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Long Zheng

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ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 1. Structural and functional characterization in vitro

Transfusion, Apr 4, 2016

BACKGROUND-Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness cause... more BACKGROUND-Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness caused by autoantibodies that decrease the activity of ADAMTS13, the von Willebrand Factor cleaving protease. Despite efficacy of plasma exchange, mortality remains high and relapse is common. Improved therapies may come from understanding the diversity of pathogenic autoantibodies on a molecular/genetic level. Cloning comprehensive repertoires of patient autoantibodies can provide the necessary tools for studying immunobiology of disease and developing animal models. STUDY DESIGN AND METHODS-Anti-ADAMTS13 antibodies were cloned from four patients with acquired TTP using phage display and characterized with respect to genetic origin, inhibition of ADAMTS13 proteolytic activity, and epitope specificity. Anti-idiotypic antisera raised to a subset of autoantibodies enabled comparison of their relatedness to each other and to polyclonal IgG in patient plasma. RESULTS-Fifty-one unique antibodies were isolated comprising epitope specificities resembling the diversity found in circulating patient IgG. Antibodies directed to both the amino terminal domains and those requiring the ADAMTS13 cysteine-rich/spacer region for binding inhibited proteolytic activity, while those solely targeting carboxy-terminal domains were noninhibitory. Anti-idiotypic antisera raised to a subset of antibody clones crossreacted with and reduced the inhibitory activity of polyclonal IgG from a set of unrelated patients. CONCLUSIONS-Anti-ADAMTS13 autoantibodies isolated by repertoire cloning display the diversity of epitope specificities found in patient plasma and provide tools for developing animal

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model

Transfusion, Apr 4, 2016

BACKGROUND-Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in w... more BACKGROUND-Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultra-large von Willebrand Factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rationale, reliable and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 variable region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory IgG in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS-Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet thrombi following focal or systemic vascular injury was examined. RESULTS-Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF

A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura

Transfusion, Jan 20, 2011

Background-Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with h... more Background-Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either pre-denatured von Willebrand factor (VWF) or small peptides derived from the VWF-A2 domain. The physiological relevance of the assay results is uncertain. Methods-We sought to develop a novel shear-based assay to assess plasma ADAMTS13 activity and inhibitor. We also compared this assay with a fluorogenic peptide assay. Results-We found that an incubation of purified plasma VWF with 0.5-1.0 μl of citrated plasma under constant vortexing at 2,500 rpm for 60 minutes in the presence of 5 mM CaCl 2 , 1.7 μM ZnCl 2 and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear-based assay appeared to be more sensitive than the guanidine-denaturization assay for determining plasma ADAMTS13 activity. Conclusions-Our fluid shear-based assay may be useful for investigating basic biological function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.

Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol–Dependent Manner

Arteriosclerosis, Thrombosis, and Vascular Biology, Feb 1, 2014

V on Willebrand factor (VWF), an ultra large (UL) or large multimeric adhesion glycoprotein in bl... more V on Willebrand factor (VWF), an ultra large (UL) or large multimeric adhesion glycoprotein in blood, is primarily synthesized in endothelial cells, megakaryocytes, and platelets. 1 The newly synthesized VWF is stored in the Weibel-Palade bodies of endothelial cells or α-granules of platelets. ULVWF is released from these storage organelles on stimulation by epinephrine, histamine, thrombin, and inflammatory cytokines or toxins. 2-4 The newly released ULVWF forms string-like structures anchored on the cell surface, 2-4 which are hyperactive and recruit flowing platelets from circulation to the site of endothelial activation or injury. Cell-bound ULVWF strings are highly susceptible to proteolysis by plasma metalloprotease ADAMTS13. 2,3 This proteolytic cleavage results in a VWF-free endothelial surface, preventing unwanted and excessive platelet adhesion/aggregation and thrombus formation after injury. However, VWF released into circulation remains large and therefore requires further processing by plasma ADAMTS13, 5 other leukocyte proteases, 6 and complement factor H. 7 An inability to cleave or process cell-bound ULVWF or circulating large VWF multimers into smaller ones results in a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP), 8,9 which is characterized by severe thrombocytopenia and microangiopathic hemolytic anemia with various degrees of organ failure. 8,9 Previous studies have demonstrated that the proteolytic cleavage of VWF by ADAMTS13 depends on the amino-terminal portion of ADAMTS13 (ie, MDTCS domains). 10-16 An extensive exosite interaction between the ADAMTS13-DTCS domains and the VWF-A2 domain 11,17 seems to be necessary for productive VWF cleavage. A

Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura

Haematologica, Aug 4, 2016

Animal models for thrombotic thrombocytopenic purpura: a narrative review

Annals of Blood

Background and Objective: Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal blood ... more Background and Objective: Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal blood disorder, resulting from severe deficiency of plasma ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13) activity. ADAMTS13 is crucial for normal hemostasis through proteolytic cleavage of ultra large von Willebrand factor (VWF). Since the discovery of ADAMTS13 in 2001, several animal models for TTP have been established. In this narrative review, we summarize the creation and characterization of the established animal models for TTP to date. Methods: We performed a literature search through PubMed from 1969 to 2022 using free text: TTP and animal model. We found 67 peer-reviewed articles but only 33 articles were included for review and 34 articles that did not discuss TTP were excluded.

ADAMTS13 Biomarkers in Management of Immune Thrombotic Thrombocytopenic Purpura

Archives of Pathology & Laboratory Medicine

Context.— Immune thrombotic thrombocytopenic purpura (iTTP) is a rare but potentially fatal blood... more Context.— Immune thrombotic thrombocytopenic purpura (iTTP) is a rare but potentially fatal blood disorder resulting from acquired deficiency of plasma ADAMTS13, a metalloprotease that cleaves endothelium-derived ultralarge von Willebrand factor. Standard of care for iTTP including therapeutic plasma exchange, caplacizumab, and immunosuppressives, known as triple therapy, has led to a significant reduction in the disease-related mortality rate. The first International Society of Thrombosis and Haemostasis TTP guideline stresses the importance of having plasma ADAMTS13 activity testing in the algorithm for diagnosis and management of iTTP. However, the predictive role of assessing plasma ADAMTS13 activity and inhibitors or other ADAMTS13-related parameters in patients with acute iTTP and during remission has not been systematically evaluated. Objective.— To review and assess the predictive values of testing plasma ADAMTS13 activity, antigen, and inhibitors or anti-ADAMTS13 immunoglob...

Synergistic effects of ADAMTS13 deficiency and complement activation in pathogenesis of thrombotic microangiopathy

Blood, 2019

This study in mice suggests a synergistic role of ADAMTS13 deficiency and complement “hyperactiva... more

Transfusion of Platelets Loaded With Recombinant ADAMTS13 (A Disintegrin and Metalloprotease With Thrombospondin Type 1 Repeats-13) Is Efficacious for Inhibiting Arterial Thrombosis Associated With Thrombotic Thrombocytopenic Purpura

Arteriosclerosis, Thrombosis, and Vascular Biology, 2018

Objective— ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cle... more Objective— ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cleaves VWF (von Willebrand factor). This process is essential for hemostasis. Severe deficiency of plasma ADAMTS13 activity, most commonly resulting from autoantibodies against ADAMTS13, causes thrombotic thrombocytopenic purpura. Therapeutic plasma exchange is the standard of care to date, which removes autoantibodies and replenishes ADAMTS13. However, such a therapy is often ineffective to raise plasma ADAMTS13 activity, and in-hospital mortality rate remains as high as 20%. Approach and Results— To overcome the inhibition by autoantibodies, we developed a novel approach by delivering rADAMTS13 (recombinant ADAMTS13 ) using platelets as vehicles. We show that both human and murine platelets can uptake rADAMTS13 ex vivo. The endocytosed rADAMTS13 within platelets remains intact, active, and is stored in α-granules. Under arterial shear (100 dyne/cm 2 ), the rADAMTS13 in platelets is relea...

Therapeutic efficacy of the platelet glycoprotein Ib antagonist anfibatide in murine models of thrombotic thrombocytopenic purpura

Blood Advances, 2016

Key Points Anfibatide potently inhibits platelet agglutination under static and arterial shear co... more

Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura

Haematologica, Nov 4, 2016

Acquired thrombotic thrombocytopenic purpura is primarily caused by deficiency of plasma ADAMTS13... more Acquired thrombotic thrombocytopenic purpura is primarily caused by deficiency of plasma ADAMTS13 activity resulting from autoantibodies against ADAMTS13. However, ADAMTS13 deficiency alone is often not sufficient to cause acute thrombotic thrombocytopenic purpura. Infections or systemic inflammation may precede acute bursts of the disease but the underlying mechanisms are not fully understood. Here, 52 patients with acquired idiopathic thrombotic thrombocytopenic purpura and 30 blood donor controls were recruited for the study. Plasma levels of human neutrophil peptides 1-3 and complement activation fragments (i.e. Bb, iC3b, C4d, and sC5b-9) were determined by enzyme-linked immunosorbent assays. Univariate analyses were performed to determine the correlation between each biomarker and clinical outcomes. We found that plasma levels of human neutrophil peptides 1-3 and Bb in patients with acute thrombotic thrombocytopenic purpura were significantly higher than those in the control (p...

Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP

Blood, Jul 7, 2016

Infection or inflammation may precede and trigger formation of microvascular thrombosis in patien... more Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood. Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 μM and 45 μM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (KD = 0.72 μM), soluble VWF (KD = 0.58 μM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼17...

ADAMTS13 and von Willebrand factor interactions

Current Opinion in Hematology, 2015

Purpose of review-ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand facto... more Purpose of review-ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity is accountable for a potentially fatal blood disorder thrombotic thrombocytopenic purpura (TTP). Understanding of ADAMTS13-VWF interaction is essential for developing novel treatments to this disorder. Recent findings-Despite the proteolytic activity of ADAMTS13 being restricted to the metalloprotease domain, the ancillary proximal C-terminal domains including the disintegrin domain, first TSP-1 repeat, cysteine-rich region, and spacer domain are all required for cleavage of VWF and its analogs. Recent studies have added to our understandings of the role of the specific regions in the disintegrin domain, the cysteine-rich domain, and the spacer domain responsible for its interaction with VWF. Additionally, regulative functions of the distal portion of ADAMTS13 including the TSP-1 2-8 repeats and the CUB domains have been proposed. Finally, fine mapping of anti-ADAMTS13 antibody epitopes have provided further insight into the essential structural elements in ADAMTS13 for VWF binding and the mechanism of autoantibody-mediated TTP. Summary-Significant progress has been made in our understandings of the structure-function relationship of ADAMTS13 in the past decade. To further investigate ADAMTS13-VWF interactions for medical applications, these interactions must be studied under physiological conditions in vivo.

Platelet-delivered ADAMTS13 inhibits arterial thrombosis and prevents murine models of thrombotic thrombocytopenic purpura

Blood, Jan 23, 2015

ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggrega... more ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggregation and arterial thrombosis. An inability to cleave ultra large VWF resulting from hereditary or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma exchange is the most effective initial therapy for TTP to date. Here, we report characterization of transgenic mice expressing recombinant human ADAMTS13 (rADAMTS13) in platelets and its efficacy in inhibiting arterial thrombosis and preventing hereditary and acquired antibody-mediated TTP in murine models. Western blotting and fluorescent resonance energy transfer (FRETS) assay detect full-length rADAMTS13 protein and its proteolytic activity, respectively, in transgenic (Adamts13(-/-)Plt(A13)), but not in wild type (WT) and Adamts13(-/-) (KO) platelets. The expressed rADAMTS13 is released upon stimulation with thrombin and collagen, but less with 2MesADP....

Novel recombinant glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 and variants for assessment of anti-ADAMTS13 autoantibodies in patients with thrombotic thrombocytopenic purpura

Thrombosis and haemostasis, 2011

Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic th... more Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). We report here a novel cell-based assay using glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 or variants expressed on cell membrane for assessment of autoantibodies in patients with TTP. We showed that IgGs from all 26 patients with acquired TTP bound to cells expressing a GPI anchored full-length ADAMTS13 (gFL) and a variant truncated after the spacer domain (gS). Also, IgGs from 25/26 (96.7%) of these TTP patients bound to cells expressing a GPI-anchored C-terminal fragment, TSP1 2-8 plus CUB (gT2C). In contrast, none of the 20 healthy blood donors showed detectable binding of their IgGs to the cells expressing gFL, gS, and gT2C. A moderate, but statistically significant correlation was observed between plasma concentrations of anti-ADAMTS13 IgG and positive cells expressing gFL (r=0.65), gS (r=0.67), and gT2C (r=0.42). These results suggest that the...

Pathogenesis of thrombotic microangiopathies

Profound thrombocytopenia and microangiopathic hemolytic anemia characterize thrombotic microangi... more Profound thrombocytopenia and microangiopathic hemolytic anemia characterize thrombotic microangiopathy, which includes two major disorders: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). TTP has at least three types: congenital or familial, idiopathic, and nonidiopathic. The congenital and idiopathic TTP syndromes are caused primarily by deficiency of ADAMTS13, owing to mutations in the ADAMTS13 gene or autoantibodies that inhibit ADAMTS13 activity. HUS is similar to TTP, but is associated with acute renal failure. Diarrhea-associated HUS accounts for more than 90% of cases and is usually caused by infection with Shiga-toxin-producing Escherichia coli (O157:H7). Diarrhea-negative HUS is associated with complement dysregulation in up to 50% of cases, caused by mutations in complement factor H, membrane cofactor protein, factor I or factor B, or by autoanti-bodies against factor H. The incomplete penetrance of mutations in either ADAMTS13 or complement regulatory genes suggests that precipitating events or triggers may be required to cause thrombotic microangiopathy in many patients. Keywords thrombotic thrombocytopenic purpura; hemolytic uremic syndrome; von Willebrand factor-cleaving metalloprotease; ADAMTS13; complement dysregulation DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura

Transfusion, 2011

Background-Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with h... more Background-Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either pre-denatured von Willebrand factor (VWF) or small peptides derived from the VWF-A2 domain. The physiological relevance of the assay results is uncertain. Methods-We sought to develop a novel shear-based assay to assess plasma ADAMTS13 activity and inhibitor. We also compared this assay with a fluorogenic peptide assay. Results-We found that an incubation of purified plasma VWF with 0.5-1.0 μl of citrated plasma under constant vortexing at 2,500 rpm for 60 minutes in the presence of 5 mM CaCl 2 , 1.7 μM ZnCl 2 and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear-based assay appeared to be more sensitive than the guanidine-denaturization assay for determining plasma ADAMTS13 activity. Conclusions-Our fluid shear-based assay may be useful for investigating basic biological function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Molecular Therapy, 2009

ADAMTS13, a member of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS) type 1 repea... more ADAMTS13, a member of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS) type 1 repeats family, 1,2 controls the sizes of von Willebrand factor (vWF) by cleaving vWF at the Tyr 1605-Met 1606 bond in the central A2 domain. 3 ADAMTS13 protein consists of a metalloprotease domain, a disintegrin domain, first thrombospondin type 1 repeat, a Cys-rich domain and a spacer domain in addition to seven thrombospondin type 1 repeats and two CUB domains. 2 The amino-terminal half of ADAMTS13 protease (up to the spacer domain) is found to be sufficient to recognize and cleave vWF under static and denatured conditions 4 or to cleave peptide substrates such as vWF73 derived from the central A2 domain of vWF. 5 The carboxyl-terminal half of ADAMTS13, however, may be required for collaborative binding and cleavage of vWF under fluid shear stress in vitro. 6,7 Deficiencies of plasma ADAMTS13 activity owing to hereditary mutations of ADAMTS13 gene 8 or acquired autoantibodies against ADAMTS13 protease 9 results in an accumulation of "unusually large" vWF multimers, 10 leading to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Profound thrombocytopenia, microangiopathic hemolytic anemia, and organ ischemia are characteristic features of TTP syndrome. 11 If left untreated, the mortality rate is as high as 80-100%. 11,12 Plasma infusion and/or plasma exchange is the only effective treatment available to date. Besides developing a TTP-like syndrome, 13 mice lacking Adamts13 gene are prothrombotic, 14,15 characterized by increased sizes of plasma vWF multimers and enhanced platelet adherence to activated endothelial cells in vivo. Intravenous infusion of recombinant human ADAMTS13 into Adamts13 −/− mice reverses the prothrombotic phenotypes and protects mice against ferric chloride-induced arterial and venous thromboses. 15 However, the short half-life of infused human ADAMTS13 into humans (t 1/2 , 2-4 days) 16 or mice (t 1/2 , 15-20 minutes) 15 makes the intravenous infusion of recombinant ADAMTS13 a less desirable strategy for a lifelong treatment of hereditary TTP. Gene therapy may offer an attractive alternative strategy to recombinant ADAMTS13 or plasma infusion for prevention and treatment of TTP, because only ~5-10% of ADAMTS13 activity (~0.05-0.1 mg/l protein) is required to induce and maintain The first two authors contributed equally to this study.

Structure–function and regulation of ADAMTS‐13 protease

Journal of Thrombosis and Haemostasis, 2013

ADAMTS13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe d... more ADAMTS13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS13 activity are emerging risk factors for developing myocardial and cerebral infarction, preeclampsia, and malignant malaria. Moreover, Adamts13 −/− mice develop more severe inflammatory responses, leading to increased ischaemia/perfusion injury and formation of atherosclerosis. Structure-function studies demonstrate that the N-terminal portion of ADAMTS13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS13 (TSP1 2-8 repeats and CUB domains) may function as a disulphide bond reductase to prevent an elongation of ultra large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure-function and autoantibody mapping studies allow us to identify an ADAMTS13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders. ADAMTS13 and potential human diseases ADAMTS13, first identified and cloned in 2001, is a member of the ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats) family (1,2). It cleaves a large polymeric adhesion protein von Willebrand factor (VWF). VWF is synthesised in vascular endothelial cells and megakaryocytes (3,4). The newly synthesised VWF is stored in intracellular organelles: Weibel-Palade bodies in endothelial cells and αgranules in megakaryocytes and platelets (3,4). VWF is released upon physiological or pathological stimulation and forms an ultra-long or ultra-large (UL) "string-like" structure on the endothelial surface (5-7). These UL-VWF "string-like" structures are hyperactive in recruiting circulating platelets to the site of endothelial activation and/or injury. Plasma ADAMTS13, which is primarily synthesised and released from hepatic stellate cells (8-10) and endothelial cells (11,12), binds and cleaves cell bound UL-VWF strings at the Tyr 1605-Met 1606 bond, thereby eliminating the UL-VWF from the endothelial surface and resulting

Multiple domains of ADAMTS13 are targeted by autoantibodies against ADAMTS13 in patients with acquired idiopathic thrombotic thrombocytopenic purpura

Haematologica, 2010

Background Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic)... more Background Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura. However, the domains of ADAMTS13 which the type G anti-ADAMT13 immunoglobulins target have not been investigated in a large cohort of patients with thrombotic thrombocytopenic purpura. Design and Methods Sixty-seven patients with acquired idiopathic thrombotic thrombocytopenic purpura were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentrations of anti-ADAMTS13 type G immunoglobulins, whereas immunoprecipitation plus western blotting determined the binding domains of these type G immunoglobulins. Results Plasma anti-ADAMTS13 type G immunoglobulins from 67 patients all bound full-length ADAMTS13 and a variant truncated after the eighth TSP1 repeat (delCUB). Approximately 97% (65/67) of patients harbored type G immunoglobulins targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients' samples reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients' type G immunoglobulins interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of type G immunoglobulins targeted against the T2-8 and/or CUB domains was inversely correlated with the patients' platelet counts on admission. Conclusions This multicenter study further demonstrated that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 type G immunoglobulins in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura. Our data shed more light on the pathogenesis of acquired thrombotic thrombocytopenic purpura and provide further rationales for adjunctive immunotherapy.

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 1. Structural and functional characterization in vitro

Transfusion, Apr 4, 2016

BACKGROUND-Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness cause... more BACKGROUND-Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness caused by autoantibodies that decrease the activity of ADAMTS13, the von Willebrand Factor cleaving protease. Despite efficacy of plasma exchange, mortality remains high and relapse is common. Improved therapies may come from understanding the diversity of pathogenic autoantibodies on a molecular/genetic level. Cloning comprehensive repertoires of patient autoantibodies can provide the necessary tools for studying immunobiology of disease and developing animal models. STUDY DESIGN AND METHODS-Anti-ADAMTS13 antibodies were cloned from four patients with acquired TTP using phage display and characterized with respect to genetic origin, inhibition of ADAMTS13 proteolytic activity, and epitope specificity. Anti-idiotypic antisera raised to a subset of autoantibodies enabled comparison of their relatedness to each other and to polyclonal IgG in patient plasma. RESULTS-Fifty-one unique antibodies were isolated comprising epitope specificities resembling the diversity found in circulating patient IgG. Antibodies directed to both the amino terminal domains and those requiring the ADAMTS13 cysteine-rich/spacer region for binding inhibited proteolytic activity, while those solely targeting carboxy-terminal domains were noninhibitory. Anti-idiotypic antisera raised to a subset of antibody clones crossreacted with and reduced the inhibitory activity of polyclonal IgG from a set of unrelated patients. CONCLUSIONS-Anti-ADAMTS13 autoantibodies isolated by repertoire cloning display the diversity of epitope specificities found in patient plasma and provide tools for developing animal

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model

Transfusion, Apr 4, 2016

BACKGROUND-Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in w... more BACKGROUND-Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultra-large von Willebrand Factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rationale, reliable and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 variable region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory IgG in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS-Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet thrombi following focal or systemic vascular injury was examined. RESULTS-Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF

A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura

Transfusion, Jan 20, 2011

Background-Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with h... more Background-Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either pre-denatured von Willebrand factor (VWF) or small peptides derived from the VWF-A2 domain. The physiological relevance of the assay results is uncertain. Methods-We sought to develop a novel shear-based assay to assess plasma ADAMTS13 activity and inhibitor. We also compared this assay with a fluorogenic peptide assay. Results-We found that an incubation of purified plasma VWF with 0.5-1.0 μl of citrated plasma under constant vortexing at 2,500 rpm for 60 minutes in the presence of 5 mM CaCl 2 , 1.7 μM ZnCl 2 and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear-based assay appeared to be more sensitive than the guanidine-denaturization assay for determining plasma ADAMTS13 activity. Conclusions-Our fluid shear-based assay may be useful for investigating basic biological function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.

Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol–Dependent Manner

Arteriosclerosis, Thrombosis, and Vascular Biology, Feb 1, 2014

V on Willebrand factor (VWF), an ultra large (UL) or large multimeric adhesion glycoprotein in bl... more V on Willebrand factor (VWF), an ultra large (UL) or large multimeric adhesion glycoprotein in blood, is primarily synthesized in endothelial cells, megakaryocytes, and platelets. 1 The newly synthesized VWF is stored in the Weibel-Palade bodies of endothelial cells or α-granules of platelets. ULVWF is released from these storage organelles on stimulation by epinephrine, histamine, thrombin, and inflammatory cytokines or toxins. 2-4 The newly released ULVWF forms string-like structures anchored on the cell surface, 2-4 which are hyperactive and recruit flowing platelets from circulation to the site of endothelial activation or injury. Cell-bound ULVWF strings are highly susceptible to proteolysis by plasma metalloprotease ADAMTS13. 2,3 This proteolytic cleavage results in a VWF-free endothelial surface, preventing unwanted and excessive platelet adhesion/aggregation and thrombus formation after injury. However, VWF released into circulation remains large and therefore requires further processing by plasma ADAMTS13, 5 other leukocyte proteases, 6 and complement factor H. 7 An inability to cleave or process cell-bound ULVWF or circulating large VWF multimers into smaller ones results in a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP), 8,9 which is characterized by severe thrombocytopenia and microangiopathic hemolytic anemia with various degrees of organ failure. 8,9 Previous studies have demonstrated that the proteolytic cleavage of VWF by ADAMTS13 depends on the amino-terminal portion of ADAMTS13 (ie, MDTCS domains). 10-16 An extensive exosite interaction between the ADAMTS13-DTCS domains and the VWF-A2 domain 11,17 seems to be necessary for productive VWF cleavage. A

Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura

Haematologica, Aug 4, 2016

Animal models for thrombotic thrombocytopenic purpura: a narrative review

Annals of Blood

Background and Objective: Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal blood ... more Background and Objective: Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal blood disorder, resulting from severe deficiency of plasma ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13) activity. ADAMTS13 is crucial for normal hemostasis through proteolytic cleavage of ultra large von Willebrand factor (VWF). Since the discovery of ADAMTS13 in 2001, several animal models for TTP have been established. In this narrative review, we summarize the creation and characterization of the established animal models for TTP to date. Methods: We performed a literature search through PubMed from 1969 to 2022 using free text: TTP and animal model. We found 67 peer-reviewed articles but only 33 articles were included for review and 34 articles that did not discuss TTP were excluded.

ADAMTS13 Biomarkers in Management of Immune Thrombotic Thrombocytopenic Purpura

Archives of Pathology & Laboratory Medicine

Context.— Immune thrombotic thrombocytopenic purpura (iTTP) is a rare but potentially fatal blood... more Context.— Immune thrombotic thrombocytopenic purpura (iTTP) is a rare but potentially fatal blood disorder resulting from acquired deficiency of plasma ADAMTS13, a metalloprotease that cleaves endothelium-derived ultralarge von Willebrand factor. Standard of care for iTTP including therapeutic plasma exchange, caplacizumab, and immunosuppressives, known as triple therapy, has led to a significant reduction in the disease-related mortality rate. The first International Society of Thrombosis and Haemostasis TTP guideline stresses the importance of having plasma ADAMTS13 activity testing in the algorithm for diagnosis and management of iTTP. However, the predictive role of assessing plasma ADAMTS13 activity and inhibitors or other ADAMTS13-related parameters in patients with acute iTTP and during remission has not been systematically evaluated. Objective.— To review and assess the predictive values of testing plasma ADAMTS13 activity, antigen, and inhibitors or anti-ADAMTS13 immunoglob...

Synergistic effects of ADAMTS13 deficiency and complement activation in pathogenesis of thrombotic microangiopathy

Blood, 2019

This study in mice suggests a synergistic role of ADAMTS13 deficiency and complement “hyperactiva... more

Transfusion of Platelets Loaded With Recombinant ADAMTS13 (A Disintegrin and Metalloprotease With Thrombospondin Type 1 Repeats-13) Is Efficacious for Inhibiting Arterial Thrombosis Associated With Thrombotic Thrombocytopenic Purpura

Arteriosclerosis, Thrombosis, and Vascular Biology, 2018

Objective— ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cle... more Objective— ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cleaves VWF (von Willebrand factor). This process is essential for hemostasis. Severe deficiency of plasma ADAMTS13 activity, most commonly resulting from autoantibodies against ADAMTS13, causes thrombotic thrombocytopenic purpura. Therapeutic plasma exchange is the standard of care to date, which removes autoantibodies and replenishes ADAMTS13. However, such a therapy is often ineffective to raise plasma ADAMTS13 activity, and in-hospital mortality rate remains as high as 20%. Approach and Results— To overcome the inhibition by autoantibodies, we developed a novel approach by delivering rADAMTS13 (recombinant ADAMTS13 ) using platelets as vehicles. We show that both human and murine platelets can uptake rADAMTS13 ex vivo. The endocytosed rADAMTS13 within platelets remains intact, active, and is stored in α-granules. Under arterial shear (100 dyne/cm 2 ), the rADAMTS13 in platelets is relea...

Therapeutic efficacy of the platelet glycoprotein Ib antagonist anfibatide in murine models of thrombotic thrombocytopenic purpura

Blood Advances, 2016

Key Points Anfibatide potently inhibits platelet agglutination under static and arterial shear co... more

Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura

Haematologica, Nov 4, 2016

Acquired thrombotic thrombocytopenic purpura is primarily caused by deficiency of plasma ADAMTS13... more Acquired thrombotic thrombocytopenic purpura is primarily caused by deficiency of plasma ADAMTS13 activity resulting from autoantibodies against ADAMTS13. However, ADAMTS13 deficiency alone is often not sufficient to cause acute thrombotic thrombocytopenic purpura. Infections or systemic inflammation may precede acute bursts of the disease but the underlying mechanisms are not fully understood. Here, 52 patients with acquired idiopathic thrombotic thrombocytopenic purpura and 30 blood donor controls were recruited for the study. Plasma levels of human neutrophil peptides 1-3 and complement activation fragments (i.e. Bb, iC3b, C4d, and sC5b-9) were determined by enzyme-linked immunosorbent assays. Univariate analyses were performed to determine the correlation between each biomarker and clinical outcomes. We found that plasma levels of human neutrophil peptides 1-3 and Bb in patients with acute thrombotic thrombocytopenic purpura were significantly higher than those in the control (p...

Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP

Blood, Jul 7, 2016

Infection or inflammation may precede and trigger formation of microvascular thrombosis in patien... more Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood. Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 μM and 45 μM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (KD = 0.72 μM), soluble VWF (KD = 0.58 μM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼17...

ADAMTS13 and von Willebrand factor interactions

Current Opinion in Hematology, 2015

Purpose of review-ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand facto... more Purpose of review-ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity is accountable for a potentially fatal blood disorder thrombotic thrombocytopenic purpura (TTP). Understanding of ADAMTS13-VWF interaction is essential for developing novel treatments to this disorder. Recent findings-Despite the proteolytic activity of ADAMTS13 being restricted to the metalloprotease domain, the ancillary proximal C-terminal domains including the disintegrin domain, first TSP-1 repeat, cysteine-rich region, and spacer domain are all required for cleavage of VWF and its analogs. Recent studies have added to our understandings of the role of the specific regions in the disintegrin domain, the cysteine-rich domain, and the spacer domain responsible for its interaction with VWF. Additionally, regulative functions of the distal portion of ADAMTS13 including the TSP-1 2-8 repeats and the CUB domains have been proposed. Finally, fine mapping of anti-ADAMTS13 antibody epitopes have provided further insight into the essential structural elements in ADAMTS13 for VWF binding and the mechanism of autoantibody-mediated TTP. Summary-Significant progress has been made in our understandings of the structure-function relationship of ADAMTS13 in the past decade. To further investigate ADAMTS13-VWF interactions for medical applications, these interactions must be studied under physiological conditions in vivo.

Platelet-delivered ADAMTS13 inhibits arterial thrombosis and prevents murine models of thrombotic thrombocytopenic purpura

Blood, Jan 23, 2015

ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggrega... more ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggregation and arterial thrombosis. An inability to cleave ultra large VWF resulting from hereditary or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma exchange is the most effective initial therapy for TTP to date. Here, we report characterization of transgenic mice expressing recombinant human ADAMTS13 (rADAMTS13) in platelets and its efficacy in inhibiting arterial thrombosis and preventing hereditary and acquired antibody-mediated TTP in murine models. Western blotting and fluorescent resonance energy transfer (FRETS) assay detect full-length rADAMTS13 protein and its proteolytic activity, respectively, in transgenic (Adamts13(-/-)Plt(A13)), but not in wild type (WT) and Adamts13(-/-) (KO) platelets. The expressed rADAMTS13 is released upon stimulation with thrombin and collagen, but less with 2MesADP....

Novel recombinant glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 and variants for assessment of anti-ADAMTS13 autoantibodies in patients with thrombotic thrombocytopenic purpura

Thrombosis and haemostasis, 2011

Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic th... more Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). We report here a novel cell-based assay using glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 or variants expressed on cell membrane for assessment of autoantibodies in patients with TTP. We showed that IgGs from all 26 patients with acquired TTP bound to cells expressing a GPI anchored full-length ADAMTS13 (gFL) and a variant truncated after the spacer domain (gS). Also, IgGs from 25/26 (96.7%) of these TTP patients bound to cells expressing a GPI-anchored C-terminal fragment, TSP1 2-8 plus CUB (gT2C). In contrast, none of the 20 healthy blood donors showed detectable binding of their IgGs to the cells expressing gFL, gS, and gT2C. A moderate, but statistically significant correlation was observed between plasma concentrations of anti-ADAMTS13 IgG and positive cells expressing gFL (r=0.65), gS (r=0.67), and gT2C (r=0.42). These results suggest that the...

Pathogenesis of thrombotic microangiopathies

Profound thrombocytopenia and microangiopathic hemolytic anemia characterize thrombotic microangi... more Profound thrombocytopenia and microangiopathic hemolytic anemia characterize thrombotic microangiopathy, which includes two major disorders: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). TTP has at least three types: congenital or familial, idiopathic, and nonidiopathic. The congenital and idiopathic TTP syndromes are caused primarily by deficiency of ADAMTS13, owing to mutations in the ADAMTS13 gene or autoantibodies that inhibit ADAMTS13 activity. HUS is similar to TTP, but is associated with acute renal failure. Diarrhea-associated HUS accounts for more than 90% of cases and is usually caused by infection with Shiga-toxin-producing Escherichia coli (O157:H7). Diarrhea-negative HUS is associated with complement dysregulation in up to 50% of cases, caused by mutations in complement factor H, membrane cofactor protein, factor I or factor B, or by autoanti-bodies against factor H. The incomplete penetrance of mutations in either ADAMTS13 or complement regulatory genes suggests that precipitating events or triggers may be required to cause thrombotic microangiopathy in many patients. Keywords thrombotic thrombocytopenic purpura; hemolytic uremic syndrome; von Willebrand factor-cleaving metalloprotease; ADAMTS13; complement dysregulation DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura

Transfusion, 2011

Background-Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with h... more Background-Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either pre-denatured von Willebrand factor (VWF) or small peptides derived from the VWF-A2 domain. The physiological relevance of the assay results is uncertain. Methods-We sought to develop a novel shear-based assay to assess plasma ADAMTS13 activity and inhibitor. We also compared this assay with a fluorogenic peptide assay. Results-We found that an incubation of purified plasma VWF with 0.5-1.0 μl of citrated plasma under constant vortexing at 2,500 rpm for 60 minutes in the presence of 5 mM CaCl 2 , 1.7 μM ZnCl 2 and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear-based assay appeared to be more sensitive than the guanidine-denaturization assay for determining plasma ADAMTS13 activity. Conclusions-Our fluid shear-based assay may be useful for investigating basic biological function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Molecular Therapy, 2009

ADAMTS13, a member of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS) type 1 repea... more ADAMTS13, a member of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS) type 1 repeats family, 1,2 controls the sizes of von Willebrand factor (vWF) by cleaving vWF at the Tyr 1605-Met 1606 bond in the central A2 domain. 3 ADAMTS13 protein consists of a metalloprotease domain, a disintegrin domain, first thrombospondin type 1 repeat, a Cys-rich domain and a spacer domain in addition to seven thrombospondin type 1 repeats and two CUB domains. 2 The amino-terminal half of ADAMTS13 protease (up to the spacer domain) is found to be sufficient to recognize and cleave vWF under static and denatured conditions 4 or to cleave peptide substrates such as vWF73 derived from the central A2 domain of vWF. 5 The carboxyl-terminal half of ADAMTS13, however, may be required for collaborative binding and cleavage of vWF under fluid shear stress in vitro. 6,7 Deficiencies of plasma ADAMTS13 activity owing to hereditary mutations of ADAMTS13 gene 8 or acquired autoantibodies against ADAMTS13 protease 9 results in an accumulation of "unusually large" vWF multimers, 10 leading to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Profound thrombocytopenia, microangiopathic hemolytic anemia, and organ ischemia are characteristic features of TTP syndrome. 11 If left untreated, the mortality rate is as high as 80-100%. 11,12 Plasma infusion and/or plasma exchange is the only effective treatment available to date. Besides developing a TTP-like syndrome, 13 mice lacking Adamts13 gene are prothrombotic, 14,15 characterized by increased sizes of plasma vWF multimers and enhanced platelet adherence to activated endothelial cells in vivo. Intravenous infusion of recombinant human ADAMTS13 into Adamts13 −/− mice reverses the prothrombotic phenotypes and protects mice against ferric chloride-induced arterial and venous thromboses. 15 However, the short half-life of infused human ADAMTS13 into humans (t 1/2 , 2-4 days) 16 or mice (t 1/2 , 15-20 minutes) 15 makes the intravenous infusion of recombinant ADAMTS13 a less desirable strategy for a lifelong treatment of hereditary TTP. Gene therapy may offer an attractive alternative strategy to recombinant ADAMTS13 or plasma infusion for prevention and treatment of TTP, because only ~5-10% of ADAMTS13 activity (~0.05-0.1 mg/l protein) is required to induce and maintain The first two authors contributed equally to this study.

Structure–function and regulation of ADAMTS‐13 protease

Journal of Thrombosis and Haemostasis, 2013

ADAMTS13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe d... more ADAMTS13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS13 activity are emerging risk factors for developing myocardial and cerebral infarction, preeclampsia, and malignant malaria. Moreover, Adamts13 −/− mice develop more severe inflammatory responses, leading to increased ischaemia/perfusion injury and formation of atherosclerosis. Structure-function studies demonstrate that the N-terminal portion of ADAMTS13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS13 (TSP1 2-8 repeats and CUB domains) may function as a disulphide bond reductase to prevent an elongation of ultra large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure-function and autoantibody mapping studies allow us to identify an ADAMTS13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders. ADAMTS13 and potential human diseases ADAMTS13, first identified and cloned in 2001, is a member of the ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats) family (1,2). It cleaves a large polymeric adhesion protein von Willebrand factor (VWF). VWF is synthesised in vascular endothelial cells and megakaryocytes (3,4). The newly synthesised VWF is stored in intracellular organelles: Weibel-Palade bodies in endothelial cells and αgranules in megakaryocytes and platelets (3,4). VWF is released upon physiological or pathological stimulation and forms an ultra-long or ultra-large (UL) "string-like" structure on the endothelial surface (5-7). These UL-VWF "string-like" structures are hyperactive in recruiting circulating platelets to the site of endothelial activation and/or injury. Plasma ADAMTS13, which is primarily synthesised and released from hepatic stellate cells (8-10) and endothelial cells (11,12), binds and cleaves cell bound UL-VWF strings at the Tyr 1605-Met 1606 bond, thereby eliminating the UL-VWF from the endothelial surface and resulting

Multiple domains of ADAMTS13 are targeted by autoantibodies against ADAMTS13 in patients with acquired idiopathic thrombotic thrombocytopenic purpura

Haematologica, 2010

Background Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic)... more Background Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura. However, the domains of ADAMTS13 which the type G anti-ADAMT13 immunoglobulins target have not been investigated in a large cohort of patients with thrombotic thrombocytopenic purpura. Design and Methods Sixty-seven patients with acquired idiopathic thrombotic thrombocytopenic purpura were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentrations of anti-ADAMTS13 type G immunoglobulins, whereas immunoprecipitation plus western blotting determined the binding domains of these type G immunoglobulins. Results Plasma anti-ADAMTS13 type G immunoglobulins from 67 patients all bound full-length ADAMTS13 and a variant truncated after the eighth TSP1 repeat (delCUB). Approximately 97% (65/67) of patients harbored type G immunoglobulins targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients' samples reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients' type G immunoglobulins interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of type G immunoglobulins targeted against the T2-8 and/or CUB domains was inversely correlated with the patients' platelet counts on admission. Conclusions This multicenter study further demonstrated that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 type G immunoglobulins in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura. Our data shed more light on the pathogenesis of acquired thrombotic thrombocytopenic purpura and provide further rationales for adjunctive immunotherapy.