Not known from ADAM(TS-13)—novel insights into the pathophysiology of thrombotic microangiopathies

Gunter Wolf

Department of Medicine, Division of Nephrology, Rheumatology and Osteology, University of Hamburg, Hamburg, Germany

Correspondence and offprint requests to: Gunter Wolf, MD, University of Hamburg, University Hospital Eppendorf, Department of Medicine, Division of Nephrology, Rheumatology and Osteology, Pavilion N26, Martinistraße 52, D-20246 Hamburg, Germany. Email: Wolf{at}uke.uni-hamburg.de

Keywords: ADAMTS-13; haemolytic–uraemic syndrome; thrombotic microangiopathy; thrombotic thrombocytopenic purpura

Introduction

Thrombotic microangiopathies encompass various disease entities that are characterized by haemolytic anaemia caused by fragmentation of erythrocytes and thrombocytopenia due to increased platelet aggregation and thrombus formation, eventually leading to disturbed microcirculation with reduced organ perfusion (Figure 1). Thrombotic thrombocytopenic purpura (TTP) and the haemolytic–uraemic syndrome (HUS) are thrombotic microangiopathies characterized by a distinct pattern of symptoms [1,2]. TTP, first described by Moschcowitz in 1924 [3], has been defined as a disease of adults characterized by severe thrombocytopenia leading to purpura, microangiopathic haemolytic anaemia with fragmented erythrocytes (so-called schistocytes), increased serum lactate dehydrogenase that is largely derived from ischaemic organs rather than from lysed erythrocytes, various neurologic abnormalities, fever and mild renal dysfunction [4]. In contrast, HUS has been considered as a syndrome attacking preferentially children and young persons, with severe acute renal failure and marked anaemia as the predominant features at presentation [4]. Ironically, the first case of TTP described by Moschcowitz in 1924 was a 16-year-old girl with petechiae, anaemia and renal involvement [3,5]. The characteristic pathological findings of both TTP and HUS are incomplete occlusive lesions of arterioles and small arteries, and associated tissue microinfarctions [2]. Characteristic periodic acid–Schiff (PAS)-positive hyaline thrombi are found in the small arteries, arterioles and capillaries of the involved organs, whereas the venous circulation is generally spared [5]. These hyaline thrombi contain platelet aggregates and small amounts of fibrin. There is also endothelial dysfunction indicated by swelling and detachment of the endothelium from the basement membrane. Subendothelial deposits of ‘fluffy material’, thought to contain degradation products of fibrin and extracellular matrix, with overlying endothelial proliferation are often found [5]. Although the organ involvement may vary between TTP (more widespread with lesions in brain, heart, pancreas, adrenals and kidney) and HUS (usually, but not always, limited to the kidneys), the basic pathological lesion is similar [2]. Consequently, it has been proposed that TTP and HUS are a single entity with many causes which initially act through the common mechanism of mediating endothelial cell damage [2]. Research in the past 5 years has provided remarkable new insights into the pathophysiology of these disorders. This review will focus on pathophysiology and will not deal in detail with potential therapeutic approaches.



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Fig. 1. Various different diseases manifest under the clinical picture of thrombotic microangiopathies. Formation of microthrombi in smaller vessels leads to organ dysfunction, consumption of platelets resulting in thrombocytopenia and mechanical fragmentation of erythrocytes inducing haemolytic anaemia.

 
TTP

A major breakthrough in deciphering the pathophysiology of TTP occurred >20 years ago when Moake and associates observed that unusually large von Willebrand factor (UL-vWF) multimers were present in the serum of patients with chronic relapsing TTP [6]. Soon afterwards, immunohistochemical analysis of TTP-associated thrombi showed an abundance of vWF and platelets in these lesions, with only little deposition of fibrin/fibrinogen [7], a finding in sharp contrast to vascular occlusions found in disseminated intravascular coagulation. vWF is an important component of the coagulation cascade and supports platelet adhesion and aggregation. A defect or deficiency in this glycoprotein resulting from gene mutations causes von Willebrand disease. The synthesis of vWF is complex. Pre-pro-vWF is produced in endothelial cells and megakaryocytes. After proteolytic removal of the signal peptide, pro-vWF monomers (280 kDa) assemble into homodimers and multimers of variable size with molecular masses ranging into millions of daltons by formation of disulfide bonds. The propeptide is removed and this highly polymerized form of vWF, also called proto-vWF or UL-vWF, is stored in the {alpha}-granules of platelets and Weibel–Palade bodies of endothelial cells [4]. Plasma vWF comes mainly from the endothelium. Upon secretion into plasma, UL-vWF is converted into smaller forms ranging from dimers up to 20mers. Under shear stress, the UL-vWF binds platelets more efficiently than the smaller forms. It is assumed that binding sites for the platelet glycoprotein Ib{alpha} are more effectively exposed on UL-vWF that unfolds on the surface of vessels than on the smaller vWF multimers (Figure 2). In 1998, Furlan et al. [8] and Tsai and Lian [9] independently reported that patients with acute non-familial TTP have inhibitory antibodies against the UL-vWF-cleaving protease. Moreover, six patients with familial TTP lacked UL-vWF-cleaving protease, but had no inhibitor, suggesting a constitutional deficiency of this enzyme [9]. As a consequence, UL-vWF cannot be cleaved, unfolds under shear stress on endothelial cells, binds platelets, and leads to thrombi.



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Fig. 2. Shear stress promotes formation of microthrombi. Uncleaved large vWF factor unfolds on the surface of injured vessels in the direction of blood flow. Thereby, these factors form a substrate for platelet adhesion and aggregation through exposing binding sites for platelet glycoprotein Ib{alpha}.

 
These breakthrough findings were important for the characterization and cloning of the UL-vWF-cleaving protease [10]. Its is a metalloproteinase called ADAMTS-13 (a disintegrin and metalloprotease, with thrombospondin-1-like domains) and cleaves peptide bonds between Tyr842 and Met843 in monomeric subunits of vWF [10]. Partial unfolding of UL-vWF by fluid shear stress increases the efficiency of cleavage by ADAMTS-13 by exposing the Tyr842–Met843 bond [11]. The gene for ADAMTS-13 is localized on chromosome 9 [10]. The glycosylated enzyme is expressed primarily in the liver, but trace amounts presumably are produced in many organs, and alternative splicing exists [12]. Members of the zinc-containing ADAMTS metalloproteinase family share several distinct structures such as a reprolysin-like metalloprotease domain with a zinc-binding motif, a disintegrin-like domain, and a thrombospondin 1 repeat, as well as cysteine-rich regions [12]. Two putative divalent cation-binding sites (Zn2+ and Ca2+) are found in ADAMTS-13. The enzyme may bind through its thrombospondin 1 domain to specific receptors on the endothelial surface (Figure 3). The only known substrate for ADAMTS-13 is UL-vWF. Little is known about the regulation of ADAMTS-13 expression under physiological conditions [12].



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Fig. 3. Scheme showing the function of ADAMTS-13. In normal individuals, ADAMTS-13 binds to the endothelial cell surface, presumably through its thrombospondin 1 domain. ADAMTS-13 locally cleaves the unusual large vWF polymers that are secreted from the Weibel–Palade bodies of the endothelium. In idiopathic TTP, an antibody against ADAMTS-13 inhibits enzyme activity, whereas in familial forms the enzyme is deficient. As a consequence, UL-vWFs are not cleaved, unfold under shear stress and aggravate platelets, leading to microthrombi. Modified after [4].

 
A severe deficiency of ADAMTS-13 activity is detected in patients with idiopathic TTP during single episodes as well as during episodes of recurrence [11]. An IgG antibody, directed against ADAMTS-13 and inhibiting its activity, was isolated from these patients [11]. Serial observations demonstrated decreased titres of this autoantibody and increased ADAMTS-13 activity during periods of remission. These findings suggest that this type of TTP is an autoimmune disease in which autoantibodies inhibit ADAMTS-13 activity leading to an increase in UL-vWF and, ultimately, microthrombi [11]. Consistent with the autoimmune nature of the disease is the observation that remissions occur after treatment with cyclophosphamide and rituximab that both suppress B cells and antibody production [13]. Furthermore, the superior effect of plasmapheresis compared with plasma infusion alone is presumably due to removal of anti-ADAMTS-13 antibodies by the former approach [14]. The cause of the defect in immune regulation is unknown, but the observation that patients with ticlodipine- or clopidogrel-associated TTP have antibodies against ADAMTS-13 suggests a potential trigger mechanism [11].

A hereditary form of TTP with severe thrombocytopenia and haemolysis is called the Schulman–Upshaw syndrome [11]. These patients have very low levels of ADAMTS-13 caused by mutations of the gene. It has been also found that patients with chronic relapsing TTP, an autosomal recessive disorder, have mutations in the ADAMTS-13 gene [15]. Different mutations have been described that are spread over the entire gene (Figure 4). Interestingly, these patients with ADAMTS-13 mutations have various clinical courses [15]. Some have episodes of TTP in infancy or childhood, but no longer in adulthood. Others have a delayed onset of chronic relapsing TTP, and occasionally patients never have an episode. Clearly, further studies on the genotype–phenotype relationship are necessary.



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Fig. 4. Different mutations (missense, deletions, nonsense mutations) are spread over the ADAMTS-13 gene resulting in dysfunctional enzyme activity found in familial forms of TTP. Modified after [15].

 
HUS

The common form of HUS (D+ HUS associated with diarrhoea) occurs in children and is associated with a bacterial infection, mainly by Escherichia coli O157:H7, that produces one or more powerful Shiga-like toxins (Stxs) [4,16]. Stxs consist of one A subunit of 33 kDa and five B subunits (7.7 kDa each). Infections through contaminated food (insufficiently cooked meat, raw milk and juices), water, and human to human transmission are the main causes [17]. After ingestion, enterohaemorrhagic bacteria bind to mucosal epithelial cells and trigger localized destruction of brush border microvilli. They induce the formation of a pedestal-like actin structure directly beneath adherent bacteria through which bacteria remain extracellularly attached to host cells. In the intestinal epithelium, Stx activates the p38 mitogen-activated pathway, leading to apoptosis [18]. In addition, Stx locally induces multiple C-X-C chemokines in intestinal epithelial cells. Transmigration of neutrophils occurs into the intestinal lumen [19]. Recent evidence indicates that such neutrophil movement is pivotal for Stx translocation in the opposite direction, i.e. across the intestinal epithelium into the bloodstream [19]. In the blood, Stx binds to neutrophils and is transported to target organs including the kidney [20]. The toxin B subunits bind to neutral glycolipid globotriasylceramide (Gb3) receptors that are expressed on glomerular endothelial, mesangial and tubular cells (Figure 5). Specific tissue damage presumably depends on expression of this receptor [21]. The toxin is internalized into the cells through receptor-mediated endocytosis. The route of Gb3 trafficking depends on the type of fatty acids associated with the receptor [21]. The A subunit undergoes partial proteolysis and is targeted to the endoplasmic reticulum where it inhibits elongation of peptide chains via depurination of the 28S rRNA [4]. This inhibition of protein synthesis results in cell death (apoptosis). In addition, Stx induces endothelial cells to secrete UL-vWF, chemokines, cytokines and reactive oxygen species as well as to upregulate the expression of adhesion molecules [4]. Sublethal Stx concentrations could change the production of endothelial-derived vasoactive factors including endothelin and nitric oxide. Finally, Stx binds to activated platelets through Gb3 receptors [4]. All these events combined cause endothelial injury and formation of microthrombi. In contrast to TTP, vWF is not prominent within these HUS-associated microthrombi where binding of fibrinogen to IIb–IIIa complexes on platelets and thrombin deposition plays a major role [4]. Indeed, a recent study shows that in E. coli O157:H7 infection, prothrombin is being converted to thrombin at an early stage of illness, when the platelet concentration and serum creatinine are still normal [22]. These findings suggest that thrombogenesis is a primary step, and may lead to renal insufficiency.



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Fig. 5. Pathogenesis of D+ HUS. Stx binds through its subunits to globotriasylceramide (Gb3) receptors that are expressed on glomerular endothelial, mesangial and tubular cells. This binding activates the release of cytokines and chemokines and also stimulates the secretion of UL-vWF. In addition, the A subunit undergoes partial proteolysis and is targeted to the endoplasmic reticulum where it inhibits elongation of peptide chains. Modified after [4].

 
Familial forms of HUS account for up to 10% of all cases (D HUS). These non-Stx-associated forms have a much poorer prognosis, with frequent relapses of the disease [5]. A subgroup of these patients suffers from an inherited factor H deficiency [23]. Factor H is a 150 kDa plasma glycoprotein consisting of 20 protein domains that has an important function in the alternative pathway of complement (Figure 6). Factor H has heparin-binding sites that may allow interaction with extracellular matrix proteins. The human factor H gene is located on the long arm of chromosome 1. It prevents the generation of the C3bBb complex and stimulates the dissociation of Bb from the C3 convertase [23]. Thereby, C3b is exposed to cleavage and inactivation by factor I. Thus, factor H is a physiological inhibitor of alternative complement activation and controls the amplification cycles of complement activation. In patients with factor H deficiency, the plasma concentration of the protein is reduced, leading to excessive activation of C3, deposition of C3 on membranes and endothelial cell injury. Different point mutations, deletions and frameshifts in the factor H gene that cluster within the most C-terminal short consensus repeat 20 have been identified [23,24]. Affinity chromatography revealed that factor H with point mutations in the C-terminal region leading to a single amino acid change binds with lower affinity to heparin [25]. Moreover, less binding of the mutated factor H to C3b/C3d and endothelial cells was found, while interaction with fluid-phase C3b was not altered [25]. These findings could explain the observation that patients with mutations in the C-terminus of factor H have complement activation on endothelial cell surfaces while serum C3 levels are only little affected.



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Fig. 6. Simplified overview of the complement system. Factor H prevents the generation of C3bBb complex and stimulates the dissociation of Bb from the C3 convertase. Therefore, factor H is a physiological inhibitor of alternative complement activation. Mutations in factor H or membrane cofactor protein (MCP), a transmembrane-spanning glycoprotein that also acts as a local inhibitor of complement activation, cause some forms of familial D HUS.

 
Recently, a mutation in a gene encoding another complement regulatory protein has been described [26]. Noris and colleagues identified a heterozygous mutation in the gene for membrane cofactor protein (MCP) in two patients with a family history of D HUS [26]. MCP is a transmembrane-spanning glycoprotein that is expressed on many cells. It acts as cofactor for factor I to cleave deposited C3b and C4b on endothelial surfaces and acts, similarly to factor H, as an inhibitor of the alternative pathway of complement activation. The mutations described create a premature stop codon, which results in severely reduced cell surface expression of MCP [26]. Since MCP is a locally expressed transmembrane protein, renal transplantation may correct the defect, at least in the kidney [26]. Presumably, other mutations in genes for complement regulatory proteins may be identified in the future that account for familial HUS not caused by factor H or MCP mutations.

Is low ADAMTS-13 activity a valid criterion to distinguish between TTP and HUS?

Taking into account the differences in pathophysiology, one would assume that TTP could be easily separated from HUS by its low or absent activity of ADAMTS-13. This issue, however, is not as straightforward as it sounds and remains highly controversial [2734]. Some studies demonstrated that severely deficient ADAMTS-13 activity is specific for TTP [27,28]. For example, Bianchi et al. reported that severe deficiency of ADAMTS-13 activity (<5%) is specific for TTP, whereas less severe reductions (between 30 and 10%) are occasionally found in other thrombocytopenic states as well, including severe sepsis, myelodysplastic syndrome and heparin-induced thrombocytopenia type 2 [27]. In another study, Tsai and colleagues found normal ADAMTS-13 activity in 16 patients with D+ HUS [28]. In fact, patients with HUS had a decrease in UL-vWF, presumably caused by enhanced proteolysis resulting from abnormal shear stress in the microcirculation [28]. ADAMTS-13 activity was normal in 29 children with D+ HUS [29]. One D+ HUS patient, who had clinical features rather typical of TTP, had a vWF-cleaving protease inhibitor producing a severe reduction in ADAMTS-13 activity [29]. The difficulties in using ADAMTS-13 as a marker to clearly separate TTP from HUS are illustrated by a prospective multicentre study including 111 cases of thrombotic microangiopathies [30]. Veyradier et al. found that ADAMTS-13 activity was normal in seven TTP and 39 HUS cases and was decreased in 59 TTP and six HUS patients [30]. This yields a 89% sensitivity and a 91% specificity for an ADAMTS-13 defect to distinguish between TTP and HUS in cases of thrombotic microangiopathy manifesting as TTP, including recurrent, intermittent and sporadic cases [30]. Remuzzi and colleagues found complete ADAMTS-13 deficiency in five of nine patients with HUS during the acute phase and even in five patients with remission [31]. HUS patients with ADAMTS-13 deficiency could not be distinguished from those with normal enzyme activity based on clinical grounds [31]. In addition, Moore et al. reported a reduced ADAMTS-13 activity in various diseases such as systemic lupus erythematosus, idiopathic thrombocytopenic purpura, disseminated intravascular coagulation and even in apparently healthy controls [32]. A moderate reduction of ADAMTS-13 activity has also been reported in women with the microangiopathic HELLP syndrome, although UL-vWF was not present in the plasma of these patients [33]. Most importantly, ADAMTS-13 deficiency should not be used as a criterion to guide plasma exchange therapy [34]. Vesely and co-workers reported that only 13% of their TTP patients were severely (<5%) deficient in ADAMTS-13 activity [34]. On the other hand, in this study, patients at all levels of ADAMTS-13 activity responded to plasma exchange therapy, including those with normal levels [34]. These observations indicate that ADAMTS-13 deficiency does not detect all patients who might benefit from plasmapheresis.

What should we make of these controversial findings? It has been argued that differences in diagnostic criteria may have contributed to these discrepant results [35]. It has also been suggested that UL-VWF should be measured in addition to ADAMTS-13 activity [35]. Only patients with low ADAMTS-13 activity and diminished cleavage of UL-VWF should be diagnosed as having TTP [36]. However, UL-VWF may also be cleaved by proteases other than ADAMTS-13 [37]. Finally, ADAMTS-13 assays are complex and not yet standardized [38]. Therefore, the diagnosis of TTP/HUS should be made exclusively on clinical grounds and pathological findings [39,40]. Determination of ADMST-13 plays no role in the initial diagnostic work-up since assays measuring ADAMTS-13 activity are lengthy and the methodology is not yet fully developed. In a given single case, it is not possible to distinguish between TTP and other microangiopathies. Measurement of ADAMTS-13 activity is therefore not recommended in current guidelines [38,39]. However, if the diagnosis remains uncertain, a citrate plasma sample should be collected before initiation of plasma exchange for the later measurement of ADAMTS-13 activity in a reference laboratory.

Conclusions

Important novel insights into the pathophysiology of TTP and HUS have accumulated over the past years. A breakthrough was the discovery that a reduction in ADAMTS-13 activity, either genetically determined or caused by an inhibiting antibody, is a key finding in TTP. However, ADAMTS-13 activity may be also reduced in other causes of thrombotic microangiopathy including HUS [40]. The pathophysiological role of Stx in the development of D+ HUS is relatively well understood. Mutations in genes of complement regulatory proteins (e.g. factor H, MCP) underlie some familial forms of D HUS. Nevertheless, important questions currently remain unanswered [40]. Why is ADAMTS-13 activity reduced in some non-TTP? Are TTP and HUS the same disease entity? What is the final common pathway despite dissimilar aetiologies? Are there additional genetic defects in patients with D HUS? How is ADAMTS-13 expression normally regulated? Do we need a novel classification of microangiopathies based on ADAMTS-13 activity?

Nevertheless, the finding that in some cases of TTP ADAMTS-13 is inhibited by an autoantibody provides for the first time a rationale to treat these patients with immunosuppressive therapy. The development of recombinant ADAMTS-13 holds great promise. The future will certainly bring many more fascinating insights into TTP and HUS. Stay tuned.

Conflict of interest statement. None declared.

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