate recognition. Binding
of the disintegrin-like domain to VWF will also activate the otherwise latent metalloprotease
domain, giving VWF access to the active-site cleft in ADAMTS13, which will eventually
result in the proteolysis of VWF (6.).
When the ADAMTS13 activity is severely impaired, the VWF multimers will start to accumulate
to which platelets can bind and form VWF/platelet-rich thrombi that cause microvascular
occlusion, resulting in the rare and life-threatening disease TTP.[6 ]
Thrombotic Thrombocytopenic Purpura
The severe deficiency of ADAMTS13 is pathognomonic of TTP and mostly occurs in adults
(90% of all cases).[7 ]
[21 ]
[37 ] The mechanism of severe ADAMTS13 deficiency can either be acquired (95% of all TTP
cases) and mainly immune-mediated (iTTP) due to the presence of anti-ADAMTS13 autoantibodies[14 ]
[38 ]
[39 ]
[40 ] or congenital (cTTP, Upshaw–Schulman syndrome, OMIM 274.150; 5% of all TTP cases)
and linked to biallelic mutations of the ADAMTS13 gene.[15 ]
[22 ]
[41 ]
[42 ]
[43 ]
[44 ]
[45 ] At presentation, TTP may either be idiopathic (50% of TTP patients) or associated
to a pre-existing condition (50% of TTP patients; infections, autoimmune diseases,
human immunodeficiency virus, pregnancy, cancer, drugs [ticlopidine and clopidogrel]).[46 ]
[47 ]
[48 ]
Immune-Mediated TTP
In iTTP, patients have developed an immune response against ADAMTS13.[38 ] However, why suddenly autoimmunity against ADAMTS13 appears and how ADAMTS13 deficiency
is precisely induced are not well understood, but both genetic as well as environmental
factors have been mentioned.[49 ] Human leukocyte antigen (HLA)-DRB1*11 is a well-established predisposing factor
for iTTP, besides female sex (2–3.5F/1M), obesity, and black ethnicity.[1 ]
[7 ]
[21 ]
[50 ] In Caucasians, the HLA-DRB1*11 and HLA-DQB1*03:01 alleles were found to be overrepresented
among iTTP patients, whereas HLA-DRB1*04 was protective.[50 ]
[51 ]
[52 ]
[53 ] A recent Italian study also suggested an association of the rs6903608 variant and
HLA-DQB1*05:03 with acquired TTP.[54 ] Interestingly, HLA-DRB1*11 and HLA-DRB1*03 positive donors could especially present
the CUB2-derived peptides, FINVAPHAR and ASYILIRD, respectively, on dendritic cells
or antigen-presenting cells (APC),[55 ]
[56 ] suggesting that carriers of those HLA genotypes could indeed be more prone to develop
anti-ADAMTS13 autoantibodies and iTTP.
Molecular mimicry between a pathogen (Influenza A , Helicobacter pylori , Brucella , Legionella, …) and ADAMTS13 has also been proposed as a possibility to evoke an immune response
against ADAMTS13.[57 ]
[58 ]
Anti-ADAMTS13 Autoantibodies
To unravel the pathophysiology of iTTP, the anti-ADAMTS13 autoantibodies in iTTP patients
have been a major source of investigation.[59 ]
[60 ]
[61 ]
[62 ]
[63 ]
[64 ] In plasma of iTTP patients, variable anti-ADAMTS13 autoantibody levels have been
detected, indicating that even little amounts of autoantibodies can already induce
ADAMTS13 deficiency.[61 ] Epitope mapping studies, using several ADAMTS13 fragments, revealed that the immune
response in iTTP patients is polyclonal, since anti-ADAMTS13 autoantibodies have been
found against all domains of ADAMTS13.[59 ]
[60 ]
[61 ]
[62 ]
[63 ]
[64 ]
[65 ]
[66 ] However, the spacer domain has been identified as an immunogenic region in ADAMTS13,
since anti-spacer autoantibodies are present in the majority of the iTTP patients.[59 ]
[60 ]
[61 ]
[62 ]
[63 ]
[64 ]
[65 ] Anti-ADAMTS13 autoantibodies are classified into inhibitory (neutralizing ADAMTS13
proteolytic activity) and noninhibitory (binding to the protease and accelerating
its clearance from plasma) autoantibodies.[38 ]
[67 ]
[68 ]
[69 ] Although inhibition was widely accepted as the major cause of ADAMTS13 deficiency,
novel findings show that antigen depletion significantly contributes to ADAMTS13 deficiency.[64 ] Interestingly, also anti-ADAMTS13 autoantibodies which cause an open ADAMTS13 in
iTTP patients have recently been identified.[70 ]
[71 ] However, further research is needed to unravel the role of those “opening” antibodies
in the pathophysiology of iTTP. Also the isotype class or subclasses of the anti-ADAMTS13
autoantibodies have been investigated, in which anti-ADAMTS13 autoantibodies are mainly
of the immunoglobulin G (IgG) isotype (with IgG4 being the most common, followed by
IgG1), but in 20% of the patients also IgAs and IgMs are found.[65 ]
[68 ]
[72 ]
[73 ]
[74 ]
[75 ] High IgA anti-ADAMTS13 antibody titers were associated with a worse prognosis as
they were correlated with lower platelet counts, an increased severity, and mortality.[72 ]
[73 ]
[74 ]
Currently, also more than 90 monoclonal anti-ADAMTS13 autoantibodies isolated from
iTTP patients have been cloned using phage display, Epstein–Barr virus immortalization,
or B cell sorting followed by antibody cloning to further understand the immune response
in iTTP patients.[76 ]
[77 ]
[78 ]
[79 ]
[80 ]
[81 ] Remarkably, almost all iTTP patients have VH1–69 encoded anti-ADAMTS13 autoantibodies
and most of the them target the spacer domain.[57 ]
[76 ]
[77 ]
[78 ]
[79 ]
[80 ] However, the role and function of anti-ADAMTS13 autoantibodies outside the spacer
domain is largely unexplored.
Immune Complexes
While almost 100% of the acute iTTP patients have free anti-ADAMTS13 autoantibodies,
ADAMTS13-specific immune complexes were found in 30 to 97%.[82 ]
[83 ]
[84 ] Interestingly, also elevated C3a and C5a complement factors have been found during
acute iTTP, which could either suggest complement activation through ADAMTS13 antigen–antibody
immune complexes (classic pathway) or through the alternative pathway.[58 ]
[82 ]
[85 ] Remarkably, ADAMTS13-specific immune complexes were also found in 93% of the patients
during remission.[83 ] It is thought that immune complexes are quickly cleared from circulation, but exceeding
the clearance capacity could cause build-up of immune complexes and lead to a proinflammatory
state.[86 ] Until now, it is not yet clear if those immune complexes and the complement system
play a major role in the etiology of iTTP.
Congenital TTP
cTTP is also rare and its prevalence uncertain (∼0.5–2 cases per million people).[87 ] cTTP is linked to compound heterozygous or homozygous mutations spread over the
complete ADAMTS13 gene,[15 ]
[22 ]
[45 ]
[87 ]
[88 ]
[89 ]
[90 ]
[91 ]
[92 ] including approximately 200 different mutations in more than 150 patients.[22 ]
[45 ]
[90 ]
[93 ]
[94 ]
[95 ] In most cases, the mutations lead to secretion deficiencies, but they can also influence
ADAMTS13 activity.[41 ]
[93 ]
[96 ]
[97 ] Additionally, also single nucleotide polymorphisms (SNPs) can influence ADAMTS13
secretion and activity and especially the interplay between different mutations and/or
SNPs can affect ADAMTS13.[41 ]
[93 ]
[96 ]
[98 ] The first episode of cTTP typically occurs during the neonatal period or early childhood
(before age 10),[22 ]
[99 ] but can also be evoked during pregnancy and is then often associated with a missense
mutation in exon 24 (p.R1060W mutation).[49 ]
[100 ]
[101 ] The severity of the disease is also variable and probably related to specific ADAMTS13 mutations and to additional genetic or environmental factors.[22 ]
[45 ]
[87 ]
[90 ]
[91 ]
[92 ]
Diagnosis of TTP
Clinical and Biological Suspicion of TTP
A severe thrombocytopenia (typically <30 × 109 /L) and a microangiopathic hemolytic anemia (schistocytes on the blood smear) are
the almost constant signs of TTP, often associated with corresponding symptoms (as
skin or mucosal hemorrhage, weakness, dyspnea) and multivisceral ischemic symptoms
targeting mainly the brain, mesenteric vessels, and the heart.[6 ]
[102 ] An elevated cardiac troponin-I (cTnI) level on admission is an independent predictor
of death or refractoriness to treatment and represents a prognostic indicator in TTP
patients.[103 ]
[104 ]
However, these clinical features are not specific for TTP, and distinguishing TTP
from other disorders can be challenging. The main differential diagnosis of TTP are
other TMA syndromes (as typical and atypical hemolytic uremic syndrome, associated
to either Shigatoxin-producing Escherichia coli or abnormalities of the alternative complement pathway), TMA syndromes associated
with pre-existing conditions (cancer, organ or hematopoietic stem cell transplantation,
sepsis, pregnancy, …), hematological cytopenia (Evans syndrome, isolated thrombocytopenia,
or hemolytic anemia), ischemic manifestations linked to autoimmune disorders (lupus,
immune thrombocytopenia, antiphospholipid syndrome…), or disseminated intravascular
coagulation.[102 ]
[105 ] Moreover, older iTTP patients usually have several comorbidities and atypical neurological
symptoms, leading to a delay in diagnosis.[106 ]
[107 ]
Two clinical scores (French score and PLASMIC score), derived from standard parameters
at presentation, are reliable to identify patients with a severe ADAMTS13 deficiency
who are most likely to benefit from therapeutic plasma exchange (TPE) in emergency
before the results of ADAMTS13 activity become available.[108 ]
[109 ]
ADAMTS13 Investigation
Since 1998, several in-house and commercial assays have been developed for ADAMTS13
investigations.
ADAMTS13 activity measurement is the first test to be performed in patients with a clinical suspicion
of TMA. A severe ADAMTS13 deficiency (activity <10%) supports the diagnosis of TTP.[37 ] A normal or an only partial deficiency in ADAMTS13 activity differentiates other
TMAs or conditions from TTP and reflects enzyme degradation, decreased synthesis,
and secretion or catalytic inhibition of the protease. Assays are based on the degradation
of a substrate (VWF full-length or synthetic peptide of VWF containing the cleavage
site of ADAMTS13) by ADAMTS13 of the tested plasma sample, and the detection of VWF
cleavage products by electrophoresis, platelet aggregation, fluorescence resonance
energy transfer (FRET), or enzyme-linked immunosorbent assays (ELISAs).[14 ]
[105 ]
[110 ]
[111 ]
[112 ]
[113 ]
[114 ]
[115 ] In-house assays using full-length VWF and FRETS-VWF73 are considered as reference
methods for ADAMTS13 activity measurement, ideally calibrated against the new World
Health Organization International Standard ADAMTS13 plasma (N = 50–150%).[110 ]
[111 ]
[112 ]
[114 ]
[116 ] However, these methods are not fully automated and limited to expert laboratories,
which can result in a time delay of 4 to 6 days before the ADAMTS13 activity results
are available. For a few years, commercial FRET and chromogenic assays have been available,[115 ]
[117 ]
[118 ]
[119 ]
[120 ]
[121 ] and the first fully automated chemiluminescence immunoassay has recently been developed.[121 ]
[122 ] Additionally, also a semiquantitative ADAMTS13 activity assay has recently been
released.[123 ]
The identification of anti-ADAMTS13 autoantibodies is the second step of investigation to document the mechanism of ADAMTS13 deficiency.[38 ]
[39 ] Inhibitory ADAMTS13 autoantibodies are screened in vitro using mixing assays of
heat-inactivated plasma samples at several dilutions, and Bethesda assays.[124 ] Anti-ADAMTS13 IgGs are also titrated in vitro using different ELISA methods (in-house
or commercial assays, performed in a specialized laboratory), using different coated
ADAMTS13 antigens and methods of detection.[38 ]
[39 ]
[72 ]
[125 ]
ADAMTS13 antigen level is less important in the diagnosis of TTP, but it has recently been linked to the
outcome of iTTP patients and therefore could be useful to measure.[126 ] In cTTP, measuring ADAMTS13 antigen levels is used for the documentation of ADAMTS13
deficiency and is dependent on the type of mutation. Different monoclonal and polyclonal
antibodies are available to quantify antigen levels using in-house or commercial ELISAs.[69 ]
[70 ]
Sequencing ADAMTS13 gene (NM_139025.4) aims at documenting sequence variations to confirm cTTP, in selected
patients.
Treatment of Thrombotic Thrombocytopenic Purpura
TTP remains a life-threatening disease with a mortality rate of 10 to 20% and a relapsing
tendency in spite of appropriate therapeutic management in patients usually admitted
at the intensive care unit (ICU).[6 ]
[10 ]
[127 ] The recent advances in the treatment of TTP have identified ADAMTS13, anti-ADAMTS13
autoantibodies, and VWF as the three therapeutic targets.[128 ]
[129 ]
[130 ]
[131 ]
[132 ]
Plasma Therapy
In 1991, Rock et al empirically used intensive TPE improving the management of acute
TTP episodes.[133 ] Today, TPE is the established front-line treatment of the acute phase and should
be started as soon as the diagnosis of TTP is established or strongly suspected until
complete remission (platelet count above 150 ×109 /L for 2 consecutive days, together with normal or normalizing lactate dehydrogenase
and clinical recovery).[7 ]
[8 ]
[133 ] In iTTP, daily TPE is more efficacious than plasma infusion, supplying ADAMTS13
and also removing circulating anti-ADAMTS13 autoantibodies and UL-VWF. Twice-daily
TPE could be considered in refractoriness (absence of response to treatment) or exacerbation
of TTP (early recurrence of iTTP within 30 days of treatment response).[134 ]
[135 ] However, immunomodulating agents are needed to definitely suppress the autoimmune
component of iTTP. In cTTP, plasma infusions are usually sufficient to supply ADAMTS13
during the acute phase and prophylactic plasma infusions every 2 to 4 weeks are appropriate
for patients with a chronic disease.[22 ]
[87 ]
[90 ]
Corticosteroids
Corticosteroids are usually associated to TPE as immunosuppressant drugs, given the
autoimmune nature of iTTP. Corticosteroids may also reduce the number of TPE and improve
the tolerance of plasma therapy, despite the paucity of high-quality evidence-based
studies.[136 ]
[137 ]
[138 ]
Rituximab
Rituximab, a humanized anti-CD20 monoclonal antibody, is used in association with
TPE, to induce a depletion of peripheral B cells and to block the production of anti-ADAMTS13
autoantibodies, with a 10 to 14-day delay to be efficient in TTP.[139 ]
[140 ]
[141 ]
[142 ]
[143 ]
[144 ]
[145 ] Rituximab was first introduced periodically in iTTP patients with a suboptimal response
to the standard treatment (four infusions of rituximab 375 mg/m2 , once or twice weekly). Recently, frontline therapy with rituximab reported shorter
hospitalization and time to treatment response, fewer relapses, but no benefit on
early deaths in iTTP patients.[143 ]
[145 ]
[146 ] Preemptive rituximab is still considered in iTTP patients with a persistent or recurrent
ADAMTS13 severe deficiency to prevent relapse. Although the preemptive effect of rituximab
reduces over time, the use of rituximab is associated with a lower mortality during
follow-up.[142 ]
[146 ]
[147 ]
[148 ]
[149 ]
[150 ]
[151 ]
[152 ]
[153 ]
[154 ] Recent studies also investigated whether the doses of rituximab can be reduced,
since multiple infusions of rituximab may expose patients to infection or other long-term
complications although treatment is generally well tolerated.[155 ]
[156 ]
Other Immunomodulatory Drugs
Vincristine, cyclosporine A, cyclophosphamide, or mycophenolate mofetil are used as
a third-line option, in patients with a refractory iTTP to stop the production of
anti-ADAMTS13 autoantibodies.[129 ]
[157 ]
[158 ]
[159 ]
[160 ] Bortezomib, a proteasome inhibitor inducing depletion of residual autoreactive and
pathogenic B-cells and plasma cells, has recently been reported in a few cases to
induce remission in refractory iTTP.[161 ]
[162 ] Splenectomy is considered and has been shown to be successful in more severe patients
with a relapsing or refractory autoimmune TTP who do not respond to other therapies.[163 ]
[164 ]
[165 ]
Caplacizumab
Caplacizumab (ALX-0081) is a nanobody derived from single-chain antibodies naturally
occurring in Camelidae . Caplacizumab targets the A1 domain of VWF and blocks the interaction of VWF multimers
with platelets. The potential of caplacizumab became evident in a safety and efficacy
study using a baboon model for TTP, in which a fast platelet recovery without a severe
bleeding risk was observed.[166 ]
[167 ] Multicenter randomized placebo-controlled clinical trials TITAN (phase II) and HERCULES
(phase III) have shown that caplacizumab administration after each TPE and for 30
days thereafter (or treatment extension for up to 4 weeks more, in cases of persistent
ADAMTS13 deficiency) may shorten the hospitalization stay, the number of TPE sessions,
and the number of days until treatment response, thereby improving preservation of
organ integrity and survival in iTTP patients. Caplacizumab presents a good safety
profile and minor bleedings, considered manageable, are reported.[168 ]
[169 ] Caplacizumab, recently approved in both Europe and United States, provides a therapeutic
bridge to effective immunosuppression in iTTP therapeutic management.[168 ]
[169 ]
[170 ]
[171 ]
[172 ]
[173 ]
[174 ]
rADAMTS13
In cTTP, recombinant ADAMTS13 (rADAMTS13; SHP655, BAX930) was evaluated in a phase
I study and considered safe and well tolerated, with a half-life of 53 hours,[24 ]
[175 ]
[176 ] similar to that reported for endogenous ADAMTS13 in plasma. After rADAMTS13 infusion,
platelet counts increased and UL-VWF decreased in cTTP patients. Currently, a phase
III randomized controlled trial is ongoing in cTTP (https://clinicaltrials.gov/ct2/show/NCT03393975 ). Since administration of rADAMTS13 in iTTP patient plasma and in a rat model for
iTTP could neutralize circulating anti-ADAMTS13 autoantibodies thereby restoring ADAMTS13
activity,[175 ]
[177 ]
[178 ] rADAMTS13 will also be evaluated in a phase II randomized controlled study in iTTP,
by saturating anti-ADAMTS13 antibodies and cleaving UL-VWF (https://clinicaltrials.gov/ct2/show/NCT03922308 ).
N-Acetylcysteine
N-Acetylcysteine (NAC), a Food and Drug Administration–approved mucolytic agent, inhibits
platelet adherence to endothelial cell-anchored soluble UL-VWF by reducing their size.
NAC also reduces intramolecular VWF–disulfide bonds as in its A1 domain, which contains
the binding site of VWF for platelet glycoprotein Ibα (GPIbα).[179 ]
[180 ]
[181 ]
[182 ]
[183 ] Although prophylactic administration of NAC was able to prevent severe TTP signs
in mice, NAC treatment was not able to resolve severe TTP signs in mice or baboons.[183 ] Currently, the use of very high doses of NAC as an adjunct therapy of TTP is investigated
in a phase I trial to assess its potential efficacy in TTP (https://clinicaltrials.gov/ct2/show/NCT01808521 ).
Long-Term Outcomes and Follow-up
Relapses
iTTP patients relapse when they are experiencing a severe decrease in ADAMTS13 activity
by the persistence or the recurrence of anti-ADAMTS13 autoantibodies more than 30
days after stopping TPE.[72 ] Clinical relapses are associated to a risk of death, TPE complications, or ICU hospitalization,[127 ] and their prevention is a concern. Especially patients with a persistently low ADAMTS13
activity should avoid external triggers (infections, pregnancy, surgery) to avoid
evoking an acute iTTP bout. In iTTP, the use of rituximab at the acute phase decreased
the relapse rate up to 1 year, but this effect diminishes over time.[151 ]
[153 ] Considering a persistent severe ADAMTS13 deficiency as a risk factor for relapse,
preemptive rituximab should be considered to restore ADAMTS13 activity together with
peripheral B cell depletion.[126 ]
[148 ]
[150 ]
In cTTP, some patients are dependent on regular prophylactic plasma infusions every
2 or 3 weeks. Prophylactic plasma therapy is also required during pregnancy to avoid
a TTP episode.[22 ]
[87 ]
Long-Term Follow-up
In iTTP, the occurrence of biological or clinical autoimmunity and late relapses are
reported many years following the recovery.[2 ]
[7 ]
[8 ]
[184 ] Long-term follow-up of iTTP patients is based on standard biology, ADAMTS13 activity
monitoring, and medical consultations to detect emerging autoimmune diseases (systemic
lupus erythematosus, Gougerot–Sjögren syndrome, connective tissue diseases), to evaluate
cognitive disturbances, arterial hypertension, and major depression.[5 ]
[8 ]
In cTTP, long-term follow-up is important and outcomes are poorly reported (chronic
kidney disease, stroke, cardiac ischemia, death).[22 ]
[45 ]
[87 ]
[90 ]
Open Questions
Unmet Needs, Gaps of Knowledge, Areas of Debate, and Controversies
Why Do Some Patients Not Relapse Despite a Persistent Severely Deficient ADAMTS13
Activity in Remission?
In clinical remission, a persistent severe ADAMTS13 deficiency predicts a risk of
relapse.[150 ]
[185 ] However, a severe ADAMTS13 deficiency is not sufficient to induce a TTP episode
and additional triggers are required and need to be identified. Some conditions increasing
plasma VWF levels such as inflammation, sepsis, or pregnancy are known to potentially
act as precipitating factors of acute TTP episodes. Recently, a reduced DNase activity
in TTP patient plasma was shown to result in the persistence of neutrophil extracellular
traps (NETs) and could serve as the second hit needed to evoke an acute TTP bout,
since human neutrophil peptides are also able to inhibit ADAMTS13 activity in vitro.[186 ]
[187 ]
[188 ]
What Is the Mechanism Leading to the Loss and Re-establishment of Self-Tolerance of
the Immune System toward ADAMTS13?
Genetic (gender, ethnicity, HLA haplotype) and/or environmental (infection, stress,
surgery, obesity) factors have been mentioned to contribute to the onset of the disease.
Autoreactivity can appear when low-affinity autoreactive CD4+ T cells escape the negative
selection in the thymus and recognize an antigen presented on an APC through an HLA
molecule.[58 ] Genetic factors such as HLA-DRB1*11 in Caucasians[51 ]
[75 ]
[189 ] are predisposing factors for iTTP. Interestingly, especially HLA-DRB1*11 and HLA-DRB1*03
positive donors could present CUB2-derived peptides (FINVAPHAR and ASYLIRD) on dendritic
cells or APC,[55 ] while reactive CD4+ T cells against those peptides have been identified in iTTP
patients,[56 ]
[189 ] suggesting that stimulation of low-affinity self-reactive CD4+ T cells might play
a role in the development of anti-ADAMTS13 autoantibodies. Other factors including
hormones, cytokines, and other mediators may play a role in the loss of self-tolerance
and the initiation of the immune response toward ADAMTS13.
Infections have been found to be an underlying cause for triggering an acute iTTP
episode. Therefore, it has been suggested that molecular mimicry between the pathogen
and ADAMTS13 could be responsible for the development of reactive anti-ADAMTS13 autoantibodies
causing iTTP.[58 ]
Further studies are required to elucidate these mechanisms leading to a loss of self-tolerance.
What Is the Role of the Open ADAMTS13 Conformation in the Loss of Self-Tolerance and
Development of Anti-ADAMTS13 Autoantibodies?
Exposure of cryptic epitopes can lead to the development of autoantibodies. Recently
it has been shown that iTTP patients have an open ADAMTS13 conformation and anti-ADAMTS13
autoantibodies against cryptic epitopes in ADAMTS13 have been identified.[70 ]
[81 ]
[190 ] It has been suggested that the development of anti-ADAMTS13 autoantibodies could
be caused by exposure of cryptic epitopes due to interaction of ADAMTS13 with certain
drugs, since several drugs (ticlopidine, clopidogrel) have been associated with the
onset of iTTP.[46 ]
[47 ]
[48 ] However, also other mechanisms like posttranslational modifications (glycosylation,
oxidation, citrullination…) could lead to the exposure of cryptic epitopes.[191 ] Indeed, changes in N-linked glycans in the CUB domains resulted in the exposure
of a cryptic epitope in the metalloprotease domain in vitro.[191 ] Interestingly, also anti-ADAMTS13 autoantibodies purified from iTTP patients are
able to change the ADAMTS13 conformation.[71 ] However, whether this open ADAMTS13 is the initial step in the loss of self-tolerance
and the development of anti-ADAMTS13 autoantibodies or whether it is a consequence
of the immune response needs further investigation.
Why Do Some Patients Frequently Relapse despite an Appropriate Immunosuppressive Therapy
or Why Are Some Patients Unresponsive to Rituximab?
Despite the same therapeutic management, some patients have a refractory iTTP to standard
treatment, or relapse after reaching treatment response. Some patients are also unresponsive
to rituximab and the mechanisms by which Bcells resist to rituximab remain unclear.
The hypothesis is based on resistance to complement-dependent cytotoxicity, to antibody-dependent
cellular toxicity, to apoptosis and downregulation, or modulation or loss of CD20
on B cells by transformation into plasma cells.[192 ]
How to Manage cTTP Patients with the p.R1060W Mutation in a Non-obstetrical Context?
The mutation p.R1060W (c.3178C > T) in the TSP1–7 domain of ADAMTS13 is reported with
a high prevalence in adult-onset cTTP, mostly in women during their first pregnancy,[22 ]
[87 ]
[101 ]
[193 ] and some cases have been reported in men.[100 ]
[194 ] The p.R1060W mutation is associated with a residual ADAMTS13 activity, supporting
the absence of a TTP episode during childhood for most patients. However, some conditions
are precipitating factors of acute TTP by increasing plasma VWF levels, as an inflammatory
context or surgery for example, and there are no guidelines on how to manage these
conditions.
Are the Diagnosis Algorithms Sufficiently Reliable to Initiate a Frontline Treatment
with TPE, Rituximab, and Caplacizumab? How to Improve a Rapid Diagnosis of TTP?
TTP is still underdiagnosed and challenging by its rarity and non-specificity of clinical
features at presentation. In the absence of rapid turnaround assays for ADAMTS13 activity
measurement, clinical scores are calculated, but are they reliable enough to initiate
a treatment with TPE, rituximab, and caplacizumab?[108 ]
[109 ] The prompt availability of ADAMTS13 activity measurement could be helpful to adjust
the front-line treatment to the severity of the disease. Recently the first fully
automated chemiluminescence immunoassay for the measurement of ADAMTS13 activity in
citrated human plasma has been developed and evaluated by the Italian group with promising
results.[121 ]
[122 ] Additionally, a semiquantitative ADAMTS13 activity assay has recently been released
to make ADAMTS13 activity measurements directly available, and is under evaluation
in several countries.[123 ]
What Is the Place of Emerging Treatment Strategies in the Management of iTTP?
From the 1990s, the standard treatment of TTP patients involved TPE and additional
immunosuppression. In the 2000s, rituximab has been used as an additional treatment
to TPE, which reduces the number of TPE procedures and additionally increases relapse-free
survival.[143 ] Recently, the use of rituximab as a frontline therapy, together with TPE and steroids,
was reported by the French, UK and U.S. groups.[143 ]
[144 ]
[195 ] However, the minimal effective dose of rituximab should be defined in a clinical
trial (https://clinicaltrials.gov/ct2/show/NCT01554514 ).[130 ]
[131 ]
[196 ] Preemptive rituximab could also be used in remission when ADAMTS13 activity drops
below 10%, to avoid iTTP relapses. However, preemptive rituximab is debatable among
physicians and there is no international consensus.
Caplacizumab is a new target therapy, used at the acute phase of iTTP and for 30 consecutive
days after the cessation of TPE. After this, the use of caplacizumab can be extended
in patients with a persistent severely deficient ADAMTS13 activity. The management
of caplacizumab therapy with the monitoring of ADAMTS13 activity and the possibility
of home therapy are attractive for the management of TTP patients. Further studies
are required to determine the optimal time for stopping caplacizumab when ADAMTS13
is no longer severely deficient (activity >20%) and its use to prevent TTP relapses.
Whether a combined therapy of rituximab and caplacizumab without the need of TPE will
be sufficient to treat TTP patients also needs further investigation. The high cost
of this treatment is a concern.
Ongoing Research
Therapeutic Management of iTTP
After the introduction of TPE 30 years ago, the management and survival of acute TTP
patients improved significantly. However, in the next few years, novel targeted therapies,
such as low-dose rituximab, caplacizumab, rADAMTS13, and NAC, may change the empiricism
of TPE, thanks to their evaluation by ongoing clinical trials and pilot studies. Bortezomib
and rituximab could be synergistic in refractory iTTP. In iTTP, rADAMTS13 could be
tested together with novel approaches using genetically engineered ADAMTS13 variants
to escape the inhibition by anti-ADAMTS13 autoantibodies,[197 ] or plasmin to bypass ADAMTS13.[198 ]
[199 ] Also other approaches like the use of high doses of magnesium sulfate (https://clinicaltrials.gov/ct2/show/NCT03237819 ), Microlyse (fusion protein between a nanobody and an enzyme that breaks down microthrombi),
efgartigimod and rozanolixizumab (antibodies that block FcRn–IgG interaction inhibiting
IgG recycling and inducing IgG clearance), or IdeS[200 ] are currently under investigation to further improve the management of TTP patients.
Adamts13−/−
mice models have been used to test different gene therapy strategies to cure cTTP,
in which ADAMTS13 -encoding lentiviral vectors, adenoviral vectors, or a non-viral sleeping beauty transposon
system were used for the expression of ADAMTS13 to cure cTTP.[201 ]
[202 ]
[203 ]
[204 ] Therefore, gene therapy may have a role in the future management of cTTP.[203 ]
[205 ]
TTP Pathophysiology
The fibrinolytic enzyme plasmin cleaves ADAMTS13, reduces its activity in vitro, and
is also able to cleave VWF. In iTTP patients, endogenous plasmin levels are increased
during the acute phase and potentially involved during a TTP episode. In mice, administration
of plasmin resolves TTP features, increasing the proteolysis of VWF.[198 ]
[199 ]
Endothelial injury, acute inflammation, or infection may trigger the formation of
microvascular thrombosis in patients with iTTP. Human neutrophil peptides 1–3 (HNPs1–3)
released from activated and degranulated neutrophils inhibit proteolytic cleavage
of VWF. HNPs1–3 may inhibit ADAMTS13 activity, suggesting a link between inflammation,
infection, and formation of microvascular thrombosis in iTTP.[186 ]
[188 ] On top of that, NETs have been found in TTP patient plasma.[186 ] The activation of the complement alternative pathway was reported in iTTP patients
and further studies have to be performed to understand the implication of the complement
pathway and potential therapeutic issues.[206 ]
[207 ]
[208 ] However, an increased complement activation was observed in patients who died during
the acute episode compared with those who achieved remission.[209 ]
Other still unknown epigenetic changes in gene expression, posttranslational modifications
related to environmental influences, should also be further explored.
Open ADAMTS13 Conformation as a Biomarker for iTTP
It has been shown that an open ADAMTS13 conformation is a unique hallmark of iTTP
patients.[70 ] Recently it has been shown that this unique hallmark is not only present during
the acute phase, but also during remission even before a severe drop in ADAMTS13 activity
(<10%).[71 ] This shows that patients should be closely monitored. However, whether the open
ADAMTS13 conformation has a predictive value and whether patients in remission with
an open conformation before a severe drop in ADAMTS13 activity (<10%), indicating
presence of anti-ADAMTS13 autoantibodies, should be treated preemptively should be
further investigated.
Use of ADAMTS13 Variants as Therapy
The immune response in iTTP patients is polyclonal, since anti-ADAMTS13 autoantibodies
against all domains in ADAMTS13 have been identified.[59 ]
[60 ]
[61 ]
[62 ]
[63 ]
[64 ]
[65 ] However, the majority of iTTP patients contain anti-ADAMTS13 autoantibodies which
target the spacer domain.[59 ]
[60 ]
[61 ]
[62 ]
[63 ]
[64 ]
[65 ] The spacer domain plays an important role in both containing the closed conformation
and the interaction with VWF. Therefore, it is under investigation whether circumventing
anti-ADAMTS13 autoantibody binding to the spacer domain, by making an ADAMTS13 spacer
variant, could be a novel therapeutic strategy.[65 ]
[197 ]
[210 ]
[211 ] Therefore, also large epitope mapping studies and further characterization of the
anti-ADAMTS13 autoantibodies are needed, since the functional significance of autoantibodies
targeting domains outside the spacer domain has been largely unexplored. The potential
of this strategy will depend on the diversity of (harmful) anti-ADAMTS13 autoantibodies
outside the spacer domain in the iTTP patients.
