Amplified endogenous plasmin activity resolves acute thrombotic thrombocytopenic purpura in mice.

Essentials Plasmin is able to proteolyse von Willebrand factor. It was unclear if plasmin influences acute thrombotic thrombocytopenic purpura (TTP). Plasmin levels are increased during acute TTP though suppressed via plasmin(ogen) inhibitors. Allowing amplified endogenous plasmin activity in mice results in resolution of TTP signs. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in the absence of ADAMTS-13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role of plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results In mice with an acquired ADAMTS-13 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 ADAMTS-13 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, although limited via suppression through α2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a back-up for ADAMTS-13 in mice and resolve TTP signs as a result of an increased proteolysis of VWF.

Preclinical assessment of a new recombinant ADAMTS-13 drug product (BAX930) for the treatment of thrombotic thrombocytopenic purpura.

UNLABELLED: 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. Lämmle present a seminar on Thrombotic Thrombocytopenic Purpura (TTP): new Insights in Pathogenesis and Treatment Modalities. SUMMARY: Background 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.

Neutralization of inhibitory antibodies and restoration of therapeutic ADAMTS-13 activity levels in inhibitor-treated rats by the use of defined doses of recombinant ADAMTS-13.

BACKGROUND: 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.

A novel flow-based assay reveals discrepancies in ADAMTS-13 inhibitor assessment as compared with a conventional clinical static assay.

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.

Recombinant ADAMTS13 normalizes von Willebrand factor-cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies.

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.

Development of a plasma- and albumin-free recombinant von Willebrand factor.

Baxter has developed a recombinant therapy for treating von Willebrand's disease. Recombinant VWF is co-expressed with the rFVIII in CHO cells used to produce the rFVIII product Advate. This rVWF is used as a drug component for a rVWF-rFVIII complex drug product. CHO cells produce partially processed and partially un-processed rVWF still containing the pro-peptide. In order to make a consistent preparation containing mature and processed rVWF only rVWF is exposed to recombinant furin to remove the pro-peptide. Recombinant VWF and furin are produced under serum- and protein-free conditions. It is highly purified by a series of chromatographic steps and formulated in a protein-free buffer and has a homogeneous multimer distribution. The specific activity is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. SDS agarose electrophoretic analysis shows the presence of ultra-high molecular weight multimers. The FVIII-binding capacity and affinity of rVWF to FVIII is comparable to VWF in plasma. Carbohydrate analysis shows an intact glycosylation pattern. Recombinant VWF binds to collagen and promotes platelet adhesion under shear stress. It stabilizes endogenous FVIII in VWF-deficient knock-out mice as seen by a secondary rise in murine FVIII.

Second international collaborative study evaluating performance characteristics of methods measuring the von Willebrand factor cleaving protease (ADAMTS-13).

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.

Cloning, expression and functional characterization of the full-length murine ADAMTS13.

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.

Recombinant von Willebrand factor: preclinical development.

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.

'Shed' furin: mapping of the cleavage determinants and identification of its C-terminus.

The human endoprotease furin is involved in the proteolytic maturation of the precursor molecules of a wide variety of bioactive proteins. Despite its localization in the membranes of the trans-Golgi system by means of a transmembrane domain, it has repeatedly been reported to form a C-terminally truncated, naturally secreted form referred to as 'shed' furin. In order to identify the cleavage site, internal deletion mutants of increasing size, N-terminal to Leu(708), and subsequently individual amino acid substitutions were introduced, and Arg(683) was identified as the prime determinant for shedding. MS analysis determined Ser(682) as the C-terminus of shed furin, suggesting that monobasic cleavage may occur N-terminal to Arg(683). Alteration of Arg(683) directs the shedding mechanism to alternative cleaving sites previously unused.

Recombinant human factor X: high yield expression and the role of furin in proteolytic maturation in vivo and in vitro.

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.

Strategies for recombinant Furin employment in a biotechnological process: complete target protein precursor cleavage.

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 x day, processing became insufficient. Stable co- and overexpression of full length Furin resulted in complete precursor cleavage in cell clones expressing 2 mug rvWF/106 cells x 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 mug rvWF/106 cells x 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 mug, and 20 mug, as well as beyond 20 mug/106 cells x 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.

Detection of false-negative results in nested primer PCR of proviral HIV-1 DNA.

A control template for a competitive nested primer PCR of the HIV-1 gag region was constructed. This construct shares the primer recognition sequences with the wild-type template and yields a 97-bp fragment after amplification (wild-type: 115 bp). To provide an internal control for the individual PCR runs, six copies of this nested primer control plasmid were introduced into a reaction tube containing the specific sample (under the PCR conditions used, this copy number reproducibly gave a positive PCR signal). The results of our study show the feasibility of this concept by analyzing a plasmid (pBH10) containing HIV-1 wild-type sequences, and examination of samples from a cohort of HIV-1-seropositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, which without the use of the control template would have yielded a false-negative result.

Quantitative multiple competitive PCR of HIV-1 DNA in a single reaction tube.

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.

Molecular analysis of two hexokinase isoenzymes from Entamoeba histolytica.

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.

Pathogenic Entamoeba histolytica: cDNA cloning of a histone H3 with a divergent primary structure.

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.

Molecular characterization of the cDNA coding for translation elongation factor-2 of pathogenic Entamoeba histolytica.

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.