Wild-type ADAMTS13 Research Articles

Disruption of ADAMTS13-Autoantibody Complexes with an ADAMTS13 Spacer Domain As a Potential Therapeutic Strategy for Immune Thrombotic Thrombocytopenic Purpura

Background: Immune thrombotic thrombocytopenic purpura (iTTP) is primarily caused by acquired immunoglobulin (Ig) G against ADAMTS13, a member of the A Disintegrin and Metalloprotease with Thrombospondin Type 1 repeats family, which specifically cleaves von Willebrand factor (VWF). Such proteolytic cleavage is essential for normal hemostasis. An inability to cleave VWF due to IgG autoantibody-mediated inhibition of plasma ADAMTS13 activity results in an accumulation of ultra large VWF multimers and disseminated microvascular thromboses, a pathognomonic feature of TTP. Previous studies have demonstrated that the spacer domain of ADAMTS13 is the primary target of IgG autoantibody; also, spacer domain is important for recognition of VWF under various conditions. Objective: In the present study, we explored if an isolated spacer domain can compete or reverse IgG autoantibody-mediated inhibition of ADAMTS13 activity, an important question to be addressed for developing a new strategy to treat iTTP. Methods:A recombinant ADAMTS13-spacer domain was expressed and purified to homogeneity; additionally, a single-chain fragment of the variable region of anti-ADAMTS13 IgG (i.e. scFv4-20) was isolated via phage display from a patient with iTTP, which was shown to specifically target the spacer domain and potently inhibit plasma ADAMTS13 activity with sub-nanomolar affinity. A functional assay with a fluorescein-labeled VWF73 substrate was used to determine the ADAMTS13 activity. Results: First, when a purified recombinant spacer domain (0-1.0 µM) was pre-incubated with scFv4-20 at various concentrations, followed by an addition of normal human plasma (NHP) that provides wild-type ADAMTS13 and a fluorescein-labeled recombinant VWF73 (F-rVWF73) substrate, we demonstrated that the inhibitory activity of scFv4-20 towards plasma ADAMTS13 decreased in a concentration-dependent manner. As a control, recombinant Cys-rich domain had no effect on blocking the scFv4-20-mediated inhibition of plasma ADAMTS13 activity under the same conditions. When scFv4-20 was pre-incubated with plasma ADAMTS13, recombinant spacer domain could also reverse the scFv4-20-mediated inhibition of ADAMTS13 activity but to a lesser degree. At the final concentration of 1.0 µM, recombinant spacer domain was able to increase ADAMTS13 activity by 2.1-fold when the recombinant spacer domain was added prior to the formation of ADAMTS13-antibody complexes but only by 1.5-fold if the recombinant spacer domain was added after the ADAMTS13 and antibody complexes were formed. Conclusion: Our results demonstrate that autoantibody-mediated inhibition of plasma ADAMTS13 is reversible and can be blocked by a molecular mimic that is the primary antibody-binding region of the full-length ADAMTS13 protein. The data suggest that it may be feasible to explore the development of agents capable of displacing inhibitory antibodies against ADAMTS13 in patients with iTTP. Disclosures Zheng: Alexion: Speakers Bureau; Ablynx/Sanofi: Consultancy, Speakers Bureau; Shire/Takeda: Research Funding; Clotsolution: Other: Co-Founder.

Enhanced activity of an ADAMTS‐13 variant (R568K/F592Y/R660K/Y661F/Y665F) against platelet agglutination in vitro and in a murine model of acute ischemic stroke

Summary Essentials ADAMTS13 requires a substrate‐induced conformational change to attain full activity in vitro.The efficacy of wild type ADAMTS13 in models of thrombosis/stroke may be enhanced by pre‐activation.A pre‐activated ADAMTS13 variant exhibits enhanced proteolysis of platelet agglutinates.This ADAMTS13 variant is protective in a murine model of stroke at a lower dose than WT ADAMTS13. SummaryBackgroundADAMTS‐13 circulates in a closed conformation, only achieving full proteolytic activity against von Willebrand factor (VWF) following a substrate‐induced conformational change. A gain‐of‐function (GoF) ADAMTS‐13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally preactivated.ObjectivesTo establish how the hyperactivity of GoF ADAMTS‐13 is manifested in experimental models mimicking the occlusive arterial thrombi present in acute ischemic stroke.MethodsThe ability of GoF ADAMTS‐13 to dissolve VWF–platelet agglutinates was examined with an assay of ristocetin‐induced platelet agglutination and in parallel‐flow models of arterial thrombosis. A murine model of focal ischemia was used to assess the thrombolytic potential of GoF ADAMTS‐13.ResultsWild‐type (WT) ADAMTS‐13 required conformational activation to attain full activity against VWF‐mediated platelet capture under flow. In this assay, GoF ADAMTS‐13 had an EC50 value more than five‐fold lower than that of WT ADAMTS‐13 (0.73 ± 0.21 nm and 3.81 ± 0.97 nm, respectively). The proteolytic activity of GoF ADAMTS‐13 against preformed platelet agglutinates under flow was enhanced more than four‐fold as compared with WT ADAMTS‐13 (EC50 values of 2.5 ± 1.1 nm and 10.2 ± 5.6 nm, respectively). In a murine stroke model, GoF ADAMTS‐13 restored cerebral blood flow at a lower dose than WT ADAMTS‐13, and partially retained the ability to recanalize vessels when administration was delayed by 1 h.ConclusionsThe limited proteolytic activity of WT ADAMTS‐13 in in vitro models of arterial thrombosis suggests an in vivo requirement for conformational activation. The enhanced activity of the GoF ADAMTS‐13 variant translates to a more pronounced protective effect in experimental stroke.

Identification of a crucial tryptophan residue in ADAMTS13 required for its secretion and enzymatic activity.

Functional deficiency of A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)13 causally assists in the pathology of thrombotic thrombocytopenic purpura (TTP). Previous study showed that the WXXW motif is very important to the enzymatic activity of ADAMTS13. However, the functional role for a single amino acid residue within WXXW motif is still undetermined. Here, Trp390 residue within WXXW motif was substituted with Ala to generate Trp390Ala (W390A) mutant ADAMTS13. We found that W390A mutant ADAMTS13 had impaired binding affinity to its substrate Von Willebrand factor (VWF). Moreover, W390A mutant retarded ADAMTS13 secretion, leading to its deposition in endoplasmic reticulum. Compared with the wild type ADAMTS13, W390A mutant also had a decreased cleavage activity for multimeric VWF under both static and shear stress conditions. These data indicate that Trp390 residue within the WXXW motif is required for ADAMTS13 secretion and enzymatic activity, which provide insight into understanding the pathological basis of TTP and opens new avenues for exploring potential treatment strategies.

A model for the conformational activation of the structurally quiescent metalloprotease ADAMTS13 by von Willebrand factor

Blood loss is prevented by the multidomain glycoprotein von Willebrand factor (VWF), which binds exposed collagen at damaged vessels and captures platelets. VWF is regulated by the metalloprotease ADAMTS13, which in turn is conformationally activated by VWF. To delineate the structural requirements for VWF-mediated conformational activation of ADAMTS13, we performed binding and functional studies with a panel of truncated ADAMTS13 variants. We demonstrate that both the isolated CUB1 and CUB2 domains in ADAMTS13 bind to the spacer domain exosite of a truncated ADAMTS13 variant, MDTCS (KD of 135 ± 1 0.1 nm and 86.9 ± 9.0 nm, respectively). However, only the CUB1 domain inhibited proteolytic activity of MDTCS. Moreover, ADAMTS13ΔCUB2, unlike ADAMTS13ΔCUB1-2, exhibited activity similar to wild-type ADAMTS13 and could be activated by VWF D4-CK. The CUB2 domain is, therefore, not essential for maintaining the inactive conformation of ADAMTS13. Both CUB domains could bind to the VWF D4-CK domain fragment (KD of 53.7 ± 2.1 nm and 84.3 ± 2.0 nm, respectively). However, deletion of both CUB domains did not prevent VWF D4-CK binding, suggesting that competition for CUB-domain binding to the spacer domain is not the dominant mechanism behind the conformational activation. ADAMTS13ΔTSP8-CUB2 could no longer bind to VWF D4-CK, and deletion of TSP8 abrogated ADAMTS13 conformational activation. These findings support an ADAMTS13 activation model in which VWF D4-CK engages the TSP8-CUB2 domains, inducing the conformational change that disrupts the CUB1-spacer domain interaction and thereby activates ADAMTS13.

The N-Linked Glycans of ADAMTS13 Are Critical Determinants of Conformation and Proteolytic Activity

Introduction: The multimeric size and haemostatic potential of Von Willebrand Factor (VWF) is regulated by the metalloproteinase ADAMTS13 that cleaves VWF within its A2 domain. Under conditions of shear stress, ADAMTS13 cleavage mediates VWF collagen and platelet binding function and therefore regulates the size of the growing thrombus. It has also recently been shown that an intra-domain interaction between the Spacer and CUB domains is present in ADAMST13 resulting in both "closed" and "open" ADAMTS13 molecules. A significant portion of the ADAMTS13 molecule is comprised of glycan structures with 10 N-linked glycosylation sites spaced throughout the molecule, while the exact functional role of these is not determined we hypothesised that they may affect ADAMTS13 conformation and proteolytic activity.Methods: Wild type and ADAMTS13 mutants lacking specific N-linked glycosylation sites were expressed in HEK293T cells as were ADAMTS13 variants truncated after the spacer domain (MDTCS), cysteine rich domain (MDTC) and the disintegrin-like domain (MD) and the isolated CUB domains. Terminal silaic acid residues and whole N-linked glycan chains were removed enzymatically with neuraminidase (As-AD13) and PNGase F (PNG-AD13) respectively. The ability of ADAMTS13 and its variants to cleave VWF was assessed under both static and flow conditions and binding between the spacer and CUB domains determined by immunoprecipitation based assays.Results: As-AD13 and PNG-AD13 both demonstrated significantly reduced cleavage of the small FRETS-76 substrate and full length VWF under static conditions and importantly under conditions of physiological shear stress. Removal of sialic acid or N-linked glycan chains from the MDTCS, MDTC and MD variants also reduced proteolysis, indicating that silylation of one or both of the N-linked glycans in the metalloproteinase domain is required for proper ADAMTS13 function. Neuraminidase digestion did not affect the binding of ADAMTS13 to VWF; however PNG-AD13 had significantly reduced binding to VWF but interestingly PNG-MDTCS bound VWF with normal affinity. Using co-immunoprecipitation we demonstrated altered interactions between the Spacer and CUB domains depending on their respective glycosylation status. We hypothesise that the N-linked glycans modulate the recently determined intra-molecular interaction within ADAMTS13, and their absence alters the conformation of ADAMTS13 in solution. To further investigate this we expressed ADAMTS13 mutants lacking specific N-linked glycosylation sites. Mutation of the conserved glycan sites in the CUB 1 (N1235) and CUB 2 (N1354) domains to glutamine did not affect the expression of ADAMTS13 but in contrast to PNG-AD13, both these mutants were gain-of-function, having significantly enhanced ability to interact with and proteolysis VWF under static and flow conditions. To assess the potential therapeutic use of these gain-of-function mutants and wild type ADAMTS13 in a thrombotic setting, we pre-formed thrombi on collagen surfaces under both anti and pro-coagulant conditions then perfused wild type or mutant ADAMTS13 over the thrombi surface and monitored the reduction in thrombi size. Surprisingly, neither wild type ADAMTS13 nor the N1235Q or N1354Q mutants were able to reduce the size of pre-formed thrombi even at high concentrations (200nM) and furthermore the presence of ADAMTS13 did not enhance the tissue plasminogen mediated reduction in fibrin rich thrombi.Conclusion: The N-linked glycans of ADAMTS13 have critical effects on ADAMTS13 function; with terminal sialic acid residues in the metalloproteinase domain being essential for proper proteolytic function and glycosylation of the Spacer and CUB domains required to modulate the open/closed conformation of ADAMTS13. In addition, our data also demonstrates that while ADAMTS13 is effective at controlling the size of the growing thrombus it is ineffective at cleaving VWF once a thrombus is formed and is thus unable to reduce the size of pre-formed thrombi. Together these data highlight the importance of N-linked glycosylation for proper ADAMTS13 function. DisclosuresNo relevant conflicts of interest to declare.

The WXXW motif in the TSR1 of ADAMTS13 is important for its secretion and proteolytic activity

IntroductionADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) is a metalloprotease enzyme that regulates the size and activity of the von Willebrand factor (VWF). ADAMTS13, like many other ADAMTSs, has a WXXW motif in its thrombospondin type 1 repeat domain (TSR1). However, the function of the WXXW motif in ADAMTSs is unclear. Materials and MethodsThe constructs of wild-type (WT) and WXXW mutant (W387A) ADAMTS13 was generated by PCR site-directed mutagenesis. The secretion of the protein was quantified with western blotting methods. The binding affinity of the WT or W387A mutant ADAMTS13 with Plasma-derived human VWF (pVWF) was investigated by using enzyme linked immunosorbent assay. The Cleaving activity of the WT or W387A mutant ADAMTS13 against full length pVWF was measured under denatured conditions or shear stress. The proteolytic activity was also validated with the FRETS-VWF73 assay. ResultsThe W387A mutant was secreted less efficiently and had a reduced binding affinity for pre-denatured pVWF in comparison to WT ADAMTS13. However, both the WT and mutant ADAMTS13 interacted equally with native pVWF. The W387A mutant showed less cleaving activity against VWF under denaturing conditions, and the same result was observed when the fluorescence resonance energy transfer substrate VWF73 (FRETS-VWF73) was used as the substrate. However, under high shear stress conditions the mutant and WT ADAMTS13 were equally able to cleave VWF. ConclusionThe WXXW motif is important for the secretion of ADAMTS13 and that it modulates the proteolytic cleavage of VWF by ADAMTS13 under denaturing conditions.

The W–x–x–W Motif in the TSR1 of ADAMTS13 Is Important for Its Secretion

Abstract 4389 Introduction:The W-x-x-W motif is commonly found in the thrombospondin type 1 repeat (TSR) of various extra cellular proteins called TSR super family proteins, including thrombospondins, ADAMTSs, F-spondin and properdin. The W–x–x–W motif is known to bind with heparin, heparan sulfate proteoglycans, collagen and transforming growth factor-β, suggesting functional significance in cell–cell interaction and/or cellular signaling. However, the function of W-x-x-W in ADAMTS13 is unclear. In this study, we investigated the role of the W-x-x-W motif of ADAMTS13. Materials and Methods:We generated a W-x-x-W mutant (W387A) construct of ADAMTS13, and expressed the mutant and the wild-type constructs in HELA cells. Percentage of the protein secretion was defined as the concentration in the culture medium divided by the concentrations in the culture medium and cell lysates, multiplied by 100%. The binding affinity of the mutant or wild-type ADAMTS13 was investigated by enzyme-linked immunosorbent assay. Measurement of ADAMTS13 proteolytic activity toward von Willebrand factor (VWF) multimers was based on the generation of a dimeric 176-kDa fragment resulting from cleavage of VWF at the Y1605-M1606 bond, under denaturing condition and high shear stress condition, analyzed by Western blots. Results:SDS-PAGE gel analysis showed that the W387A mutant was secreted less efficiently relative to the wild-type construct. As for the binding affinity for the VWF multimer, there was no difference between the wild-type and mutant ADAMTS13. The W387A mutant was less active under denaturing condition; the same result was reproduced when FRETS-VWF73 was used as the substrate. However, under high shear stress condition, the mutant was as efficient as the wild-type ADAMTS13. Conclusions:The W–x–x–W motif is necessary for efficient secretion of ADATMS13. Further studies are needed to determine the contribution of the motif to the VWF cleave activity of ADAMTS13. Disclosures:No relevant conflicts of interest to declare.

Characterization of Coding Synonymous and Non-Synonymous Variants in ADAMTS13 Using Ex Vivo and In Silico Approaches

Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these “silent” variations can have a significant impact on protein expression and function and should no longer be considered “silent”. Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.

Gain-of-Function ADAMTS13 Variants Resistant to Anti-ADAMTS13 Autoantibody Inhibition: A Potential Novel Therapy for Acquired Autoimmune Thrombotic Thrombocytopenic Purpura

Abstract 1188Spacer domain of ADAMTS13 is essential for recognition and proteolytic cleavage of von Willebrand factor. It also contains major antigenic epitope targeted by anti-ADAMTS13 autoantibodies in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). Through site-directed mutagenesis and recombinant protein strategy, we identified two novel ADAMTS13 variants, which exhibited enhanced specific activity, but reduced inhibition by anti-ADAMTS13 autoantibodies. Among twenty-three ADAMTS13 variants tested, ADAMTS13-M4 (R660K/F592Y/R568K /Y661F) and ADAMTS13-M5 (R660K/F592Y/R568K /Y661F/Y665F) exhibited increased specific activity by 4∼5 fold and 12∼16 fold in cleavage of FRETS-VWF73 and plasma-derived multimeric VWF under denaturing conditions, respectively. Moreover, these two novel ADAMTS13 variants (i.e. M4 and M5) were resistant to inhibition by anti-ADAMTS13 IgG autoantibodies from 10 out of 12 acquired TTP patients tested. In contrast, proteolytic activity of wild-type ADAMTS13 and several other mutants (i.e. M1: R660K, M2: R660K/F592Y, and M3: R660K/F592Y/R568K) were significantly inhibited by same amount of anti-ADAMTS13 IgGs in all 12 patients’ plasmas. The resistance to inhibition of these variants was the result of reduced binding interaction between the ADAMTS13 variants (M4 and M5) and the anti-ADAMTS13 IgGs. These findings demonstrate for the first time that an exosite modification in the spacer domain may be a viable strategy for identification of novel ADAMTS13 variants with more desirable enzymatic properties. The results provide a potential tool for treatment of acquired (autoimmune) TTP and perhaps other arterial thrombotic disorders. Disclosures:No relevant conflicts of interest to declare.

Inhibitory Spleen-Derived Anti-ADAMTS13 Antibodies Are Characterized by a Limited Number of Variable Heavy Chain CDR3 Signatures in Patients with Relapsing Acquired Thrombotic Thromobocytopenic Purpura

Abstract 194 Background.Acquired Thrombotic thromobcytopenic purpura (TTP) is the result of autoantibodies neutralizing and-or accelerating clearance of ADAMTS13. We have previously isolated mononuclear cells of two spleens donated to our laboratory by patients (A and B) splenectomized for frequently relapsing TTP to generate human monoclonal anti-ADAMTS13 antibodies. Preliminary characterization of the patient’s entire IgG4 repertoire using phage display technology and Epstein Barr virus (EBV) transformation of switched memory B cells (CD19+, CD27+, IgG+) showed that the memory repertoire of Rituximab-resistant (B) and non-resistant (A) anti-ADAMTS13 IgG used the same restricted VH germline genes, namely VH1-3 (55%), VH1-69 (17%), VH3-30 (7%) and 4–28 (21%). In contrast, peripheral blood-derived IgG1 anti-ADAMTS13 antibody repertoire from 2 acquired TTP patients, inhibiting ADAMTS13 activity by maximal 15–40% used VH1-69 germline gene segment only (Luken et al, JTH 2006 and Pos et al, JTH 2009). To evaluate if the generated spleen-derived monoclonal anti-ADAMTS13 antibodies are of pathological relevance we started with their functional characterization. Methods.Both patients (A and B) were suffering from acute TTP with high titers of anti-ADAMTS13-Ab and therefore needed to be splenectomized because of frequent relapses, which occurred even after several courses of Rituximab (B). Both patients have remained in remission since splenectomy. Previous screening of the IgGk-IgGλ IgG4-Fab phage-display libraries and EBV-transformed clones on recombinant ADAMTS13 by ELISA revealed high ADAMTS13 specificity for 16/34 and 13/234 antibodies, respectively. Functional characterization of the phage-display clones included (a) determination of their inhbitiory capacity by functional FRETS inhibitor assay, (b) assessment of their affinity performed by either fluid-phase competition ELISA and/or plasma surface resonance (Biacore), and (c) epitope mapping by dot blot analysis testing their recognition of recombinant full-length wildtype ADAMTS13 and a truncated ADAMTS13 variant. Results.Detailed sequence analysis of all human monoclonal anti-ADAMTS13 analyzed showed seven unique CDR3 signatures in the variable heavy region of all clones generated using both techniques (phage display and EBV-transformation), three of which were seen in both patients. These CDR3 signatures are different from signatures found in peripheral blood-derived antibodies (Luken et al, JTH 2006 and Pos et al, Blood 2010). Strikingly, the anti-ADAMTS13 antibodies of EBV-transformed memory B cells used λ light chain exclusively, whereas the phage-display-derived clones showed usage of either κ- (A: 6/11 and B: 3/5) or λ-light chain (A: 5/11 and B: 2/5). Anti-ADAMTS13 IgG in supernatants of EBV-immortalized switched memory B-cell clones were mainly of subclass IgG4 (A: 60%; B: 25%) and IgG1 (A: 40%; B: 75%) thus IgG1 being the prevailing subclass in patient B, who relapsed despite several courses of Rituximab. Functional characterization of the anti-ADAMTS13 IgG4 antibodies revealed that 15/16 phage display clones are strongly inhibitory, showing 0–100% (A) and 60–100% (B) inhibition at 200 nM and even 0–92% (A) and 0–16% (B) inhibition at 20 nM. This represents a 5–6 times increase of inhibitory capacity when compared to previouse findingy by others, in the range of 2×10−7-7×10−10 M (A) and 1×10−9-6×10−10M (B) as measured by Biacore or/and competition ELISA. Epitope mapping revealed that 3/16 clones recognized a novel epitope at the C-terminus of ADAMTS13, in contrast to the previously reported N-terminally located spacer domain. Conclusions.The identification of several spleen-derived anti-ADAMTS13 clones displaying highly similar CDR3 variable heavy chains, in 2 unrelated acquired TTP patients is striking and novel. Our data points at the discovery of unique CDR3 signatures that characterize the pathological relevant, inhibitory antibodies paving thus the road to define their triggering ADAMTS13 epitope(s), while providing insight into the mode of success of splenectomy in relapsing TTP patients. Further functional characterization of the anti-ADAMTS13 EBV-derived clones and confirmation of our data by analysis of the immunological memory from two additional spleens are underway. Disclosures:No relevant conflicts of interest to declare.

Characterization of Conformation-Sensitive Antibodies to ADAMTS13, the von Willebrand Cleavage Protease

BackgroundThe zinc metalloprotease ADAMTS13 is a multidomain protein that cleaves von Willebrand Factor (VWF) and is implicated in Thrombotic Thrombocytopenic Purpura (TTP) pathogenesis. Understanding the mechanism of this protein is an important goal. Conformation sensitive antibodies have been used to monitor protein conformation and to decipher the molecular mechanism of proteins as well as to distinguish functional and non-functional mutants.Methodology/Principal FindingsWe have characterized several antibodies against ADAMTS13, both monoclonal and polyclonal. We have used flow cytometry to estimate the binding of these antibodies to ADAMTS13 and demonstrate that antibodies raised against the TSP and disintegrin domains detect conformation changes in the ADAMTS13. Thus for example, increased binding of these antibodies was detected in the presence of the substrate (VWF), mainly at 37°C and not at 4°C. These antibodies could also detect differences between wild-type ADAMTS13 and the catalytically deficient mutant (P475S). The flow cytometry approach also allows us to estimate the reactivity of the antibody as well as its apparent affinity.Conclusions/SignificanceOur results suggest that these antibodies may serve as useful reagents to distinguish functional and non-functional ADAMTS13 and analyze conformational transitions to understand the catalytic mechanism.

Characterization of Five Homozygous ADAMTS13 Mutations in Hereditary Thrombotic Thrombocytopenic Purpura – Towards a Phenotype-Genotype Correlation?

Homozygous or compound heterozygous mutations in the ADAMTS13 gene cause hereditary thrombotic thrombocytopenic purpura (TTP), an inherited disorder characterized by severe deficiency of plasma ADAMTS13 activity resulting in occlusive microvascular thrombosis. More than eighty ADAMTS13 mutations have been identified, of which 15 have been found in homozygous state. Homozygous mutations are of special interest to unravel a correlation between the ADAMTS13 genotype and the often variable clinical phenotype in hereditary TTP. We studied 5 ADAMTS13 mutations found in homozygous state in 6 patients with hereditary TTP. Detailed patients' histories were recorded and plasma ADAMTS13 activity and antigen levels were determined. Recombinant ADAMTS13 mutants were transiently expressed in COS-7 cells and stably transfected HEK293 cell lines were generated for all five mutants. The biosynthesis, secretion and specific enzymatic activity under both static and flow conditions were studied. Intracellular localization of the mutants was also determined by immunofluorescent microscopy. Patients homozygous for W688X, R692C or C804R became manifest with acute TTP as neonates and several siblings had died of a TTP-like disorder. Plasma ADAMTS13 activity was < 1% of the normal and ADAMTS13 antigen was undetectable. In patients with homozygous R1060W mutation, milder courses with adult onset of TTP after strong triggering factors were observed. One patient presented with the first TTP attack at the age of 19 years during her first pregnancy while a second patient experienced a first bout at the age of 61 years during a severe pneumonia. Both patients had 3% of residual plasma ADAMTS13 activity and 6% of antigen of normal human plasma. Homozygous mutation G1239V was found in a patient with frequent TTP bouts in childhood starting at 3 years of age and an attenuated course in adulthood with attacks triggered by excessive alcohol consumption. His plasma ADAMTS13 activity was <1% along with undetectable antigen. Recombinant mutants R692C, C804R, R1060W and G1239V migrated at 190 kDa like wild-type ADAMTS13. Secretion of all 4 missense mutants was severely reduced and was highest for R1060W. The truncated mutant W688X was efficiently secreted with a molecular weight of 97 kDa. This mutant could cleave the VWF73 peptide as did wild-type ADAMTS13. Proteolytic activity towards multimeric von Willebrand factor (VWF) under static conditions was moderately impaired and even substantially reduced under fluid shear stress as compared to wild-type. Immunofluorescent staining showed the mutant ADAMTS13 proteases within endoplasmatic reticulum and Golgi apparatus, consistent with the distribution of secretory proteins. We conclude that all 5 investigated ADAMTS13 mutations are causative for hereditary TTP. The homozygous missense mutations in the carboxyl-terminal part of ADAMTS13 severely impair the secretion of the protease while the truncating mutation affects the proteolytic activity towards multimeric VWF under shear stress. Our findings highlight the significance of the middle and distal carboxylterminal domains of ADAMTS13 for secretion and enzymatic activity in vivo. This study of homozygous patients showed an early onset and severe course of TTP for mutations in the 2nd and 3rd TSP-1 repeat while the mutations in the 7th TSP-1 and the first CUB domain led to a later onset and a milder course of TTP with bouts requiring strong triggering factors. The two patients homozygous for R1060W showed low levels of residual plasma ADAMTS13 activity and antigen and the mildest clinical courses which is consistent with the cell culture studies. Together, our findings suggest a phenotype-genotype correlation in hereditary TTP.

Mild Congenital ADAMTS13 Deficiency in a Patient with Familial Recurrent Ischemic Stroke

A severe functional deficiency in the ADAMTS13 protease, characteristic for patients suffering from thrombotic thrombocytopenic purpura (TTP), results in persisting prothrombotic ultra-large von Willebrand factor (ULVWF) multimers, which can lead to systemic microvascular thrombosis. The congenital form of TTP is attributed to the presence of homozygous or double heterozygous mutations in the ADAMTS13 gene leading to impaired secretion and/or a functional defect of the protease. Decreased ADAMTS13 activity has been also described for several physiological and pathological conditions. Emerging evidence indicates mild ADAMTS13 deficiency might also increase risk for arterial thrombosis such as for ischemic stroke. We therefore investigated a possible causal relation between moderate ADAMTS13 deficiency and ischemic stroke in one patient selected with the lowest ADAMTS13 activity amongst a cohort of 70 patients with ischemic stroke. At age 34 and 46 years the patient had suffered from idiopathic ischemic strokes with negative common prothrombotic risk factors. We found slightly elevated VWF antigen (161%), ULVWF multimers and reduced ADAMTS13 activity (17%). Genetic analysis identified five nucleotide exchanges in the ADAMTS13 gene leading to one heterozygous missense mutation P353L and four homozygous amino acid polymorphisms (R7W, Q448E, P618A, A732V). Her asymptomatic son presented with normal VWF antigen (99%) and an ADAMTS13 activity of 58% transmitted by a mutant maternal allele and a paternal allele harboring the single Q448E polymorphism. Recombinant expression studies indicated P353L alone causes an impact on ADAMTS13 secretion that is further pronounced in the context of the four polymorphisms. Secreted ADAMTS13 containing P353L or P353L combined with the four polymorphisms presented with similar binding interaction in a binding assay with immobilized multimeric VWF, however, the binding affinity was slightly less than that of wild-type ADAMTS13 or polymorphic rADAMTS13 proteins containing either Q448E alone or all four polymorphisms combined. ADAMTS13 activity analysis suggested that mainly the secretion process or the protein stability rather than the proteolytic activity was affected by the mutations. Co-transfections mimicking the patient's genotype indicated homozygosity of the four polymorphisms in the patient led to an attenuation of the adverse effect of the heterozygous P353L point mutation resulting in the moderate ADAMTS13 deficiency in the patient. Our results suggest moderate familial ADAMTS13 deficiency might be causally involved in the pathogenesis of ischemic stroke.

Prevalence of the ADAMTS-13 missense mutation R1060W in late onset adult thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor-cleaving protease (ADAMTS-13) activity. ADAMTS-13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear. We sought to identify common ADAMTS-13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood. We detected a missense mutation (C3178T) in exon 24 of ADAMTS-13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls (n = 100). Three of the patients had pregnancy-associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1-7 domain of ADAMTS-13. In vitro expression of mutant and wild-type ADAMTS-13 demonstrated that R1060W caused severe intracellular retention of ADAMTS-13 (<5% secretion) without affecting its metalloprotease activity. One homozygous and five heterozygous patients were identified. No other causative mutations were discovered, yet all six patients had ADAMTS-13 activity levels <5% at presentation (normal: 66-126%). Antibodies/inhibitors to ADAMTS-13 were detected in three/five heterozygous patients, and all six patients had subnormal antigen levels. Six asymptomatic first-degree relatives, including those of two probands with antibodies, were also heterozygous for C3178T; all but one had subnormal ADAMTS-13 activity. The high prevalence of R1060W ADAMTS-13 in adult onset TTP, together with its absence in childhood congenital TTP cases reported elsewhere, suggests it may be a factor in the development of late onset TTP.

Prevalence of the ADAMTS-13 missense mutation R1060W in late onset adult thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor-cleaving protease (ADAMTS-13) activity. ADAMTS-13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear. We sought to identify common ADAMTS-13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood. We detected a missense mutation (C3178T) in exon 24 of ADAMTS-13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls (n = 100). Three of the patients had pregnancy-associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1-7 domain of ADAMTS-13. In vitro expression of mutant and wild-type ADAMTS-13 demonstrated that R1060W caused severe intracellular retention of ADAMTS-13 (<5% secretion) without affecting its metalloprotease activity. One homozygous and five heterozygous patients were identified. No other causative mutations were discovered, yet all six patients had ADAMTS-13 activity levels <5% at presentation (normal: 66-126%). Antibodies/inhibitors to ADAMTS-13 were detected in three/five heterozygous patients, and all six patients had subnormal antigen levels. Six asymptomatic first-degree relatives, including those of two probands with antibodies, were also heterozygous for C3178T; all but one had subnormal ADAMTS-13 activity. The high prevalence of R1060W ADAMTS-13 in adult onset TTP, together with its absence in childhood congenital TTP cases reported elsewhere, suggests it may be a factor in the development of late onset TTP.

Phenotypic Correction of Adamts13-Deficient Mice by Early Intra-Amniotic Gene Transfer of Lentiviral Vector Encoding ADAMTS13 Genes.

ADAMTS13, a member of A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, is mainly synthesized in the hepatic stellate cells, endothelial cells and megakaryocytes or platelets. It controls the sizes of von Willebrand factor (VWF) multimers by cleaving VWF at the Tyr1605-Met1606 bond. Genetic deficiency of plasma ADAMTS13 activity results in hereditary thrombotic thrombocytopenic purpura (TTP), also named Upshaw-Schülman syndrome. To develop a potential gene therapy approach and to determine the domains of ADAMTS13 required for recognition and cleavage of VWF in vivo, a self-inactivating lentiviral vector encoding human wild-type ADAMTS13 or variant truncated after the spacer domain (construct MDTCS) was administrated by intra-amniotic injection on embryonic day 8. Direct stereomicroscopy and immunofluorescent microscopic analysis revealed that the green fluorescent protein (GFP) reporter, ADAMTS13 and MDTCS were predominantly expressed in the heart, kidneys and skin. The synthesized ADAMTS13 and truncated variant were detectable in mouse plasma by immunoprecipitation and Western blot, as well as by proteolytic cleavage of FRETS-VWF73 substrate. The levels of proteolytic activity in plasma of mice expressing ADAMTS13 and MDTCS were 5 ± 7% and 60 ± 70%, respectively using normal human plasma as a standard, and this proteolytic activity persisted for at least 24 weeks in Adamts13−/−mice and 42 weeks in wild-type mice tested (the duration of observation). The mice expressing both recombinant ADAMTS13 and MDTCS showed a significantly decreased ratio of plasma VWF collagen-binding activity to antigen and a reduction in VWF multimer sizes as compared to those in the controls. Moreover, the mice expressing ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time (9.0 ± 0.6 and 25.2 ± 3.2 min, respectively) as compared to the Adamts13−/− mice expressing GFP alone (5.6 ± 0.5 min) (p<0.01). The ferric chloride-induced carotid occlusion time in Adamts13−/− mice expressing ADAMTS13 was almost identical to that in wild type mice with same genetic background (C56BL/6) (8.0 ± 0.2 min) (p>0.05). The data demonstrate the correction of the prothrombotic phenotype in Adamts13−/−mice by gene transfer to the fetus by viral vectors encoding human wild type ADAMTS13 and the carboxyl terminal truncated variant (MDTCS), supporting the feasibility of developing a gene therapy based treatment for hereditary TTP. The discrepancy in the proteolytic activity of MDTCS between in vitro (Zhang P et al. Blood, 2007 in press) and in vivo in the present study suggests the potential cofactors in murine circulation that may rescue the defective proteolytic activity of the carboxyl-terminal truncated ADAMTS13 protease seen in vitro.

O-Fucosylation Is Required for ADAMTS13 Secretion

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [(3)H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-beta1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDP-fucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.