Unraveling antibody-induced structural dynamics in the ADAMTS13 CUB1-2 domains via HDX-MS.

Allosteric regulation of ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type-1 motif, member 13) activity involves an interaction between its Spacer (S) and CUB1-2 domains to keep the enzyme in a closed, latent conformation. Monoclonal antibodies (mAb) uncouple the S-CUB interaction to open the ADAMTS13 conformation and thereby disrupt the global enzyme latency. The molecular mechanism behind this mAb-induced allostery remains poorly understood.

ADAMTS13 inhibition to treat acquired von Willebrand syndrome during mechanical circulatory support device implantation.

Background: Acquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear-induced proteolysis of von Willebrand factor (VWF) by A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13 (ADAMTS13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired hemostasis that could explain the bleeding complications that are frequently observed in these patients.

Antithrombin p.Thr147Ala: The First Founder Mutation in People of African Origin Responsible for Inherited Antithrombin Deficiency.

Background:  Hereditary antithrombin deficiency is a rare autosomal-dominant disorder predisposing to recurrent venous thromboembolism (VTE). To date, only two founder mutations have been described.

Objectives:  We investigated the antithrombin p.

von Willebrand factor increases in experimental cerebral malaria but is not essential for late-stage pathogenesis in mice.

Background: Cerebral malaria (CM) is the most severe complication of malaria. Endothelial activation, cytokine release, and vascular obstruction are essential hallmarks of CM. Clinical studies have suggested a link between von Willebrand factor (VWF) and malaria pathology.

Open ADAMTS13, induced by antibodies, is a biomarker for subclinical immune-mediated thrombotic thrombocytopenic purpura.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma.

Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function.

Plasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF.

Anti-ADAMTS13 autoantibodies in immune-mediated thrombotic thrombocytopenic purpura do not hamper ELISA-based quantification of ADAMTS13 antigen.

Background: The biological diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) is based on determination of ADAMTS13 activity (<10%) and anti-ADAMTS13 autoantibodies. ADAMTS13 antigen levels are not routinely measured in iTTP patients, but studies have shown that antigen levels are a valuable prognostic factor.

Objectives: To (a) report the validation of our in-house developed ADAMTS13 antigen enzyme-linked immunosorbent assay (ELISA) and determine ADAMTS13 antigen in a large cohort of healthy donor and iTTP patient plasma samples; and (b) to investigate whether ADAMTS13 antigen determination is not disturbed by the presence of anti-ADAMTS13 autoantibodies.

Generation of anti-idiotypic antibodies to detect anti-spacer antibody idiotopes in acute thrombotic thrombocytopenic purpura patients.

In autoantibody-mediated autoimmune diseases, autoantibody profiling allows patients to be stratified and links autoantibodies with disease severity and outcome. However, in immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, stratification according to antibody profiles and their clinical relevance has not been fully explored. We aimed to develop a new type of autoantibody profiling assay for iTTP based on the use of anti-idiotypic antibodies.

Differences in von Willebrand factor function in type 2A von Willebrand disease and left ventricular assist device-induced acquired von Willebrand syndrome.

Background: Patients with von Willebrand disease (VWD) type 2A or acquired von Willebrand syndrome (aVWS) as a consequence of implantation of left ventricular assist devices (LVAD) are both characterized by a loss of von Willebrand factor (VWF) function. Loss of VWF function is however more severe in VWD type 2A than in LVAD patients.

Objectives: To compare VWF function in patients with VWD type 2A and LVAD-induced aVWS to highlight the differences in VWF activity and to stress the importance of VWF multimer analysis for correct diagnosis of aVWS in LVAD patients.

Anti-ADAMTS13 Autoantibodies against Cryptic Epitopes in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency, the presence of anti-ADAMTS13 autoantibodies and an open ADAMTS13 conformation with a cryptic epitope in the spacer domain exposed. A detailed knowledge of anti-ADAMTS13 autoantibodies will help identifying pathogenic antibodies and elucidating the cause of ADAMTS13 deficiency. We aimed at cloning anti-ADAMTS13 autoantibodies from iTTP patients to study their epitopes and inhibitory characteristics.

Child-onset thrombotic thrombocytopenic purpura caused by p.R498C and p.G259PfsX133 mutations in ADAMTS13.

Introduction: Patients suffering from congenital thrombotic thrombocytopenic purpura (cTTP) have a deficiency in ADAMTS13 due to mutations in their ADAMTS13 gene.

Objective: The aim of this study was to determine ADAMTS13 parameters (activity, antigen, and mutations), to investigate if the propositus suffered from child-onset cTTP, and to study the in vitro effect of the ADAMTS13 mutations.

Methods: ADAMTS13 activity and antigen were determined using the FRETS VWF73 assay and ELISA and ADAMTS13 mutations via sequencing of the exons.

Anti-ADAMTS13 Antibodies and a Novel Heterozygous p.R1177Q Mutation in a Case of Pregnancy-Onset Immune-Mediated Thrombotic Thrombocytopenic Purpura.

In this study, we investigated a case of pregnancy-onset thrombotic thrombocytopenic purpura (TTP). The patient had severely decreased ADAMTS13 ( isintegrin nd etalloprotease with hrombo pondin type 1 motif, member 13) activity levels during acute phase and the presence of inhibitory anti-ADAMTS13 autoantibodies was demonstrated, which led to the diagnosis of immune-mediated TTP. However, ADAMTS13 activity was only mildly restored during remission, although inhibitory anti-ADAMTS13 antibodies were no longer detected.

High and long-term von Willebrand factor expression after Sleeping Beauty transposon-mediated gene therapy in a mouse model of severe von Willebrand disease.

Unlabelled: Essentials von Willebrand disease (VWD) is the most common inherited bleeding disorder. Gene therapy for VWD offers long-term therapy for VWD patients. Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice.

Reduced ADAMTS13 levels in patients with acute and chronic cerebrovascular disease.

Von Willebrand Factor (VWF) plays a major role in thrombosis and hemostasis and its thrombogenicity is controlled by ADAMTS13. Whereas increasing evidence shows a clear association between VWF levels and acute ischemic stroke, little is known about a correlation with ADAMTS13. Therefore, the aim of this study was to compare plasma levels of ADAMTS13 between 85 healthy volunteers (HV), 104 patients with acute ischemic stroke and 112 patients with a chronic cerebrovascular disease (CCD).

Long-Term Prevention of Congenital Thrombotic Thrombocytopenic Purpura in ADAMTS13 Knockout Mice by Sleeping Beauty Transposon-Mediated Gene Therapy.

Objective: Severe deficiency in the von Willebrand factor-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) because of mutations in the gene can lead to acute episodes of congenital thrombotic thrombocytopenic purpura (TTP), requiring prompt treatment. Current treatment consists of therapeutic or prophylactic infusions of fresh frozen plasma. However, lifelong treatment with plasma products is a stressful therapy for TTP patients.

-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.

Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy linked to a deficiency in the metalloprotease ADAMTS13. In the current study, a novel mouse model for acquired TTP was generated to facilitate development and validation of new therapies for this disease. Therefore, a large panel (n = 19) of novel anti-mouse ADAMTS13 (mADAMTS13) monoclonal antibodies (mAbs) of mouse origin was generated.

Platelet-derived VWF is not essential for normal thrombosis and hemostasis but fosters ischemic stroke injury in mice.

Von Willebrand factor (VWF) is a key hemostatic protein synthesized in both endothelial cells and megakaryocytes. Megakaryocyte-derived VWF is stored in α-granules of platelets and is enriched in hyperactive "ultra-large" VWF multimers. To elucidate the specific contribution of platelet VWF in hemostasis and thrombosis, we performed crossed bone marrow transplantations between C57BL/6J and Vwf(-/-) mice to generate chimeric mice.

Inhibition of von Willebrand factor-platelet glycoprotein Ib interaction prevents and reverses symptoms of acute acquired thrombotic thrombocytopenic purpura in baboons.

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) can be explained by the absence of active ADAMTS13, leading to ultra-large von Willebrand factor (UL-VWF) multimers spontaneously interacting with platelets. Preventing the formation of UL-VWF-platelet aggregates therefore is an attractive new treatment strategy. Here, we demonstrate that simultaneous administration of the inhibitory anti-VWF monoclonal antibody GBR600 and the inhibitory anti-ADAMTS13 antibody 3H9 to baboons (prevention group) precluded TTP onset as severe thrombocytopenia and hemolytic anemia were absent in these animals.

MRI assessment of blood outgrowth endothelial cell homing using cationic magnetoliposomes.

The use of contrast material to stimulate magnetic resonance imaging (MRI) of migrating cells has become an important area of research. In the present study, cationic magnetoliposomes (MLs) were used to magnetically label human blood outgrowth endothelial cells (BOECs) and follow their homing by magnetic resonance imaging (MRI). The biodistribution and functional integration capacity of BOECs, which have shown extensive promise as gene delivery vehicles, have thus far only rarely been investigated.

The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo.

Background: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified.

Methods: Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro.

Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus).

Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factor-cleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n = 6) induced severe thrombocytopenia (< 12 × 10(9)/L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase.

Mutation of the H-bond acceptor S119 in the ADAMTS13 metalloprotease domain reduces secretion and substrate turnover in a patient with congenital thrombotic thrombocytopenic purpura.

Hereditary thrombotic thrombocytopenic purpura is caused by mutations in a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS13) resulting in defective processing of von Willebrand factor (VWF) that causes intravascular platelet aggregation culminating in thrombocytopenia with shistocytic anemia. In this study the functional and structural role of a recently identified ADAMTS13 metalloprotease domain mutation S119F was investigated. Secretion from heterologous cells was hampered but not completely eliminated.

The novel S527F mutation in the integrin beta3 chain induces a high affinity alphaIIbbeta3 receptor by hindering adoption of the bent conformation.

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor alphaIIbbeta3 in a patient with an ill defined platelet disorder: one in the beta3 gene (S527F) and two in the alphaIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant alphaIIbbeta3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function.