Pediatr Nephrol DOI 10.1007/s00467-017-3744-y

REVIEW

The role of von Willebrand factor in thrombotic microangiopathy Damien G. Noone 1,2

&

Magdalena Riedl 3 & Christoph Licht 1,2,3,4,5

Received: 29 January 2017 / Revised: 5 June 2017 / Accepted: 21 June 2017 # IPNA 2017

Abstract Thrombotic microangiopathy (TMA) is caused by thrombus formation in the microvasculature. The disease spectrum of TMA includes, amongst others, thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS). TTP is caused by defective cleavage of von Willebrand factor (VWF), whereas aHUS is caused by overshooting complement activation and subsequent endothelial cell (EC) injury. Despite their distinct pathophysiology, the clinical manifestation of TTP and aHUS consisting of microangiopathic haemolytic anaemia and thrombocytopenia is often similar and difficult to distinguish. Recent evidence hints at both a genetic and functional link between TTP and aHUS, especially between VWF and the complement system. There is novel in vitro evidence that complement activation not only results in VWF release from ECs, but that VWF also functions as a negative complement regulator, thus protecting the EC surface from ongoing complement attack. Although contrary to previous experimental work suggesting that complement can be activated on VWF multimers, there may be an

explanation in vivo that rationalizes these apparently contradictory findings, whereby a system primarily meant to regulate becomes overwhelmed or pathologic in the disease state. The importance of unravelling these recent findings for our understanding of TMA pathology becomes even more evident considering that glomerular ECs express VWF in a heterogeneous pattern with an overall decreased expression level, thus potentially leaving the glomerular ECs vulnerable to complement-mediated injury. Taken together, these findings support the concept that TTP and aHUS represent two extreme ends of a TMA disease spectrum rather than isolated disease entities. Keywords Thrombotic microangiopathy . Thrombotic thrombocytopenic purpura . Von Willebrand factor . ADAMTS13 . (Atypical) haemolytic uraemic syndrome . Complement

Introduction * Damien G. Noone [email protected]

1

Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada

2

Department of Paediatrics, University of Toronto, Toronto, ON, Canada

3

Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada

4

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada

5

Institute of Medical Science, University of Toronto, Toronto, ON, Canada

Thrombotic microangiopathy (TMA) is a pathological condition arising from the formation of intravascular thrombi in the microvasculature, leading to a microangiopathic haemolytic anaemia, occlusion of the vessels and ischaemia to the tissues downstream of the occluding thrombi [1]. It is defined by the occurrence of swollen, damaged microvascular endothelial cells (ECs) coupled with platelet-rich microthrombi with capillary and arteriolar thrombosis [2]. TMA is a multisystem disease and can, in principle, affect any organ system, including the heart, lungs, brain, liver, pancreas, skin, bones and kidneys, with effects ranging from sudden death to progressive organ damage and loss. It can be an acute and lifethreatening disease with serious long-term sequelae and, in some forms, a risk of recurrence [3–6].

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There is an increasing spectrum of diseases, or ‘syndromes’ with a defined cause [2], associated with a TMA which, in concert with the traditional definitions of TMA diagnoses, will likely require a new classification system in the future based on our expanding mechanistic insight [2, 7, 8]. For now, broadly speaking, the TMAs can be categorized as being either hereditary or acquired/ secondary [2]. Specific causes of hereditary TMA include: (1) thrombotic thrombocytopenic purpura (TTP) secondary to ADAMTS13 mutations, (2) atypical haemolytic uremic syndrome (aHUS) due to mutations in proteins involved in the regulation or activation of the complement alternative pathway, (3) the inherited metabolic disorders of cobalamin, such as methyl malonic acidemia and homocystinuria [9] or (4) mutations in other EC genes involved in coagulation, such as thrombomodulin [10] or the recently discovered DGKε [11]. Acquired/secondary forms of TMA include, but are not limited to: (1) TTP secondary to autoantibodies against ADAMTS13, (2) aHUS secondary to autoantibodies to the regulator complement factor (CF) H, (3) drug-related TMAs, such as those associated with cyclosporine or quinine use [12, 13], (4) pregnancy-related TMA [14], (5) transplant-associated TMA, particularly in association with antibody-mediated rejection [15, 16] and post stem cell transplantation [17, 18] and (6) infection-related TMA, such as Shiga toxin Escherichia coli-mediated HUS and Streptococcus pneumoniaassociated HUS. Although there is an emerging spectrum of diseases leading to a TMA, TTP and HUS are the two quintessential diseases associated with TMA [2]. Whether TTP and HUS are distinctly separate entities or rather represent a spectrum and share certain commonalities has been the subject of debate since their first descriptions. Symmers proposed the term ‘thrombotic microangiopathy’ in 1952 to indicate Bthe location and the most striking feature of the characteristic histological lesions without mentioning inconsistent and controversial features of this clinico-pathological entity^ [19]. Although somewhat indistinguishable pathologically, the presentation was not so easy to separate, with significant clinical overlap often apparent. With the discovery of the pathogenic role of uncleaved von Willebrand Factor (VWF) multimers in TTP, and the role of Shiga toxin E. coli and aberrant complement regulation in the typical and atypical forms of HUS, respectively, it seemed that there was indeed a clear dichotomy between TTP and HUS. However, more recently there has been a significant improvement in our understanding of the link between complement and coagulation that seems to reconcile how these two apparently distinct conditions can have so much overlap. Herein, we review this exciting new experimental data, especially those data linking VWF and the complement system.

Thrombotic thrombocytopenic purpura Moschcowitz first described TTP in a 16-year-old girl who died within 2 weeks of presenting with haemolytic anaemia, thrombocytopenia, fever and neurological symptoms. At the post-mortem examination, hyaline thrombosis of the capillaries was found [20]. Almost 60 years passed before unusually large VWF multimers, a hemostatic protein, were identified in the plasma of TTP patients [21]. Sixteen years later the reason for the presence of these unusually large VWF multimers in the plasma of TTP patients was discovered to be a defect in the VWF cleaving protease ADAMTS13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13). This is now known to be either an acquired phenomenon via an inhibiting antibody or due to a mutation of the gene encoding ADAMTS13 [22, 23]. TTP occurs either as the hereditary Upshaw–Schulman syndrome [24] that presents in its most severe form as congenital thrombocytopenia, or later in life [25], and sometimes with a precipitant such as pregnancy [26], where there are either homozygous or compound heterozygous mutations in ADAMTS13, or autoantibodies directed against it (Moschcowitz disease) [27–31]. Apart from the severe congenital form, Upshaw–Schulman syndrome, TTP is more common in adults, manifesting clinically with a non-immune microangiopathic haemolytic anaemia, thrombocytopenia, altered neurological status, kidney failure and fever. These symptoms make up the classically described pentad of TTP [32]. Activity levels of ADAMTS13 of <10% with additional inhibiting antibodies or a genetic mutation corroborate the diagnosis [2].

Atypical HUS Haemolytic uremic syndrome is a life-threatening disease, typically of childhood, with the child developing a TMA with anaemia, thrombocytopenia and subsequent kidney failure after infection by an enterohaemorrhagic, Shiga toxinproducing E. coli (STEC) strain [33–35]. About 15% of children infected with STEC will progress to HUS 7–10 days after ingestion of the bacteria, usually in association with bloody diarrhoea. The triad of a non-immune, microangiopathic haemolytic anaemia, thrombocytopenia and acute kidney injury (AKI) characterizes HUS. Historically this was classified as diarrhoea-positive HUS, and occasionally it is associated with epidemics or outbreaks [33]. Although STEC HUS can present with a severe illness, recovery is usually spontaneous, there are no relapses and the overall prognosis is good [36]. Following the recognition of HUS as a new disease entity came the realization that there was a subtype of HUS that was ‘atypical’, in that it was either recurrent [37, 38] or familial and had a more severe presentation and worse

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outcome [39, 40]. Early in the history of aHUS, disturbance and activation of the complement system was noted [41–43]. Low levels of the complement regulator Factor H (CFH) had been described in aHUS patients [44, 45], but the major breakthrough came when Warwicker et al. mapped the inherited form of HUS to the region on chromosome 1q that encoded CFH and confirmed genetic deficiency in this complement regulator as being the cause [46, 47]. Warwicker and Goodship were the first to propose a Bcomplement-based theory of microangiopathy^ [48]. To date, mutations in several other complement proteins and regulators have been detected, as well as autoantibodies altering the CFH function [8, 49].

The clinical overlap between TTP and HUS The principal distinction between TTP and HUS has always been attributed to the involvement of the neurological system and relative sparing of the kidney in TTP and the predominant renal phenotype in HUS. It is of course now known that although unusual in TTP, renal disease can occur in a substantial number of patients and that its presence certainly does not exclude a diagnosis of TTP [50, 51]. In a cohort of 92 TTP patients (ADAMTS13 <10%), 54 patients (58.7%) presented with AKI as defined by the 2012 Kidney Disease: Improving Global Outcomes (KDIGO) guidelines (http://www. kdigo.org/clinical_practice_guidelines/pdf/CKD/ KDIGO_2012_CKD_GL.pdf) and 42.6% progressed to have mild-moderate chronic kidney disease at the 6month follow up. Furthermore, a lower C3 count was independently associated with AKI by multivariate analysis [50]. Other groups had previously reported less frequent AKI, but this is likely due to how AKI was defined [52, 53]. Data from the Oklahoma TTP–HUS Registry found that ten of 60 (16.7%) patients with ADAMTS13 levels of <10% had severe renal failure, defined as a serum creatinine rise of ≥0.5 mg/dL/day on two consecutive days or a serum creatinine of ≥4 mg/dL with the patient needing dialysis. However, there are limitations to interpreting these data because HUS and TTP patients are n o t c l e a r l y d i ff e r e n t i a t e d i n t h e r e g i s t r y [ 5 1 ] . Neurological involvement also fails to clearly differentiate between TTP and HUS. Neurological involvement in TTP is certainly more common, occurring in up to 50% of patients, but rates in the region of 10–30% have been reported in HUS, both in the typical and atypical forms [54]. A more definitive, but again not perfect, means of distinguishing TTP from HUS involves measuring plasma ADAMTS13 activity by various fluorescence assays [55, 56]. Levels of ADAMTS13 of <10% are considered to be highly suggestive of TTP and are generally used as a diagnostic guide [57].

VWF and ADAMTS13 Von Willebrand factor is synthesized in the endoplasmic reticulum of megakaryocytes, platelets and ECs as a pre-proVWF, which is transported to the Trans-Golgi after glycosylation and removal of the signalling peptide. There, after more modification steps (glycosylation, sulfation) the pro-VWF starts to multimerize. VWF multimers may either be released at a low level into the circulation, or VWF multimers are packaged and stored in the so-called Weibel–Palade body (WPB) of ECs and α-granules of platelets. The cigar-shaped WPBs can be up to 5 μm in length and are not only used for storage but also for transportation to the plasma membrane. The multimerization process in WPBs forms ultra large VWF (ULVWF) that are >10,000 kDa in size [58]. Stimuli for the exocytosis of VWF are elevated levels of intracellular calcium or elevated cAMP. For release, WPBs fuse with the membrane, and the accumulation of actin and actomysosin contraction allows the release of their contents [59]. In most cases this involves the release of VWF, although only the release of small proteins has been reported [60].In addition, ULVWF can be released from a single WPB or from a ‘secretory pod’ of coalesced WPBs [58]. Under the influence of blood flow, globular VWF elongates into strings of between 100 and 500 μm in length [61], which trap and bind platelets, thus initiating clot formation [58, 59, 62, 63]. VWF strings anchored to ECs are much more efficient in adhering platelets after shear stress exposes their A1 domain [58, 64]. When released from ECs these multimers can form (bundles of) strings which unfold in the circulation under the influence of shear flow, thus unveiling the glycoprotein (GP) 1b platelet-binding site [59]. Normally these strings are subject to proteolysis by ADAMTS13; alternatively, released VWF can bind to collagen [65]. Secreted VWF differs from that in the circulation, which is present in a globular state, thus maintaining both the platelet GP1b binding site and the ADAMTS13 cleavage site hidden [59]. Additionally, VWF is also stored in the α-granules of platelets and released upon secretory stimulus [66]. It was initially recognized that VWF in the circulation was cleav ed b y a zinc pro tea se [67, 68], termed ADAMTS13 [30, 31]. ADAMTS13 prevents VWF multimers from becoming excessively large by cleaving the VWF [69] at a peptide bond in its A2 domain [70], and VWF multimers attached to the ECs undergo structural alterations under shear flow that enable ADAMTS13 to cleave them [71, 72]. When ADAMTS13 is congenitally deficient or inhibited by an autoantibody, then ULVWF and platelet-rich thrombi can form and occlude the microcirculation. Platelets avidly bind and are firmly fixed to ULVWF multimers that have unfolded following the shear forces of the circulation, thus forming a nidus for clot formation [23, 27, 31, 73, 74].

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A link between complement and coagulation Phylogenesis links complement and coagulation The coagulation and complement systems have been partners in protecting their hosts for millions of years, although a greater understanding of the extent of the interaction between the two systems continues to evolve [75–77]. This partnership of complement and coagulation in phylogenesis has enhanced immune defence by compartmentalizing damaged or infected segments of the vasculature. A bidirectional crosstalk between both proteolytic cascades has evolved and is evident even in the horseshoe crab, with both systems capable of activating, amplifying and even regulating the other [78]. The convergence of both systems has recently come to the foreground, especially with the finding of an additional C3-independent pathway of complement activation by thrombin factor IIa, a proteolytic serine protease integral to coagulation [79]. This was followed by the finding that thrombomodulin (CD141), a cofactor for thrombin, also functioned as a cofactor for CFImediated C3 cleavage, thereby acting as a complement regulator, and that certain heterozygous missense mutations could be linked to patients with aHUS [10]. In addition to thrombin, other components of the coagulation system directly activate complement by cleaving both C3 and C5 into their respective anaphylatoxins, such as the serine protease plasmin and coagulation factors FXa, FIXa and FXIa [80, 81]. Evidence for a complement–coagulation interface in patients It has emerged that complement may play a role in TTP, blurring the lines of separation between aHUS and TTP [82–86], although available clinical data is still somewhat limited. Sera from TTP patients can activate the complement alternative pathway (AP), leading to EC cytotoxicity in vitro [84]. The anaphylatoxin C3a and soluble C5b-9 levels were also found to be higher in 23 TTP patients during acute disease as compared to healthy controls [85]. Although the levels of C3a and soluble C5b-9 came down, as expected, with plasma exchange relative to those of the healthy controls, the clinical significance of these complement byproducts in the sera, or what they mean, is not known, and how they compare to aHUS patients with active disease, a biologically more relevant control group, is also indeterminate [85]. In another cohort of 38 TTP patients, 33 (87%) had evidence of complement activation at presentation that was even more pronounced in those patients that died, although again, there was no inclusion of a control group, such as active aHUS patients, to quantify the ‘degree’ of complement activation there is or to determine how relevant these complement biomarkers are in TTP [86]. Recently, the link between complement and coagulation in aHUS has been expanded to involve VWF and its cleaving

protease, ADAMTS13. In a recent study involving 29 aHUS patients, Feng et al. found additional ADAMTS13 polymorphisms associated with decreased ADAMTS13 activity [87]. Among these 29 patients, 80% also carried at least one nonsynonymous change in ADAMTS13, and 38% had multiple ADAMTS13 variations. Some of these variations were common variants having minor allele frequencies of >1%, but three of these single nucleotide polymorphisms were much rarer and possibly significant, considering that measured ADAMTS13 activity was <60% in half of the patients studied [87]. Molecular interactions of VWF and complement proteins Alongside the clinical observations, there have been a number of experimental studies linking CFH, ADAMTS13 and VWF. Firstly, in vitro evidence has emerged showing the assembly and activation of components of the complement AP, especially C3 and C5, on ULVWF strings secreted from and anchored to ECs in static, non-fluidic, conditions [88]. The experimental rationale for the static conditions was to allow accumulation of the VWF on the ECs rather than having it washed away under fluidic conditions. This of course only recapitulates the in vivo setting if there is perhaps an occlusive microthrombus, as otherwise there should be flow. Furthermore, under fluidic or shear conditions, more akin to the in vivo situation, and in the absence of ADAMTS13, C3 was found to be bound to histamine-induced VWF strings, to VWF adherent platelets and to the EC that secreted the VWF [89]. However, the functional downstream effect of this binding on the actual EC was not determined [89]. It has recently been postulated, based on the static experiments, that VWF contributes to amplifying complement activation, which would be very pertinent to TMA and aHUS [90]. However, other experimental evidence has also linked the principal complement AP regulator FH to VWF. FH can bind to ULVWF strings attached to the EC [88]. FH has been shown to be a reductase for large soluble VWF (LsVWF) multimers, the product of ADAMTS13 cleavage of ULVWF secreted from ECs, further facilitating the degradation of VWF, and perhaps in vivo, where the concentration of FH is significantly higher than that used in these experiments [91], of VWF-rich thrombi. These LsVWF cannot be further cleaved by ADAMTS13 but can still bind platelets [91]. In addition, the binding of FH via its C-terminus has also been shown to enhance ADAMTS13 cleavage of ULVWF in vitro [92]. By favouring VWF cleavage, this might prevent complement assembly to the VWF strings and thereby prevent complement recruitment on the EC surface. Finally, FH cofactor activity for factor Imediated inactivation of C3b, and therefore complement regulation, may be enhanced by an interaction between low molecular weight VWF and FH, but not the ULVWF [93]. How might this be relevant or translate in vivo though?

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To further address this controversy and to study the interaction between complement and VWF, we studied blood outgrowth endothelial cells (BOECs) isolated from type 3 VWD (VWD BOECs) patients, who are naturally devoid of VWF, and exposed them to complement activation in vitro under static and fluidic conditions [94]. BOECs are endothelial progenitor cells that can be isolated with high efficiency from small volumes of peripheral blood using cell separation methods and direct culture, and they have close affinities to their donor’s

vascular ECs [95]. Using BOEC we were able to take advantage of a naturally occurring EC line with negligent amounts of VWF, essentially equating to a VWF knockout. Specifically, we wanted to study whether VWF had a complement-amplifying role [96, 97]. First we confirmed that complement induces EC activation and VWF release (Fig. 1), likely via elevation of intracellular calcium [98–100]. After exposing VWD BOECs to complement, applying a sensitization protocol whereby we block the membrane complement regulator CD59 (that inhibits the binding of C9 to C5b-8 and thus the final membrane attack complex) and apply normal human serum to the cells [94], we demonstrated increased C3b deposition on their surface as compared with control BOECs. This was biologically relevant, at least in vitro, as VWF-deficient BOECs exhibited decreased survival to complement-mediated cytotoxicity. These VWD BOECs expressed similar amounts of the surface-bound complement regulators CD46/MCP, CD55/DAF and CD59, leading to the intriguing conclusion that VWF release from complement challenged ECs in vitro is protective and shields the ECs from complement deposition. There are potential limitations to our

Fig. 1 Under static conditions, blood outgrowth endothelial cells (BOECs) exposed to 10 and 30 min of complement activation, respectively, were fixed and stained for extracellular von Willebrand factor (VWF, red staining). Subsequently, cells were permeabilized and stained

for intracellular VWF (green). During complement challenge BOECs release VWF (reflected by a decrease in the intracellular VWF pool, green) as VWF strings (increase of extracellular red staining). See also Noone et al. [94]. Scale bar = 10μm

Complement products seem to attach to ULVWF strings and may even continue to be active [88], however, ADAMTS13 and FH act in concert to cleave these strings, likely allowing them to be cleared by the circulation. FH may further regulate complement on these smaller VWF strings. However, in the setting of low ADAMTS13 or FH, or if a microthrombus leads to cessation of blood flow, then complement activation on ULVWF might well become detrimental to the underlying endothelium. VWF—A complement activator or regulator?

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Fig. 2 In a microfluidic chamber (Bioflux®; Fluxion Biosciences, Inc., South San Francisco, CA) BOECs were exposed to prolonged complement activation and shear stress of 1 dyne/cm2 for 1 h followed by the introduction of platelets. Terminal complement complex C5b-9 staining (C5b-9, red) of the right cell confirmed complement activation. Complement activation resulted in von Willebrand factor (VWF) release (green) with VWF depletion of the intracellular pool of this cell. Excreted VWF unfolds under shear stress and allows for platelets (green, arrow) to adhere. Of note, the left cell does not show complement activation and consequently has intact intracellular VWF pools, absent extracellular VWF and no platelet adhesion

work, namely that the experimental conditions served only to activate complement on the ECs to study the downstream effects on the ECs. Unlike in aHUS where it is assumed that there is just selective AP activation, in our experimental system both the classical and alternative pathways are activated. Also, we did not reproduce the defective VWF cleavage and the accumulation of ULVWF multimers that occur in TTP. Finally, the clinical relevance of these findings are as yet unknown.

Fig. 3 a. Complement challenge leads to endothelial cell (EC) activation, von Willebrand factor (VWF) release, platelet adhesion and binding of complement activation products. VWF serves as a negative complement regulator and supports maintenance of EC homeostasis. b. Persistent complement challenge in association with deficient or overpowered complement regulators and/or reduced VWF leads to EC injury and functional defects allowing— amongst others—for the formation of platelet microthrombi (i.e. thrombotic microangiopathy)

Considering the emerging evidence linking VWF and complement, a further question is posed, namely that of whether cells either expressing or containing less VWF are more vulnerable to complement-mediated injury. It is known that there is significant heterogeneity between ECs, with different microvascular endothelial beds often having distinctly different transcriptomes, properties and functions, with the fenestrated glomerular endothelium being the perfect example of this. This EC heterogeneity is also known to include VWF expression [101–104]. The fenestrated glomerular endothelium shows patchy positivity for VWF, as detected in one study by immunohistochemistry on biopsy and post mortem specimens [105]. Looking at cultured human glomerular ECs we also observed a heterogeneous expression of VWF [94]. This could partially explain why the glomerular endothelium is vulnerable to complement attack in aHUS and perhaps why in TTP the kidney is classically spared [94]. Taken together these observations could lead to a new understanding of aHUS pathogenesis, or a two-phase view of the sequence of events that might lead to TMA: –

–

Phase 1: Complement activation leads to calcium influx, EC activation and WPB exocytosis. The release of VWF facilitates platelet adhesion and may form a platform on which complement products adhere (Fig. 2). In the presence of efficient and normal levels of ADAMTS13 these VWF multimers, laden with complement products, are cleaved and released into the circulation, thus helping to maintain EC homeostasis (Fig. 3a). Phase 2: In the presence of ongoing or persistent complement challenge, especially in association with deficient complement regulators or potent amplifiers of the AP, or if

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there is diminished clearance of the ULVWF multimers, either because of deficient ADAMTS13 activity or decreased blood flow as might occur after the formation of a thrombus in the microvasculature, the initially protective effect of VWF can be overcome, leading to progression towards a TMA phenotype (Fig. 3b). This might help explain the recent finding of ADAMTS13 mutations in aHUS patients associated with decreased ADAMTS13 activity [87].

Therapies targeted towards VWF Establishment of the role of VWF in complement-mediated TMA may just open the door to novel therapeutic targets for not just TTP, but also aHUS, that serve as adjuncts to anticomplement therapy and target VWF and the associated microthrombi. They might also become important in the treatment of those forms of aHUS in which complement mutations have not been shown, for example DGKε [11], where there is a prothrombotic endothelium and no clear role for complement or complement inhibition as a therapy. Currently, therapy for TTP in the acute setting revolves around plasma exchange, which removes autoantibodies directed against ADAMTS13 and replenishes ADAMTS13 and immunosuppression, with many agents, including vincristine, cyclosporine and cyclophosphamide, being used successfully [106]. In the autoimmune form of TTP, most success has been with the use of corticosteroids, and rituximab, the anti-CD20 B-cell depleting agent, has an additional role. More recently, the proteasome inhibitor bortezomib, which also targets B cells by leading to cell cycle arrest, has also been successfully used in refractory TTP. To date, about a dozen patients have received bortezomib, with 11 having survived off plasma exchange [107]. Future therapies on the horizon focus on either (1) cleaving ULVWF multimers or (2) inhibiting platelet–VWF binding. In the search for ways to cleave VWF, there have been promising preclinical animal studies using recombinant ADAMTS13 [108, 109]. Another potential method of VWF cleavage involves plasminogen. Plasmin has been shown to cleave VWF in vitro and has been postulated as being very relevant in TTP, where hypoxia-activated ECs can produce plasminogen, activate it via urokinase-type plasminogen activator to plasmin, which then mediates VWF cleavage, thereby enhancing ADAMTS13-mediated cleavage of VWF [110, 111]. The recent finding of mutations in PLG, the plasminogen gene, resulting in decreased plasminogen expression in aHUS might corroborate this finding that plasmin-mediated cleavage of VWF multimers may become relevant as microvascular thrombosis progresses [112]. The second proposed method of treating TTP involves inhibiting the platelet-VWF interaction directly. To this end,

two agents are in the forefront, namely N-acetylcysteine (NAC) and caplacizumab. NAC reduces disulphide bonds via its free sulfhydryl group, and both in vitro and preclinical studies have demonstrated proof of concept that it may reduce the size of VWF multimers [113, 114]. Published case reports, however, report variable success of NAC in vivo, and further work is necessary before this agent can be recommended [115–117]. Caplacizumab is a nanobody directed against the platelet binding site of VWF, the A1, that interacts with platelets via their glycoprotein Ib receptor [118]. Preclinical success in a baboon model of TTP has recently been repeated in the TITAN study, where although it failed to present relapse, at least in the short term, it was associated with an acceleration in time to platelet count normalization [119].

Conclusion Although TTP and HUS are both TMAs with distinctly different etiologies, there still remains a significant overlap in terms of clinical presentation. These disease entities remain inextricably linked, however, as the two biological systems at play, both the complement system and coagulation, cannot be so easily separated or compartmentalized. VWF might represent a string that ties these two entities together, and its role certainly needs more clarification. It makes ‘biological sense’ that ECs would have a protective use for VWF, but like all biological systems, its protective role may be overcome, or it may not be sufficiently cleared by both ADAMTS13 and plasmin, thereby allowing further complement activation and ischaemia to the endothelium. It is also intriguing to consider that EC heterogeneity, in particular to VWF, may explain the disparate brain and kidney involvement in the two conditions, although much work remains to confirm this concept.

Compliance with ethical standards Conflict of interest DN, MR or CL have no conflicts of interest to delare that are relevant to this review.

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