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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.

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.

SummaryBackground Acquired thrombotic thrombocytopenic purpura (TTP) is caused by an autoantibody-mediated deficiency of the von Willebrand factor-cleaving protease ADAMTS-13. Acute episodes of the disease are treated with a combination of immunosuppression and repeated cycles of plasma exchange to remove anti-ADAMTS-13 autoantibodies and, at the same time, replenish functional ADAMTS-13. Although this is often effective, the mortality rate has remained between 10% and 20%, highlighting the need for safer treatment options. Objectives We previously showed that, in vitro, human recombinant ADAMTS-13 (rADAMTS-13) is able to override neutralizing antibodies and restore ADAMTS-13 activity in plasma from patients with acquired TTP. In the present study, we assessed the in vivo feasibility of this strategy by using a rat model. Methods Wild-type rats were adjusted to an ADAMTS-13 inhibitor (inhibitor) titer of ~ 10 BU mL−1 with goat anti-ADAMTS-13 IgG, and treated with increasing doses of rADAMTS-13. Blood samples were drawn and analyzed for ADAMTS-13-specific parameters, including FRETS-VWF73 activity, inhibitor, and ADAMTS-13-specific immune complexes (ICs). The pharmacokinetics of ADAMTS-13 activity and inhibitors were evaluated. Results Administration of inhibitor titer-adjusted doses of rADAMTS-13 to inhibitor-treated rats predictably restored activity. Inhibitors were readily neutralized through formation of ADAMTS-13-specific ICs, which were cleared at a higher rate than the free inhibitor. Surplus protease was enzymatically active in plasma, and showed similar pharmacokinetics to ADAMTS-13 in not inhibitor-treated rats. Conclusions Defined doses of rADAMTS-13 neutralized circulating anti-ADAMTS-13 antibodies and enabled reconstitution of ADAMTS-13 activity in plasma in our model, indicating that the protease may be a promising candidate for further exploration in treating acute episodes of acquired TTP.

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.

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.

Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in absence of ADAMTS13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role for plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results Also in mice with an acquired ADAMTS13 deficiency, plasmin is generated during TTP as reflected by increased plasmin-α2-antiplasmin (PAP)-complex levels. However, mice still developed TTP suggesting that this increase is not sufficient to control the pathology. As mice with TTP also had increased plasminogen activator inhibitor 1 (PAI-1) levels, we investigated whether blocking the plasmin(ogen) inhibitors would result in the generation of sufficient plasmin to influence TTP outcome in mice. Interestingly, when amplified plasmin activity was allowed (α2-antiplasmin-/- mice with inhibited PAI-1) in mice with an acquired ADAMTS13 deficiency, a resolution of TTP signs was observed as a result of an increased proteolysis of VWF. In line with this, in patients with acute TTP, increased PAP-complex and PAI-1 levels were also observed. However, neither PAP-complex levels nor PAI-1 levels were related to TTP signs and outcome. Conclusions In conclusion, endogenous plasmin levels are increased during acute TTP, though limited via suppression through α2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a backup for ADAMTS13 in mice and TTP signs are resolved due to an increased proteolysis of VWF. This article is protected by copyright. All rights reserved.

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 however not always effective and therefore new treatment methods are highly necessary. N-acetylcysteine (NAC), an FDA-approved anti-mucolytic agent, could be a possible new treatment strategy for TTP as it was demonstrated to reduce disulfide bonds in VWF, thereby decreasing VWF multimer size and hence its 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 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 pre-existing TTP signs; thrombocytopenia, hemolytic anemia and organ damage could not be reversed, as thrombus resolution could not be 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, is effective in preventing severe TTP signs in mice but NAC is not effective in resolving acute TTP signs in mice and baboons. ispartof: Blood vol:129 issue:8 pages:1030-1038 ispartof: location:United States status: published

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.

Summary. Background: Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non-inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme. Objectives: To explore the use of recombinant ADAMTS13 (rADAMTS13) as a potential therapeutic agent in acquired TTP, we investigated its efficacy in normalizing VWF-cleaving activity in the presence of ADAMTS13 inhibitors. Methods: Thirty-six plasma samples from TTP patients were adjusted to predefined inhibitor titers, and recovery of ADAMTS13 activity was analyzed following supplementation with rADAMTS13. Results: We showed a linear relation between the inhibitor titer measured and effective rADAMTS13 concentration necessary for reconstitution of VWF-cleaving activity in the presence of neutralizing autoantibodies. Conclusions: Our results support the further investigation of the potential therapeutic applicability of rADAMTS13 as an adjunctive therapy in acquired TTP.

Essentials ADAMTS-13-deficiency is a cause of thrombotic thrombocytopenic purpura (TTP). Preclinical safety of recombinant human ADAMTS-13 (BAX930) was shown in animal models. Preclinical efficacy of BAX930 was shown in a mouse model of TTP. BAX930 showed advantageous efficacy over fresh frozen plasma, the current standard of care. Click to hear Dr Cataland and Prof. Lammle present a seminar on Thrombotic Thrombocytopenic Purpura (TTP): new Insights in Pathogenesis and Treatment Modalities. SummaryBackground Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder characterized by microthrombosis in small blood vessels of the body, resulting in a low platelet count. Baxalta has developed a new recombinant ADAMTS-13 (rADAMTS-13) product (BAX930) for on-demand and prophylactic treatment of patients with hereditary TTP (hTTP). Objectives To evaluate the pharmacokinetics, efficacy and safety of BAX930 in different species, by use of an extensive preclinical program. Methods The prophylactic and therapeutic efficacies of BAX930 were tested in a previously established TTP mouse model. Pharmacokinetics were evaluated after single intravenous bolus injection in mice and rats, and after repeated dosing in cynomolgus monkeys. Toxicity was assessed in rats and monkeys, safety pharmacology in monkeys, and local tolerance in rabbits. Results BAX930 was shown to be efficacious, as demonstrated by a stabilized platelet count in ADAMTS-13 knockout mice that were thrombocytopenic when treated. Prophylactic efficacy was dose-dependent and comparable with that achieved by treatment with fresh frozen plasma, the mainstay of hTTP treatment. Therapeutic efficacy was treatment interval-dependent. Safety pharmacology evaluation did not show any deleterious effects of BAX930 on cardiovascular and respiratory functions in monkeys. The compound's pharmacokinetics were similar and dose-proportional in mice, rats, and monkeys. BAX930 was well tolerated in rats, monkeys, and rabbits, even at the highest doses tested. Conclusions These results demonstrate that BAX930 has a favorable preclinical profile, and support the clinical development of rADAMTS-13 for the treatment of hTTP.

SummaryWe 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 –1420ng/ml (median 1080ng/ml) resulting in an ADAMTS13 activity to antigen ratio of 0.48 to 1.68 U/µg. 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.

Summary. Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.

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. (Blood. 2004;103:4514-4519)

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.

Summary. 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.

Summary Background Several static Bethesda-type assays are routinely used to determine ADAMTS-13-neutralizing autoantibodies in acquired thrombotic thrombocytopenic purpura (TTP), but the inhibitory activity of these antibodies has not been thoroughly evaluated under the more physiologic condition of flow. Objectives We investigated whether ADAMTS-13 inhibitor assessment with the FRETS-VWF73 assay is predictive for evaluation under flow. Methods Anti-ADAMTS-13 autoantibodies were purified from patients with acquired TTP by chromatography involving an ADAMTS-13 affinity matrix and/or protein G. ADAMTS-13 activity was measured with the FRETS-VWF73 assay and a novel flow assay determining the ADAMTS-13-mediated decrease in platelet aggregate surface coverage, caused by perfusion of a suspension containing platelets, erythrocytes and von Willebrand factor (VWF) over a surface coated with extracellular matrix components. The neutralizing activities of ADAMTS-13 inhibitors were compared under static conditions and under flow by use of the two assays. Results The suitability of the flow-based ADAMTS-13 activity assay for quantification of ADAMTS-13 inhibitors could be demonstrated by reversibility of the ADAMTS-13-dependent decrease in surface coverage upon addition of goat ADAMTS-13 antiserum. Testing the neutralizing activity of purified autoantibodies from six patients in the flow assay according to their FRETS-VWF73-based inhibitor titers gave rise to vastly different inhibitory effects, indicating a discrepancy in inhibitor assessment between static and flow conditions. Conclusions Anti-ADAMTS-13 autoantibodies may show inhibitory properties in vivo that are not consistent with the ADAMTS-13 inhibitor levels determined in routine static assays, possibly because certain epitopes are selectively exposed under shear. Consequently, the course of disease and treatment efficacy may vary among TTP patients, despite common inhibitor titers.

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.

Von Willebrand factor (vWF) is a multimeric glycoprotein (GP) that attracts platelets to the site of vascular injury, mediates platelet-platelet interaction, and stabilizes factor VIII (FVIII) in the circulation. Quantitative and qualitative defects of vWF result in von Willebrand disease (vWD), manifested by modest to severe bleeding episodes. Substitution therapy, with plasma-derived FVIII/vWF complex concentrates, is used for patients suffering the more severe forms of vWD. Efficacy of these preparations is often unsatisfactory because inadvertent proteolytic degradation during the manufacturing process causes them to lack the hemostatically most active high-molecular-weight multimers. In contrast, recombinant vWF (r-vWF), which is constitutively expressed at high yields in Chinese hamster ovary (CHO) cells and secreted into the conditioned medium under perfusion fermentation in "protein-free" medium, has high-molecular-weight multimers of extraordinary structural integrity. Functional analysis has shown that r-vWF promotes ristocetin cofactor-mediated platelet aggregation, collagen interaction and FVIII binding, and platelet-collagen adhesion under shear stress. Infusing vWF-deficient animals with r-vWF corrected vWF concentration and reduced blood loss, subsequently stabilizing endogenous FVIII associated with the reduction of bleeding time. Compared with plasma-derived vWF preparations, r-vWF was found to have a prolonged half-life, further enhancing the potential value of r-vWF as a therapeutic agent for treating patients suffering from vWD.

Mycoplasma contamination of tissue culture cells easily evades detection and, thus, represents a continous threat to cell biologists. In cases where infected cell can not simply be replaced, attempts have to be made to eradicate mycoplasma from the tissue culture cells. A variety of anti-microbial agents have been shown to be toxic to mycoplasma strains; however, cell associated mycoplasmas are often protected from antibiotics at concentrations shown to be effectivein vitro. Antibiotic concentrations high enough to be lethal to cell asso|ciated mycoplasmas frequently are also detrimental to the host cells, while moderately increased antibiotic levels tolerated by the host cells often lead to only temporary growth suppression and/or to the emergence of mycoplasma strains resistant even to high concentrations of the antibiotic applied. Here, a genetic approach for the elimination of mycoplasma from tissue culture cells that overcomes these limitations is described. By expression of a selection marker conferring resistance to an otherwise toxic agent,Acholeplasma laidlawii infected BHK-21 cells used as the model system were enabled to temporarily tolerate antibiotic concentrations high enough to be lethal to cell associated mycoplasma while leaving the host cells unharmed. Upon successful mycoplasma eradication, cultivation of the cured host cells in the absence of the selective agent yielded revertant cell clones that had regained susceptibility to the toxic agent. Cessation of the selection marker expression was shown to result from the loss of the selection marker DNA, which is a consequence of the fact that the stable and permanent integration of foreign DNA in eucaryotic cell chromosomes is highly inefficient. Thus, the cells were cured from mycoplasma yet remained biochemically unaltered.

Factor X/Xa plays a pivotal role in the coagulation cascade and exhibits a therapeutic potential for the treatment of factor X-deficient as well as FVIII and FIX inhibitor patients. This report describes the establishment of Chinese hamster ovary cell clones expressing recombinant human factor X up to 120 microg/mL x day and 78 microg/10(6) cells x day, that is to 100-fold higher levels than reported previously. Although propeptide removal and single chain precursor to light and heavy chain processing as well as vitamin K-dependent gamma-carboxylation became impaired at these expression levels, up to 25% of the recombinant human factor X produced was active. This represents the highest functional activity ever reported for a vitamin K-dependent protein at such an expression level. Expression of recombinant human factor X in Chinese hamster ovary cells lacking the endoprotease Furin revealed that propeptide removal still occurred, whereas single chain precursor to light/heavy chain processing was abolished. This suggests that a protease different from Furin mediates propeptide removal, a unique finding compared with the other vitamin K-dependent coagulation factors. In contrast, exposure of incompletely processed rFX molecules to soluble recombinant Furin in vitro mediated both of these cleavage reactions despite the absence of a typical argP4-xP3-lys/argP2-argP1 Furin cleavage site in the propeptide, indicating relaxed specificity in vitro. Concomitantly with the degree of processing, the functional activity of recombinant human factor X increased. Interestingly, Furin was shown to even perform correct N-terminal proteolytic trimming of FX molecules truncated amino-terminal to the P3 residue in vitro. Depending on the absence or presence of warfarin in the culture media, as well as on the processing state, four distinct recombinant human factor X light chain isoforms were observed and their structure characterized. One of these light chain forms correlated with the functional activity. Finally, the distribution of the individual light chain isoforms suggests that gamma-carboxylation may be a prerequisite for propeptide removal.

Coagulation factors, amongst many other proteins, often require posttranslational endoproteolytic processing for maturation. Upon high yield expression of recombinant forms of these proteins, processing frequently becomes severely limiting, resulting in a hampered function of the protein. In this report, the human endoprotease Furin was used to achieve complete propeptide removal from recombinant von Willebrand Factor (rvWF) precursors in CHO cells. At expression beyond 200 ng rvWF/106 cells × day, processing became insufficient. Stable co- and overexpression of full length Furin resulted in complete precursor cleavage in cell clones expressing 2 μg rvWF/106 cells × day. Rather than occuring intracellularly, processing was found to be mediated by a naturally secreted form of rFurin, present in 100 fold higher concentrations than endogenous Furin and accumulating in the cell culture supernatant. Attempts to increase rFurin yield by amplification, in order to ensure complete rvWF precursor processing at expression rates beyond 2 μg rvWF/106 cells × day, failed. Truncation of the trans-membrane domain resulted in immediate secretion of rFurin and approximately 10 fold higher concentrations in the conditioned medium. In cases where these high rFurin concentrations are not sufficient to ensure complete processing, an in vitro downstream processing procedure has to be established. Secreted affinity epitope-tagged rFurin derivatives were constructed, the fate of which, at expression, was dependent on the size of the C-terminal truncation and the type of the heterologous epitope added. A suitable candidate was purified by a one step affinity procedure, and successfully used for in vitro processing. This allows complete proteolytic processing of large amounts of precursor molecules by comparably small quantities of rFurin. Complete precursor cleavage of a target protein at expression rates of up to approximately 200 ng, 2 μg, and 20 μg, as well as beyond 20 μg/106 cells × day can thus be anticipated to be accomplished by endogenous Furin, additional expression of full length rFurin, co-expression of truncated and hence secreted rFurin, and a protein-chemical in vitro procedure, respectively.

A quantitative multiple competitive PCR (QMC-PCR) for determination of DNA copy numbers is described. Four competitive DNA templates for the env region of HIV-1 were constructed with sizes longer (187 and 163 bp) or shorter (122 and 105 bp) than the 142 bp of the wild-type PCR product. Varying amounts of each of these competitors are introduced together with the sample into a single reaction tube. Since competitors and wild-type fragments share the same primer recognition sequence (SK68/SK69), amplification occurs according to the rate of the introduced copy numbers. The PCR products are run on an agarose gel, and the copy number of the sample is determined by analyzing the bands with a video densitometer and calculating the equivalence point in a linear regression plot.

The zymodemes, electrophoretic patterns of hexokinase, phosphoglucomutase and glucose phosphate isomerase isoenzymes, have been widely used to determine the pathogenicity of Entamoeba histolytica isolates. Although pathogenic and nonpathogenic forms of E. histolytica differ clearly in sequences of many homologous genes, a conversion between pathogenic and nonpathogenic zymodemes has been reported by several laboratories. To approach the question what might be the basis for the observed conversion, we examined the molecular biology of the hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzymes in pathogenic E. histolytica. We isolated two different cDNAs pHXK1 and pHXK2 coding for polypeptides with significant sequence similarity to hexokinases and deduced molecular masses of 49.8 kDa and 49.4 kDa. The two hexokinase sequences differed by 11% on the amino acid and by 8% on the nucleotide level. Expression of the cDNAs in Escherichia coli as nonfusion proteins gave two polypeptides with hexokinase activity. The recombinant Hxk1 and Hxk2 polypeptides comigrated with the more basic and more acidic isoforms of pathogenic amoebae in starch gel electrophoresis, as well as in low and high resolution isoelectric focussing gels. This identified the observed hexokinase isoenzymes of pathogenic E. histolytica as the products of two genes, hxk1 and hxk2.

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.

Entamoeba histolytica has an unusual nuclear structure characterized by a low degree of chromatin condensation and the absence of stainable metaphase chromosomes. Although nucleosome-like particles were observed, no information about histones was available so far. In this paper we describe a cDNA clone with significant homology to H3 histones that was isolated from a library of pathogenic E. histolytica. The complete cDNA encodes a 15-kDa polypeptide, which like the histone sequence from Volvox carteri is shorter by one residue than the human homologue. The amino acid sequence has only 69% identity with human H3.3 histone and 67% identity with the human H3.1 histone. This is the highest degree of sequence divergence observed for any eukaryote H3 histone sequence. Our results indicate that this divergence may contribute to the unusual chromatin structure of E. histolytica.

Cyanelle containing organisms, notably Cyanophora paradoxa, best studied among them, are unique with respect to the occurrence of peptidoglycan (murein) within an eukaryotic cell. Enzyme activities involved in the biosynthesis of UDP-N-acetyl-muramylpentapeptide could be localized within the cyanelle compartment. Some of the enzymes performing later steps of murcin biosynthesis were detected in the postcyanelle supernatant rather than in the cyanelle lysate. This is taken to reflect a ‘periplasmic’ location of these enzymes that are partially liberated upon rupture of the cyanelle outer membrane.

Summary. Background: Over the last 4 years ADAMTS-13 measurement underwent dramatic progress with newer and simpler methods. Aims: Blind evaluation of newer methods for their performance characteristics. Design: The literature was searched for new methods and the authors invited to join the evaluation. Participants were provided with a set of 60 coded frozen plasmas that were prepared centrally by dilutions of one ADAMTS-13-deficient plasma (arbitrarily set at 0%) into one normal-pooled plasma (set at 100%). There were six different test plasmas ranging from 100% to 0%. Each plasma was tested ‘blind’ 10 times by each method and results expressed as percentage vs. the local and the common standard provided by the organizer. Results: There were eight functional and three antigen assays. Linearity of observed-vs.-expected ADAMTS-13 levels assessed as r2 ranged from 0.931 to 0.998. Between-run reproducibility expressed as the (mean) CV for repeated measurements was below 10% for three methods, 10–15% for five methods and up to 20% for the remaining three. F-values (analysis of variance) calculated to assess the capacity to distinguish between ADAMTS-13 levels (the higher the F-value, the better the capacity) ranged from 3965 to 137. Between-method variability (CV) amounted to 24.8% when calculated vs. the local and to 20.5% when calculated vs. the common standard. Comparative analysis showed that functional assays employing modified von Willebrand factor peptides as substrate for ADAMTS-13 offer the best performance characteristics. Conclusions: New assays for ADAMTS-13 have the potential to make the investigation/management of patients with thrombotic microangiopathies much easier than in the past.

The platelet dependent-clotting process is a highly complex pathway that needs to be tightly controlled by a variety of soluble and cellular components. Failure to regulate the activity of multimeric von Willebrand factor (VWF) by ADAMTS13 for instance causes thrombotic thrombocytopenia purpura (TTP). Regulation is achieved through the established function of ADAMTS13 as a VWF-cleaving protease; however, it has been reported that ADAMTS13 may additionally curtail VWF multimer fibrillation by forming disulfide bridges with VWF. Since the latter mechanism remained ill-defined, we aimed to directly visualize disulfide bond formation between ADAMTS13 and VWF using multimer gel electrophoresis. Covalent interaction between ADAMTS13 and VWF was time-, concentration-, temperature-, and shear-stress dependent. The covalent linkage was independent of the proteolytic activity of ADAMTS13 and could be assigned to a C-terminal fragment comprising TSP1 domains 5 to 8 plus the CUB domains. This interaction was blocked by thiol-reactive agents, indicating that association was accomplished by a thiol/disulfide exchange reaction. Furthermore, a polyclonal anti-ADAMTS13 antibody that inhibited the protease in a FRETS-VWF73 activity assay also inhibited the formation of VWF-ADAMTS13 adducts. Using a coupled reversed phase HPLC-ESI-QTOF-MS system, several partially reduced free thiols were identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent. Together with the observation that a C1275S point mutation retained the ability to form covalent linkages with VWF, this result indicated that ADAMTS13 has disulfide plasticity. The same conditions that led to VWF complex formation also caused a hitherto undetected homo-oligomerization of ADAMTS13. An even more pronounced oligomerization was noted in the presence of thiol-sensitive agents such as glutathione. We propose a dynamic network of redundant, partially free thiols in ADAMTS13 that undergo intra- and inter-molecular redox reactions capable not only of depolymerizing VWF, but also to prevent further VWF multimerization by facilitating the aggregation of ADAMTS13 around the monomers. Disclosures Rottensteiner: Baxalta Innovations GmbH: Employment. Skalicky:Baxalta Innovations GmbH: Employment. Seyfried:Baxalta Innovations GmbH: Employment. Kaufmann:Baxalta Innovations GmbH: Employment. Fiedler:Baxalta Innovations GmbH: Employment. Hasslacher:Baxalta Innovations GmbH: Employment. Plaimauer:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.

Thrombotic thrombocytopenic purpura (TTP) is characterized by a functional deficiency in the plasma metalloprotease ADAMTS13, caused by mutations in the ADAMTS13 gene or by autoantibody inhibition. ADAMTS13 is the key regulator of the hemostatic activity of von Willebrand factor (VWF), accomplished by cleavage of a single site within the A2 domain of VWF. The catalytic domain of ADAMTS13 possesses various binding sites for metallic cations including one Zn2+ binding site composed of three histidine residues within the sequence HEXXHXXGXXHD and up to three putative calcium ion-binding sites. The dependence of ADAMTS13 activity on zinc and calcium ions is reflected in its inactivation by chelating agents such as EDTA and doxycycline. Although replenishment with an appropriate metallic cation is thought to fully restore the proteolytic activity of the enzyme, the stability of ADAMTS13 in a Ca2+-depleting environment has not yet been explored. This aspect, however, is clinically relevant, as citrated human plasma serves as the standard source for testing ADAMTS13-specific parameters, where the chelator citrate not only prevents activation of the coagulation cascade, but also renders ADAMTS13 inactive. Here, we addressed the stability of plasma ADAMTS13 in the presence or absence of citrate (0.38%) at various temperatures. While ADAMTS13 proved stable at 4-8°C and at room temperature for up to 24 h irrespective of the presence of citrate, a time-dependent decrease in activity was observed at 37°C in the presence but not in the absence of citrate. No decrease in activity was seen when heparinized plasma was used as source of ADAMTS13, but the addition of 0.38% citrate again caused ADAMTS13 instability at 37°C. Similar results were observed when using the purified recombinant protein as source for ADAMTS13. Higher order structural analyses using Fourier-transformed infrared spectroscopy and dynamic light scattering demonstrated citrate-dependent structural changes in ADAMTS13 at 37°C that are typical for less-ordered protein structures. Addition of 5 mM Ca2+, Zn2+, or both metal ions prior to incubation completely restored ADAMTS13 stability, but these compounds failed to restore activity when added post incubation even after prolonged periods, suggesting that the loss in activity was irreversible. We conclude that Zn2+ and/or Ca2+ ions are required to stabilize the structure of ADAMTS13 at physiological temperature. This finding needs to be considered when using citrated plasma as source of ADAMTS13 in clinical settings such as for plasma exchange to treat acute episodes of TTP. Disclosures Grillberger: Baxalta Innovations GmbH: Employment. Kaufmann:Baxalta Innovations GmbH: Employment. Kink:Baxalta Innovations GmbH: Employment. Matthiessen:Baxalta Innovations GmbH: Employment. Plaimauer:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment. Rottensteiner:Baxalta Innovations GmbH: Employment.

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 αIIbβ3 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.

Von Willebrand factor (vWF) is a multimeric plasma glycoprotein that promotes platelet aggregation, mediates platelet adhesion to the subendothelium, and stabilizes coagulation factor VIII (FVIII). Recombinant vWF (rvWF) was constitutively expressed at high yield in stable CHO cell clones (CHO-rvWF). Carbohydrate analysis of rvWF and plasma derived (pd) vWF revealed common and divergent structures. The absence of terminal high mannose residues indicated intact and complete glycosylation. Alteration of terminal carbohydrate structures by α (2,6) sialyltransferase coexpression did not influence rvWF mediated platelet aggregation and collagen binding, both of which require appropriate glycosylation and are sensitive to glycosylation changes. Upon increasing rvWF expression by amplification, from 100 ng to 20 μg rvWF/106 cells × day, proteolytic propeptide removal had become incomplete resulting in impaired interaction with FVIII. Complete propeptide cleavage could be accomplished by employing recombinant Furin, a ubiquitous endoprotease, and derivatives thereof, either by coexpression in vivo or by treatment in vitro. Multimerization, also crucial to vWF function, could be significantly improved by cell culture medium modification.

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.

The activity of ADAMTS13 (a von Willebrand factor–cleaving metalloprotease) is decreased in congenital and acquired thrombotic thrombocytopenic purpura (TTP) either because of a gene defect or because of transient inhibition by an autoantibody.[1][1],[2][2] Current diagnosis in patients is based

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.

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 designatedADAMTS13, 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.

Abstract 1086 Introduction: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening generalized disorder, characterized by classic “pentad”. Since 1998, it has been characterized by severe deficiency of ADAMTS13 activity (ADAMTS13:AC), due to genetic abnormalities or acquired autoantibodies (ADAMTS13:INH) to this enzyme. A drug-induced form of TTP, ticlopidine-associated (tc)- TTP, is also associated with severe deficiency of ADAMTS13:AC and ADAMTS13:INH, although unlike acquired idiopathic (ai)-TTP, spontaneous relapses do not occur. A first-line treatment of ai-and tc-TTP is plasma exchange (PE) that remarkably reduced the mortality. However, a certain population of the ai-TTP patients experiences a new drop in platelet count during the treatment. In 2011, we have reported that these ai-TTP patients were frequently associated with a tremendous increase of ADAMTS13:INH titers with PE, and termed “inhibitor boosting” (Isonishi et al. ISTH 2011). However, no systematic studies on this topic have been done. In this study, we analyzed ADAMTS13:INH boosting in Japan-Nara TMA registry. Patients and Methods: Between Jan 2004 and Dec 2011, 215 patients were diagnosed with ai-TTP (100 males/115 females) and 14 tc-TTP (7m/7f) in our registry. For analyzing of ADAMTS13:INH boosting, we evaluated patients in whom both ADAMTS13:AC and ADAMTS13:INH were analyzed more than 3 times within 14 hospital days after PE initiation (selected patients). The number of selected patients with ai-TTP was 56 (24m/32f) and tc-TTP was 5 (3m/2f). Assays for ADAMTS13:AC and ADAMTS13:INH were performed by chromogenic act-ELISA, and the ADMTS13:INH titers were expressed by the Bethesda units (BU). ADAMTS13:INH boosting was defined by fulfilling the followings: 1) patients must have ADAMTS13:INH titer of more than 1 BU/ml before PE; 2) ADAMTS13:INH levels increased more than those before PE, during PE or within 14 days after PE initiation. Autoantibody titers for anti-ADAMTS13 IgG, IgM, and IgA isotypes and IgG1-4 subclasses were determined as previously described (Ferrari et al, JTH 2009). Results: (1) Frequency of the boosting: In ai-TTP, 174 out of 215 (81%) patients showed severely decreased ADAMTS13:AC under 0.5% of the normal. All 56 selected ai-TTP patients had severe deficiency of ADAMTS13:AC, of which ADAMTS13:INH boosting was identified in 23 patients (23/56, 41%). The frequency of inhibitor boosting versus the inhibitor titers before PE was the followings: 4/17 (24%) with ADAMTS13:INH titers of 1- (2) Characterization of inhibitor autoantibodies: We analyzed the anti-ADAMTS13 immunoglobulin isotypes and IgG subtypes in 8 selected patients with ai-TTP (6 with the boosting, and 2 without) and 2 patients with tc-TTP. All 6 ai-TTP patients with boosting exhibited IgG antibodies, and 3 had additional IgA antibodies; none had IgM antibodies. As for the IgG subclasses, the following combinations were found: G1 alone (one patient), G1+G2 (one patient), G1+G2+G4 (three patients), and G1+G4 (one patient). On the other hand, among 2 ai-TTP patients without boosting, both had IgG antibodies, one had the additional IgA antibodies, and none had IgM antibodies. As for the IgG subclasses, the following combinations were found: G1 alone (one patient), and G1+G2+G4 (one patient). Thus, we did not identify any specific difference between patients with versus without the ADAMTS13:INH boosting. Further, in 2 tc-TTP patients, both had IgG+IgA antibodies. As for the IgG subclasses, one patient had G1+G2+G4, and the other had G1+G2+G3. (3) Effect of rituximab: Five ai-TTP patients with the boosting were treated with rituximab (375 mg/m2weekly 3–5 times), which remarkably suppressed high levels of ADAMTS13:INH and achieved clinical remission. Conclusion: In this study, we identified that ai-TTP patients with severe deficiency of ADAMTS13:AC with ADAMTS13:INH titers more than 2 BU/ml before PE are prone to develop the inhibitor boosting during PE, and that rituximab therapy is potentially very useful in this setting, due to suppression of IgG autoantibodies. Interestingly, the inhibitor boosting was not seen in tc-TTP patients with severe deficiency of ADAMTS13:AC with its autoantibodies. Disclosures: Matsumoto: Alexion Pharma: Membership on an entity's Board of Directors or advisory committees. Plaimauer:Baxter BioScience: Employment. Fujimura:Baxter BioScience: Membership on an entity's Board of Directors or advisory committees; Alexion Pharma: Membership on an entity's Board of Directors or advisory committees.

Abstract 4361 A severe functional deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13 results in the accumulation of uncleaved, highly adhesive VWF multimers in plasma leading to increased platelet aggregation and thrombus formation in the microcirculation. Anti-ADAMTS13 autoantibodies that either neutralize ADAMT13’s activity or enhance protein clearance are the major cause of severe ADAMTS13 deficiency in acquired thrombotic thrombocytopenic purpura (TTP). Circulating anti-ADAMTS13 immune complexes that may play a role in disease pathogenesis have been described in plasma of TTP patients. We have developed a new ELISA assay to specifically determine the levels of ADAMTS13-specific circulating immune complexes (CIC) in which the antigen portion is immobilized by a polyclonal anti-ADAMTS13 IgG and the immunoglobulin component detected by a class-specific antibody. An inverse correlation between levels of circulating anti-ADAMTS13 autoantibodies and ADAMTS13-specific CIC has been observed in plasma of acquired TTP patients. The correlation between free anti-ADAMTS13 titer and the level of CIC and insight into the immunoglobulin (sub) classes involved may provide valuable prognostic information about disease severity and progression, and aid patient treatment monitoring. Disclosures: Plaimauer: Baxter Innovations GmbH: Employment. Ferrari:Baxter Innovations GmbH: Employment. Gruber:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

To investigate the humoral immune response of patients with amoebic dysentry against Entamoeba histolytica, immunoglobulin G (IgG)-immunopositive cDNA clones from the pathogenic strain SFL-3 were examined. A large part of the IgG-positive cDNA clones obtained with one serum encoded highly conserved intracellular proteins. A clone was found that was homologous to translation elongation factor-2 (EF-2). Sequence analysis of the EF-2 cDNA showed 63.6% amino acid sequence identity with the human homologue. The deduced protein sequence has a length of 840 amino acid residues with a molecular mass of 93.3 kD. The 3' and 5' untranslated regions of the mRNA are relatively short as shown for other genes of E. histolytica. A genomic clone was used to analyze the region upstream of the translation initiation codon. The codon distribution of EF-2 and other published E. histolytica sequences reflects the high A/T content. The codons for different amino acids are biased to a widely differing extent.

Thrombotic thrombocytopenic purpura (TTP) is characterized by systemic microvascular thrombosis leading to life-threatening ischemia of multiple organs, and is associated with a severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13, which permits highly adhesive ultra-large VWF multimers to accumulate in the circulation. Inhibitory and less frequent non-inhibitory anti-ADAMTS13 autoantibodies have been detected in patients suffering from acquired idiopathic TTP. Current treatment with plasma exchange therapy is considered to remove the autoantibodies while concomitantly supplying plasma with the deficient protease. Plasma therapy greatly reduces mortality, nevertheless, still about 10% of the patients die from refractory TTP. To explore the cause of treatment failure in a fatal case of idiopathic acquired TTP we investigated retrospectively the anti-ADAMTS13 immunological profile of a 70-year-old female patient during the course of disease progression. At admittance, she presented with schistocytes, thrombocytopenia and severe neurological disturbances. ADAMTS13 antigen and activity were borderline low in the presence of initially non-inhibiting anti-ADAMTS13 IgG and IgM antibodies, no ADAMTS13 gene aberrations were detected. She had had no previous episodes of TTP. During treatment, the patient received repeated plasma exchanges, supportive extracorporal immunoadsorption and corticosteroid therapy. Despite ongoing treatment anti-ADAMTS13 antibodies still developed and ADAMTS13 activity remained less than 0.1U/ml. After 5 weeks of therapy, during which time the patient was seriously ill with striking neurological limitations, her platelet count dropped and a splenectomy was performed. The patient’s state improved despite a complicated postoperative phase. During the next 4 weeks, her platelet count increased gradually up to 780 G/L, a moderately low ADAMTS13 activity of 0.24U/ml (0.57ng/ml ADAMTS13 antigen) was detected and ADAMTS13-specific antibodies were not measurable. However, within a few days her platelet count dramatically dropped to 20G/L, anti-ADAMTS13 antibodies reappeared and ADAMTS13 activity again was undetectable. The patient’s state deteriorated and she died 2 days later from multiple organ failure. We show the results of the fluctuating anti-ADAMTS13 antibody profile (functional inhibitors and total IgG, IgG subtypes, IgA and IgM) and the pattern of the ADAMTS13 domain-specific reactivity superimposed with the common clinical laboratory data over the patient’s 9-week clinical course.

Thrombotic thrombocytopenic purpura (TTP) is associated with a severe functional deficiency of the ADAMTS13 metalloprotease resulting in accumulation of highly adhesive ultra-large von Willebrand factor (ULVWF) multimers. ULVWF promote spontaneous platelet clumping eventually leading to life-threatening widespread microvascular thrombosis in multiple organs. Inhibitory and non-inhibitory anti-ADAMTS13 autoantibodies that result in functional deficiency and/or accelerated in vivo clearance of ADAMTS13 have been detected in patients suffering from acquired idiopathic TTP. Current treatment with plasma exchange therapy is considered to remove the autoantibodies and to replenish plasma with the deficient protease. While researching therapeutic recombinant ADAMST13 (rADAMTS13) as a possible treatment modality for TTP patients, we investigated the in vitro potency of rADAMTS13 to overwhelm and neutralize circulating anti-ADAMTS13 antibodies and concurrently to reinstate plasma ADAMTS13 to normal. Mixing and incubating patient plasma with normal reagent plasma and subsequent FRETSVWF73 activity analysis was used to calculate the individual anti-ADAMTS13 inhibitor titers in 36 untreated idiopathic TTP patient plasma samples with severe ADAMTS13 deficiency (

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