Evidence of protective effects of recombinant ADAMTS13 in a humanized model of sickle cell disease.

Sickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/reoxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the firsttime evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events.

N-acetylcysteine in preclinical mouse and baboon models of thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder diagnosed by thrombocytopenia and hemolytic anemia, associated with a deficiency in von Willebrand factor (VWF)-cleaving protease ADAMTS13. Current treatment is based on plasma infusion for congenital TTP, or plasma exchange, often in combination with immunosuppressive agents, for acquired TTP. These treatment methods are not always effective; therefore, new treatment methods are highly necessary. N-acetylcysteine (NAC), an FDA-approved anti-mucolytic agent, is a possible new treatment strategy for TTP, as it was demonstrated to reduce disulfide bonds in VWF, thereby decreasing VWF multimers size and hence their prothrombotic potential. We investigated whether NAC, without concurrent plasma exchange and immunosuppressive therapy, is effective in preventing and resolving TTP signs, using well-established murine and baboon models for TTP. In mice, prophylactic administration of NAC was effective in preventing severe TTP signs. This in vivo finding was supported by in vitro data demonstrating the VWF multimer-reducing properties of NAC in solution. Nonetheless, in both mice and baboons, administration of NAC was not effective in resolving preexisting TTP signs; thrombocytopenia, hemolytic anemia, and organ damage could not be reversed, as thrombus resolution was not achieved. Failure to improve clinical outcome occurred even though a reduction in VWF multimers was observed, demonstrating that NAC was efficient in reducing disulfide bonds in circulating VWF multimers. In conclusion, prophylactic administration of NAC, without concurrent plasma exchange, was effective in preventing severe TTP signs in mice, but NAC was not effective in resolving preexistent acute TTP signs in mice and baboons.

ADAMTS13-mediated thrombolysis of t-PA-resistant occlusions in ischemic stroke in mice.

Rapid vascular recanalization forms the basis for successful treatment of cerebral ischemia. Currently, tissue plasminogen activator (t-PA) is the only approved thrombolytic drug for ischemic stroke. However, t-PA does not always result in efficient thrombus dissolution and subsequent blood vessel recanalization. To better understand thrombus composition, we analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. Thrombi contained on average 20.3% ± 10.1% VWF, and this was inversely correlated with thrombus red blood cell content. We hypothesized that ADAMTS13 can exert a thrombolytic effect in VWF-containing thrombi in the setting of stroke. To test this, we generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. Interestingly, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA-resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (P < .005). Delayed ADAMTS13 administration 60 minutes after occlusion was still effective but to a lesser extent (P < .05). These data show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke.

Potential for Recombinant ADAMTS13 as an Effective Therapy for Acquired Thrombotic Thrombocytopenic Purpura.

OBJECTIVE: The metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) regulates the size of von Willebrand factor multimers. A deficiency in ADAMTS13 activity is associated with the life-threatening disease thrombotic thrombocytopenic purpura (TTP). The vast majority of patients have acquired TTP, where circulating anti-ADAMTS13 autoantibodies are causative for the decreased ADAMTS13 activity. Current treatment consists of plasma exchange, but improved therapies are highly warranted. APPROACH AND RESULTS: We have developed a new rat model mimicking various aspects of acquired TTP to investigate the therapeutic efficacy of human recombinant ADAMTS13. A polyclonal antibody against ADAMTS13 completely blocked endogenous rat ADAMTS13 activity in Sprague-Dawley rats. When TTP was triggered using recombinant von Willebrand factor, the animals displayed severe TTP-like symptoms, such as thrombocytopenia, hemolytic anemia, and von Willebrand factor-rich thrombi in the kidneys and brain. Subsequent injection of 400, 800, or 1600 U/kg recombinant ADAMTS13 prevented full development of these symptoms. Analysis of plasma samples confirmed that recombinant ADAMTS13 was able to override circulating anti-ADAMTS13 inhibitory antibodies, resulting in restoration of ADAMTS13 activity and degradation of ultralarge von Willebrand factor multimers. CONCLUSIONS: Recombinant ADAMTS13 was shown to be effective in averting severe acquired TTP-like symptoms in rats and holds promising value for the treatment of this severe and life-threatening disease in humans.

Poor responder to plasma exchange therapy in acquired thrombotic thrombocytopenic purpura is associated with ADAMTS13 inhibitor boosting: visualization of an ADAMTS13 inhibitor complex and its proteolytic clearance from plasma.

BACKGROUND: Plasma exchange (PE) is the first-line treatment for primary acquired thrombotic thrombocytopenic purpura (aTTP) with severe deficiency of ADAMTS13 activity (ADAMTS13:AC). Some patients are poor responders to PE, raising concern over multiple pathogenetic pathways. STUDY DESIGN AND METHODS: Based on 52 aTTP patients in our national cohort study, we monitored plasma levels of ADAMTS13, clinical and laboratory findings, and outcomes. In a representative poor responder to PE, we examined an ADAMTS13 inhibitor (ADAMTS13:INH) complex in plasma milieu, by means of a large-pore isoelectric focusing (IEF) analysis. RESULTS: Of 52 aTTP patients, 20 were good responders and 32 were poor responders. In the latter group, plasma ADAMTS13:AC levels never increased to more than 10% of normal during 14 days after PE initiation. Mean (±SD) plasma ADAMTS13:INH titers (Bethesda unit/mL) were 5.7 (±4.5) before PE, but decreased to 1.4 (±0.8) on the fourth PE day and then remarkably increased to 14.8 (±10.0) on the 10th PE day, termed "inhibitor boosting," and then slowly decreased to undetectable level over 1 month. On admission, none of the routinely available clinical and laboratory markers differentiated these two groups. However, elevated pre-PE levels of ADAMTS13:INH were correlated with a poor response. We visualized an ADAMTS13:INH (immunoglobulin G) complex in a patient plasma by an IEF analysis and found proteolytic fragment of ADAMTS13 antigen by a two-dimensional IEF and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. CONCLUSION: Findings from this cohort of aTTP patients demonstrated that inhibitor boosting often occurs in aTTP patients in Japan. Poor responders could be predicted by elevated pre-PE ADAMTS13:INH levels on admission, but not by routinely collected clinical or laboratory data.

Persistence of circulating ADAMTS13-specific immune complexes in patients with acquired thrombotic thrombocytopenic purpura.

Anti-ADAMTS13 autoantibodies are the main cause of acquired thrombotic thrombocytopenic purpura. Binding of these antibodies to ADAMTS13 eventually results in the formation of antigen-antibody immune complexes. Circulating ADAMTS13-specific immune complexes have been described in patients with acquired thrombotic thrombocytopenic purpura, although the prevalence and persistence of these immune complexes over time have hitherto remained elusive. Here, we analyzed a large cohort of patients with acquired thrombotic thrombocytopenic purpura for the presence of free and complexed anti-ADAMTS13 antibodies. In the acute phase (n=68), 100% of patients had free IgG antibodies and 97% had ADAMTS13-specific immune complexes. In remission (n=28), 75% of patients had free antibodies (mainly IgG) and 93% had ADAMTS13-specific immune complexes. Free antibodies were mainly of subclasses IgG1 and IgG4, whereas IgG4 was by far the most prevalent in ADAMTS13-specific immune complexes. Comparison of ADAMTS13 inhibitor and anti-ADAMTS13 IgG (total and subclasses) antibody titers in acute phase and in remission samples showed a statistically significant decrease in all parameters in remission. Although non-significant, a trend towards reduced or undetectable titers in remission was also observed for ADAMTS13-specific immune complexes of subclasses IgG1, IgG2 and IgG3. No such trend was discernible for IgG4; IgG4 immune complexes persisted over years, even in patients who had been treated with rituximab and who showed no features suggesting relapse.

Treatment with recombinant ADAMTS13, alleviates hypoxia/reoxygenation-induced pathologies in a mouse model of human sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is an inherited red blood cell disorder with a causative substitution in the beta-globin gene that encodes beta-globin in hemoglobin. Furthermore, the ensuing vasculopathy in the microvasculature involves heightened endothelial cell adhesion, inflammation, and coagulopathy, all of which contribute to vaso-occlusive crisis (VOC) and the sequelae of SCD. In particular, dysregulation of the von Willebrand factor (VWF) and a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in human SCD pathology. OBJECTIVES: To investigate the beneficial potential of treatment with recombinant ADAMTS13 (rADAMTS13) to alleviate VOC. METHODS: Pharmacologic treatment with rADAMTS13 in vitro or in vivo was performed in a humanized mouse model of SCD that was exposed to hypoxia/reoxygenation stress as a model of VOC. Then, pharmacokinetic, pharmacodynamic, and behavioral analyses were performed. RESULTS: Administration of rADAMTS13 to SCD mice dose-dependently increased plasma ADAMTS13 activity, reduced VWF activity/antigen ratios, and reduced baseline hemolysis (free hemoglobin and total bilirubin) within 24 hours. rADAMTS13 was administered in SCD mice, followed by hypoxia/reoxygenation stress, and reduced VWF activity/antigen ratios in parallel to significantly (p < .01) improved recovery during the reoxygenation phase. Consistent with the results in SCD mice, we demonstrate in a human in vitro system that treatment with rADAMTS13 counteracts the inhibitory activity of hemoglobin on the VWF/ADAMTS13-axis. CONCLUSION: Collectively, our data provide evidence that relative ADAMTS13 insufficiency in SCD mice is corrected by pharmacologic treatment with rADAMTS13 and provides an effective disease-modifying approach in a human SCD mouse model.

Systemic antithrombotic effects of ADAMTS13.

The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 deficiency is linked to a life-threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet-rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in activated microvenules of Adamts13-/- mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi formation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in alphaIIbbeta3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our findings reveal that ADAMTS13 has a powerful natural antithrombotic activity and recombinant ADAMTS13 could be used as an antithrombotic agent.

Identification of cysteine thiol-based linkages in ADAMTS13 in support of a non-proteolytic regulation of von Willebrand factor.

BACKGROUND: ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) to regulate its function. Additionally, ADAMTS13 is thought to regulate lateral association of VWF multimers to form fibrillar structures through its free thiols. OBJECTIVE: The purpose of the present study is to obtain direct evidence for ADAMTS13 to engage in thiol/disulfide exchange reactions. METHODS: Covalent complexes between ADAMTS13 and VWF were determined by agarose gel electrophoresis under nonreducing conditions. Free thiols in ADAMST13 were identified by a reversed phase high-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry system. RESULTS: We demonstrate formation of covalent linkage between ADAMTS13 and VWF, which is time, concentration, temperature, and shear dependent. This interaction is independent of proteolytic activity of ADAMTS13 but depends on the C-terminal domains comprising the fifth through eighth thrombospondin type 1 repeats and C1r/C1s, Uegf, Bmp1 (CUB) domains. The interaction can be blocked by thiol-reactive agents, indicating that association is accomplished through disulfide bridge formation. Several partially reduced free thiols are identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent, although a point mutation (C1275S) in ADAMTS13 does not alter its ability to form covalent linkages with VWF. This suggests functionally relevant disulfide plasticity in ADAMTS13. Interestingly, ADAMTS13 also forms homo-oligomers under the same conditions as required for the generation of hetero-oligomeric complexes of ADAMTS13 and VWF. CONCLUSIONS: Our results suggest that a dynamic network of free thiols in ADAMTS13 undergoing intra- and inter-molecular redox reactions may add another layer of regulation to VWF function under various conditions.

Temperature-dependent irreversible conformational change of recombinant ADAMTS13 upon metal ion chelation.

BACKGROUND: The catalytic domain of ADAMTS13 possesses one Zn2+ and up to three putative Ca2+ binding sites and can be inactivated by chelating agents. Although replenishment with an appropriate metallic cation is thought to restore the enzyme's proteolytic activity fully, ADAMTS13 stability in a metal ion-depleting environment has not been explored. OBJECTIVES: To address the stability of ADAMTS13 in citrated human plasma. METHODS: ADAMTS13 activity was measured using the FRETS-VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Higher-order structural changes were analyzed using Fourier-transformed infrared spectroscopy and dynamic light scattering. RESULTS: ADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time-dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca2+ or Zn2+ prior to but not postincubation prevented the activity decrease of the enzyme. The SEC-ICP-MS analyses showed that ADAMTS13 only bound Zn2+ and that its reduced activity correlated with a gradual loss of bound Zn2+ . Concomitant higher-order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less-ordered protein structures. CONCLUSIONS: Zn2+ is required to stabilize ADAMTS13 structure at physiologic temperature, thereby preventing irreversible loss of enzyme activity. This finding is particularly important to consider when using citrated human plasma as a source of ADAMTS13 in clinical settings.

Relation between ADAMTS13 activity and ADAMTS13 antigen levels in healthy donors and patients with thrombotic microangiopathies (TMA).

We have established a new, enzyme-linked immunosorbent assay (ELISA) for the detection of ADAMTS13 antigen using purified polyclonal rabbit anti-human ADAMTS13 IgG. Normal plasma ADAMTS13 antigen levels span a concentration of 740-1420 ng/ml (median 1080 ng/ml) resulting in an ADAMTS13 activity to antigen ratio of 0.48 to 1.68 U/mug. In a cohort of HUS patients, ADAMTS13 antigen was in the normal range, whereas in hereditary TTP patients antigen levels were low to undetectable, in concordance with severe deficient ADAMTS13 activity. Plasma of acquired TTP patients was found to contain free as well as autoantibody-bound ADAMTS13. We also present evidence for circulating anti-ADAMTS13 antibody/ADAMTS13 antigen immune complexes not only in acutely ill or actively treated patients but also in patients who have already achieved clinical remission. This new developed ADAMTS13 antigen ELISA assay allows rapid determination of ADAMTS13 antigen levels in human plasma but is of limited predictive value for the diagnosis or treatment of acquired TTP due to the detection of ADAMTS13 in antibody complexes.

Expression and characterization of recombinant human ADAMTS-13.

Thrombotic thrombocytopenic purpura (TTP) is a severe disease associated with unusually large, hemostatically hyperactive von Willebrand factor (VWF) and severe deficiency in ADAMTS-13, the protease responsible for the proteolytic degradation of VWF in plasma. ADAMTS-13 prevents inappropriate microvascular platelet aggregation by cleaving VWF between Tyr1605 and Met1606 thereby producing dimers of 176 kd and 140 kd and smaller multimers. Identification of the ADAMTS13 gene and cloning of the corresponding cDNA allowed for the application of recombinant techniques, such as genetic engineering of ADAMTS13 cDNA, cell culture expression, and in vitro activity studies to analyze the functional relationship between ADAMTS-13 and the pathophysiology of ADAMTS-13 deficiency. In vitro expression and characterization of recombinant ADAMTS-13 (rADAMTS-13) clearly established that ADAMTS-13 is deficient in congenital TTP and inhibited in acquired TTP. Recent studies have contributed greatly to our current understanding of the molecular mechanism leading to congenital and acquired TTP. Apart from being a useful tool, availability of rADAMTS-13 raised the prospect of developing a recombinant substitution therapy to improve TTP treatment and allowing present diagnostic assays to be simplified. Here we report on recent advances in cell culture expression and functional characterization of human rADAMTS-13.

Modulation of ADAMTS13 secretion and specific activity by a combination of common amino acid polymorphisms and a missense mutation.

Sequence analysis of the ADAMTS13 locus of 2 patients with hereditary thrombotic thrombocytopenic purpura (TTP) revealed the homozygous presence of 4 single nucleotide polymorphisms (SNPs) (R7W, Q448E, P618A, A732V) and a rare missense mutation (R1336W). Analysis of the individual effect of any amino acid exchanges showed that several sequence variations can interact with each other, thereby altering the phenotype of ADAMTS13 deficiency. Introduction of polymorphisms R7W, Q448E, and A732V had no or only minor effects on ADAMTS13 secretion. In contrast, P618A, R1336W, and the A732V-P618A combination strongly reduced ADAMTS13-specific activity and antigen levels. Surprisingly, R7W and Q448E were positive modifiers of ADAMTS13 secretion in the context of P618A and A732V but neither could rescue the severely reduced specific activity conferred by P618A. However, in the context of R1336W, polymorphisms R7W and Q448E enhanced the detrimental effect of the missense mutation and led to undetectable enzyme activity. We show that dependent on the sequence context, the same polymorphisms might be either positive or negative modifiers of gene expression. Our results might therefore be widely relevant to understanding the influence of polymorphisms on the phenotypic expression of complex diseases.

ADAMTS13 autoantibodies in patients with thrombotic microangiopathies and other immunomediated diseases.

Autoantibodies neutralizing human ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif), the metalloprotease that physiologically cleaves von Willebrand factor, are a major cause of severe deficiency of the protease and of acquired thrombotic thrombocytopenic purpura (TTP). We evaluated prevalence of anti-ADAMTS13 antibodies in 59 patients with thrombotic microangiopathies (TMAs) and in 160 patients with immunologic or thrombocytopenic diseases different from TTP, using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G (IgG) antibodies directed against ADAMTS13 were found in 97% of untreated patients with acute acquired TMA who had plasma levels of ADAMTS13 activity below 10%. The corresponding prevalence of IgM antibodies was 11%. In contrast, anti-ADAMTS13 antibodies of G or M isotypes were detected in 20% of patients with TMA with ADAMTS13 activity above 10%. The ELISA was more sensitive than the standard functional inhibitor assay for detecting antibodies against ADAMTS13. Patients with thrombocytopenia from various causes (n = 50), systemic lupus erythematosus (SLE; n = 40), and the antiphospholipid antibody syndrome (APS; n = 55) had prevalences of IgG antibodies of 8%, 13%, and 5% respectively, only slightly higher than the prevalence in 111 healthy donors (4%). A rather high prevalence of anti-ADAMTS13 IgM antibodies was found in patients with SLE and APS (18% each). The clinical significance of IgM antibodies in these groups is unclear. In conclusion, the ELISA method detected anti-ADAMTS13 IgG antibodies in a very large proportion of patients with acquired TMA associated with severe ADAMTS13 deficiency, and was more sensitive than the inhibitor assay.

ADAMTS13 gene defects in two brothers with constitutional thrombotic thrombocytopenic purpura and normalization of von Willebrand factor-cleaving protease activity by recombinant human ADAMTS13.

Genetic analysis of the ADAMTS13 locus identified six mutations in the ADAMTS13 genes of two brothers suffering from constitutional thrombotic thrombocytopenic purpura (TTP): a stop codon leading to a truncated protein on the paternal ADAMTS13 allele and five amino acid exchanges on the maternal allele, three of which were single nucleotide polymorphisms. The other two mutations, not detected in 230 sequenced alleles of healthy control subjects, are, therefore, probably responsible, alone or as part of a combination, for the severe ADAMTS13 deficiency. We also investigated the feasibility of using recombinant ADAMTS13 (rADAMTS13) for normalization of von Willebrand factor-cleaving protease (VWF-cp) activity in plasma of the two congenitally deficient patients. Addition of rADAMTS13 to their plasma restored the VWF-processing pattern to normal, suggesting the potential usefulness of rADAMTS13 for therapy and prophylaxis of familial TTP.

Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura.

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 can lead to thrombotic thrombocytopenic purpura (TTP), a disease associated with the widespread formation of platelet-rich thrombi in many organs. Autoantibodies that inactivate ADAMTS13 are the most frequent cause of acquired TTP. Little is known about epitope specificity and reactivity of anti-ADAMTS13 antibodies. In this study, a series of ADAMTS13 domains were expressed in Escherichia coli, and the reactivity of purified recombinant fragments with anti-ADAMTS13 auto-antibodies from 25 patients with severe ADAMTS13 deficiency was evaluated in vitro. All TTP plasmas contained antibodies directed against the cysteine-rich spacer (cys-rich/spacer) domain of ADAMTS13. In the plasma of 3 patients, antibodies were detected that reacted exclusively with the cys-rich/spacer domain, underscoring the importance of this region for functional activity of ADAMTS13. In 64% of the plasmas, antibodies reacted with the 2 CUB domains, and in 56% they reacted with the isolated first thrombospondin type 1 (TSP-1) repeat and with the compound fragment consisting of the catalytic, the disintegrin-like, and the TSP1-1 domain. Less frequently, in 28% of the plasmas, antibodies reacted with the TSP1 repeats 2 to 8. Unexpectedly, antibodies reacted with the propeptide region in 20% of the plasmas. In conclusion, this study shows that even though anti-ADAMTS13 autoantibodies react with multiple domains of the protease, the cys-rich/spacer domain is consistently involved in antibody reactivity.

Nonneutralizing IgM and IgG antibodies to von Willebrand factor-cleaving protease (ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura.

Acquired thrombotic thrombocytopenic purpura (TTP) has been linked to severe deficiency of ADAMTS-13 activity caused by autoantibodies inhibitory to ADAMTS-13. We report data on a patient with confirmed TTP who had severely reduced ADAMTS-13 activity but showed no ADAMTS-13 inhibition in a widely used fluid phase activity assay. With a newly developed enzyme-linked immunosorbent assay, using immobilized recombinant ADAMTS-13, we found high titers of IgM and IgG antibodies that bound to ADAMTS-13, but did not neutralize protease activity. These autoantibodies probably influenced the half-life of ADAMTS-13 or its binding to the endothelial cell surface, thereby compromising ADAMTS-13 activity in vivo. Given that ADAMTS-13 may interact physiologically with various receptors or ligands, the occurrence, distribution, and the epitope mapping of nonneutralizing antibodies will be an important area for future research.

Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13).

Deficient von Willebrand factor (VWF) degradation has been associated with thrombotic thrombocytopenic purpura (TTP). In hereditary TTP, the specific VWF-cleaving protease (VWF-cp) is absent or functionally defective, whereas in the nonfamilial, acquired form of TTP, an autoantibody inhibiting VWF-cp activity is found transiently in most patients. The gene encoding for VWF-cp has recently been identified as a member of the metalloprotease family and designated ADAMTS13, but the functional activity of the ADAMTS13 gene product has not been verified. To establish the functional activity of recombinant VWF-cp, we cloned the complete cDNA sequence in a eukaryotic expression vector and transiently expressed the encoded recombinant ADAMTS13 in HEK 293 cells. The expressed protein degraded VWF multimers and proteolytically cleaved VWF to the same fragments as those generated by plasma VWF-cp. Furthermore, recombinant ADAMTS13-mediated degradation of VWF multimers was entirely inhibited in the presence of plasma from a patient with acquired TTP. These data show that ADAMTS13 is responsible for the physiologic proteolytic degradation of VWF multimers.