Publications by authors named "Hanspeter Rottensteiner"

59 Publications

Modulation of the liver immune microenvironment by the adeno-associated virus serotype 8 gene therapy vector.

Mol Ther Methods Clin Dev 2021 Mar 4;20:95-108. Epub 2020 Nov 4.

Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, Calgary, AB T2N 4N1, Canada.

Adeno-associated viruses (AAVs) are emerging as one of the vehicles of choice for gene therapy. However, the potential immunogenicity of these vectors is a major limitation of their use, leading to the necessity of a better understanding of how viral vectors engage the innate immune system. In this study, we demonstrate the immune response mediated by an AAV vector in a mouse model. Mice were infected intravenously with 4 × 10 copies (cp)/kg of AAV8, and the ensuing immune response was analyzed using intravital microscopy during a period of weeks. Administration of AAV8 resulted in the infection of hepatocytes, and this infection led to a moderate, but significant, activation of the immune system in the liver. This host immune response involved platelet aggregation, neutrophil extracellular trap (NET) formation, and the recruitment of monocytes, B cells, and T cells. The resident liver macrophage population, Kupffer cells, was necessary to initiate this immune response, as its depletion abrogated platelet aggregation and NET formation and delayed the recruitment of immune cells. Moreover, the death of liver cells produced by this AAV was moderate and failed to result in a robust, sustained inflammatory response. Altogether, these data suggest that AAV8 is a suitable vector for gene therapy approaches.
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http://dx.doi.org/10.1016/j.omtm.2020.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7750493PMC
March 2021

Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency.

Mol Ther Methods Clin Dev 2020 Dec 22;19:486-495. Epub 2020 Oct 22.

Herman B Wells Center for Pediatric Research, Indiana University, Indianapolis, IN 46202, USA.

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized nucleotide sequences were employed. A comprehensive immunogenicity assessment using stimulation assays and various preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.
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http://dx.doi.org/10.1016/j.omtm.2020.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708868PMC
December 2020

BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression.

Blood 2021 Feb;137(6):763-774

Gene Therapy Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC.

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608.
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http://dx.doi.org/10.1182/blood.2019004625DOI Listing
February 2021

Development of an Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release.

Mol Ther Methods Clin Dev 2020 Jun 17;17:581-588. Epub 2020 Mar 17.

Baxalta Innovations GmbH, a member of the Takeda group of companies, Uferstraße. 15, A-2304 Orth an der Donau, Austria.

Gene therapy product release requires reliable and consistent demonstration of biopotency. In hemophilia B vectors, this is usually determined by measuring the plasma levels of the expressed human factor IX (FIX) transgene product in FIX knockout mice. To circumvent this laborious assay, we developed an method in which the HepG2 human liver cell line was infected with the vector, and the resulting FIX activity was determined in the conditioned medium using a chromogenic assay. The initial low sensitivity of the assay, particularly toward adeno-associated viral serotype 8 (AAV8), increased approximately 100-fold and allowed linear measurement in a broad range of multiplicities of infection. Statistical parameters indicated high assay repeatability (relative standard deviation (RSD) < 5%) and intra-assay reproducibility (RSD < 20%). To compare the performance of the and biopotency assay, we applied statistical analyses including regression techniques and variation decomposition to the results obtained for 25 AAV8-FIX vector lots (BAX 335). These showed a highly significant correlation, with the cell culture-based assay demonstrating less variation than the test. The assay thus constitutes a viable alternative to using animals for lot release testing.
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http://dx.doi.org/10.1016/j.omtm.2020.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139127PMC
June 2020

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

J Thromb Haemost 2019 12 3;17(12):2099-2109. Epub 2019 Sep 3.

Baxalta Innovations GmbH, a member of the Takeda group of companies, Vienna, Austria.

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.
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http://dx.doi.org/10.1111/jth.14602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916347PMC
December 2019

Evaluation of Factor VIII Polysialylation: Identification of a Longer-Acting Experimental Therapy in Mice and Monkeys.

J Pharmacol Exp Ther 2019 10 31;371(1):95-105. Epub 2019 Jul 31.

Baxalta Innovations GmbH, a Member of the Takeda Group of Companies, Vienna, Austria

Extended half-life (EHL) factor therapies are needed to reduce the burden of prophylaxis and improve treatment adherence in patients with hemophilia. BAX 826 is a novel polysialylated full-length recombinant factor VIII [polysialyic acid (PSA) rFVIII] with improved pharmacokinetics (PK), prolonged pharmacology, and maintained safety attributes to enable longer-acting rFVIII therapy. In factor VIII (FVIII)-deficient hemophilic mice, PSArFVIII showed a substantially higher mean residence time (>2-fold) and exposure (>3-fold), and prolonged efficacy in tail-bleeding experiments (48 vs. 30 hours) compared with unmodified recombinant FVIII (rFVIII), as well as a potentially favorable immunogenicity profile. Reduced binding to a scavenger receptor (low-density lipoprotein receptor-related protein 1) and von Willebrand factor (VWF) as well as a largely VWF-independent circulation time in mice provide a rationale for prolonged BAX 826 activity. The significantly improved PK profile versus rFVIII was confirmed in cynomolgus monkeys [mean residence time: 23.4 vs. 10.1 hours; exposure (area under the curve from time 0 to infinity): 206 vs. 48.2 IU/ml⋅h] and is in line with results from rodent studies. Finally, safety and toxicity evaluations did not indicate increased thrombogenic potential, and repeated administration of BAX 826 to monkeys and rats was well tolerated. The favorable profile and mechanism of this novel experimental therapeutic demonstrated all of the requirements for an EHL-rFVIII candidate, and thus BAX 826 was entered into clinical assessment for the treatment of hemophilia A. SIGNIFICANCE STATEMENT: Prolongation of FVIII half-life aims to reduce the burden of prophylaxis and improve treatment outcomes in patients with hemophilia. This study shows that polysialylation of PSArFVIII resulted in prolongations of rFVIII circulation time and procoagulant activity, together with a favorable nonclinical safety profile of the experimental therapeutic.
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http://dx.doi.org/10.1124/jpet.119.260067DOI Listing
October 2019

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

J Thromb Haemost 2019 06 21;17(6):995-1002. Epub 2019 Apr 21.

Baxalta Innovations GmbH, a member of the Takeda group of companies, Vienna, Austria.

Background: The catalytic domain of ADAMTS13 possesses one Zn and up to three putative Ca 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 Ca or Zn prior to but not postincubation prevented the activity decrease of the enzyme. The SEC-ICP-MS analyses showed that ADAMTS13 only bound Zn and that its reduced activity correlated with a gradual loss of bound Zn . Concomitant higher-order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less-ordered protein structures.

Conclusions: Zn 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.
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http://dx.doi.org/10.1111/jth.14440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850365PMC
June 2019

Development of Methods for the Selective Measurement of the Single Amino Acid Exchange Variant Coagulation Factor IX Padua.

Mol Ther Methods Clin Dev 2018 Sep 28;10:29-37. Epub 2018 Jun 28.

Shire, Global Research, Donau-City-Str. 7, Vienna, Austria.

The description of hyper-functional factor IX (FIX) Padua triggered the development of BAX 335, an AAV8-based hemophilia B gene therapy vector designed to compensate for low FIX protein expression levels by expressing the FIX Padua variant, thereby reducing the exposure to viral vector. The presence of inactive FIX protein at baseline hindered conventional FIX:Ag ELISA from contributing to a more profound understanding of clinical data from the BAX 335 Phase 1/2 study (ClinicalTrials.gov: NCT01687608). By applying phage display technology, a Fab2 mini-antibody selectively binding to FIX Padua was developed and used to establish a FIX Padua-specific ELISA. The assay adequately performed, utilizing human and monkey plasma samples, and enabled the selective quantification of FIX Padua protein in human plasma samples from the BAX 335 trial. The mini-antibody also allowed the development of a chromogenic FIX Padua-specific activity assay, which adequately performed in human and mouse plasma. Collectively, the isolated FIX Padua-specific mini-antibody enabled the development of transgene-product-specific assays, which should improve the monitoring of hemophilia B gene therapies. The approach applied here for FIX Padua could be leveraged to develop variant-specific activity assays for other therapies where highly active enzymes are instrumental in achieving therapeutic levels of the transgene product.
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http://dx.doi.org/10.1016/j.omtm.2018.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039963PMC
September 2018

A bio-inspired method for direct measurement of local wall shear rates with micrometer localization using the multimeric protein von Willebrand factor as sensor molecule.

Biomicrofluidics 2017 Jul 30;11(4):044117. Epub 2017 Aug 30.

Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9, A-1060 Vienna, Austria.

Wall shear rates are critical for a broad variety of fluidic phenomena and are taken into account in nearly every experimental or simulation study. Generally, shear rates are not observable directly but rather derived from other parameters such as pressure and flow, often assuming somehow idealized systems. However, there is a biological system which is able to constantly measure the wall shear as a part of a regulatory circuit: The blood circulation system takes advantage of shear rate sensor (protein)molecules (multimeric forms of von Willebrand Factor, VWF), which are dissolved in the blood plasma and dramatically change their conformation under shear conditions. The conformational changes are accompanied by several functional variations and therefore interplay with the regulation of the coagulation system. In this study, we use a recombinantly produced and therefore well-defined multimeric form of VWF as a sensor which directly responds to shear rates. Shear rates, up to 32.000 s, were obtained using a kind of micro-plate-to-plate rheometer capable of adsorbing shear-stretched VWF oligomeric molecules on a surface to conserve their differently stretched conformation and so allow detection of their elongation by atomic force microscopy. The laminar flow in this geometrically simple device has been characterized by adopting classical fluid dynamical models, in order to ensure well-known, stable shear rates which could be correlated quantitatively with an observed stretching of sensor molecules.
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http://dx.doi.org/10.1063/1.5000503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577009PMC
July 2017

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

Arterioscler Thromb Vasc Biol 2017 05 2;37(5):836-844. Epub 2017 Mar 2.

From the Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Belgium (S.V., N.V., I.P., H.D., S.F.D.M., K.V.); Mobile DNA, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany (Z.I.); and Shire, Gene Therapy, Vienna, Austria (H.R.).

Objective: Severe deficiency in the von Willebrand factor-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) because of mutations in the gene can lead to acute episodes of congenital thrombotic thrombocytopenic purpura (TTP), requiring prompt treatment. Current treatment consists of therapeutic or prophylactic infusions of fresh frozen plasma. However, lifelong treatment with plasma products is a stressful therapy for TTP patients. Here, we describe the use of the nonviral sleeping beauty (SB) transposon system as a gene therapeutic approach to realize lifelong expression of ADAMTS13 and subsequent protection against congenital TTP.

Approach And Results: We demonstrated that hydrodynamic tail vein injection of the SB100X system expressing murine ADAMTS13 in mice resulted in long-term expression of supraphysiological levels of transgene ADAMTS13 over a period of 25 weeks. Stably expressed ADAMTS13 efficiently removed the prothrombotic ultralarge von Willebrand factor multimers present in the circulation of mice. Moreover, mice stably expressing ADAMTS13 were protected against TTP. The treated mice did not develop severe thrombocytopenia or did organ damage occur when triggered with recombinant von Willebrand factor, and this up to 20 weeks after gene transfer.

Conclusions: These data demonstrate the feasibility of using SB100X-mediated gene therapy to achieve sustained expression of transgene ADAMTS13 and long-term prophylaxis against TTP in mice.
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http://dx.doi.org/10.1161/ATVBAHA.116.308680DOI Listing
May 2017

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

PLoS One 2016 1;11(8):e0160388. Epub 2016 Aug 1.

Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy linked to a deficiency in the metalloprotease ADAMTS13. In the current study, a novel mouse model for acquired TTP was generated to facilitate development and validation of new therapies for this disease. Therefore, a large panel (n = 19) of novel anti-mouse ADAMTS13 (mADAMTS13) monoclonal antibodies (mAbs) of mouse origin was generated. Inhibitory anti-mADAMTS13 mAbs were identified using the FRETS-VWF73 assay. Four mAbs strongly inhibited mADAMTS13 activity in vitro (∼68-90% inhibition). Injecting a combination of 2 inhibitory mAbs (13B4 and 14H7, 1.25 mg/kg each) in Adamts13+/+ mice resulted in full inhibition of plasma ADAMTS13 activity (96 ± 4% inhibition, day 1 post injection), leading to the appearance of ultra-large von Willebrand factor (UL-VWF) multimers. Interestingly, the inhibitory anti-mADAMTS13 mAbs 13B4 and 14H7 were ideally suited to induce long-term ADAMTS13 deficiency in Adamts13+/+ mice. A single bolus injection resulted in full ex vivo inhibition for more than 7 days. As expected, the mice with the acquired ADAMTS13 deficiency did not spontaneously develop TTP, despite the accumulation of UL-VWF multimers. In line with the Adamts13-/- mice, TTP-like symptoms could only be induced when an additional trigger (rVWF) was administered. On the other hand, the availability of our panel of anti-mADAMTS13 mAbs allowed us to further develop a sensitive ELISA to detect ADAMTS13 in mouse plasma. In conclusion, a novel acquired TTP mouse model was generated through the development of inhibitory anti-mADAMTS13 mAbs. Consequently, this model provides new opportunities for the development and validation of novel treatments for patients with TTP. In addition, these newly developed inhibitory anti-mADAMTS13 mAbs are of great value to specifically study the role of ADAMTS13 in mouse models of thrombo-inflammatory disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160388PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968830PMC
August 2017

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

Blood 2016 05 29;127(19):2337-45. Epub 2016 Feb 29.

Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium;

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.
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http://dx.doi.org/10.1182/blood-2015-08-662650DOI Listing
May 2016

Low ADAMTS13 activity is associated with an increased risk of ischemic stroke.

Blood 2015 Dec 28;126(25):2739-46. Epub 2015 Oct 28.

Department of Hematology.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin motif repeats 13) has antithrombotic properties because it cleaves von Willebrand factor (VWF) in smaller, less active multimers. The aim of our study was to investigate prospectively the association between ADAMTS13 activity and ischemic stroke. We included 5941 individuals ≥55 years without a history of stroke or transient ischemic attack (TIA) of the Rotterdam Study, a population-based cohort study. ADAMTS13 activity was measured at inclusion with the FRETS-VWF73 assay and VWF antigen (VWF:Ag) levels by enzyme-linked immunosorbent assay. We assessed the association among ADAMTS13 activity, VWF:Ag levels, and ischemic stroke by Cox proportional hazard analysis. The added value of ADAMTS13 activity above the traditional risk factors for ischemic stroke risk prediction was examined by the C-statistic and the net reclassification improvement index (NRI). All individuals were followed for incident stroke or TIA. Over a median follow-up time of 10.7 years (56,403 total person-years), 461 participants had a stroke, 306 of which were ischemic. After adjustment for cardiovascular risk factors, individuals with ADAMTS13 activity in the lowest quartile had a higher risk of ischemic stroke (absolute risk, 7.3%) than did those in the reference highest quartile (absolute risk, 3.8%; hazard ratio, 1.65; 95% confidence interval [CI], 1.16-2.32). Adding ADAMTS13 to the model in prediction of ischemic stroke, increased the C-statistic by 0.013 (P = .003) and provided 0.058 (95% CI, -0.002 to 0.119) NRI. Low ADAMTS13 activity is associated with the risk of ischemic stroke and improves the accuracy of risk predictions for ischemic stroke beyond traditional risk factors.
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http://dx.doi.org/10.1182/blood-2015-05-643338DOI Listing
December 2015

Shear-Dependent Interactions of von Willebrand Factor with Factor VIII and Protease ADAMTS 13 Demonstrated at a Single Molecule Level by Atomic Force Microscopy.

Anal Chem 2015 Oct 30;87(20):10299-305. Epub 2015 Sep 30.

Institute of Chemical Technologies and Analytics, Vienna University of Technology , Getreidemarkt 9/164, A-1060 Vienna, Austria.

Vital functions of mammals are only possible due to the behavior of blood to coagulate most efficiently in vessels with particularly high wall shear rates. This is caused by the functional changes of the von Willebrand Factor (VWF), which mediates coagulation of blood platelets (primary hemostasis) especially when it is stretched under shear stress. Our data show that shear stretching also affects other functions of VWF: Using a customized device to simulate shear conditions and to conserve the VWF molecules in their unstable, elongated conformation, we visualize at single molecule level by AFM that VWF is preferentially cleaved by the protease ADAMTS13 at higher shear rates. In contrast to this high shear-rate-selective behavior, VWF binds FVIII more effectively only below a critical shear rate of ∼30.000 s(-1), indicating that under harsh shear conditions FVIII is released from its carrier protein. This may be required to facilitate delivery of FVIII locally to promote secondary hemostasis.
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http://dx.doi.org/10.1021/acs.analchem.5b02078DOI Listing
October 2015

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

Arterioscler Thromb Vasc Biol 2015 Nov 3;35(11):2336-42. Epub 2015 Sep 3.

From the Laboratory for Thrombosis Research, KU Leuven, Campus Kulak Kortrijk, Kortrijk, Belgium (C.T., S.F.D.M., K.V.); and Baxalta Innovations GmbH, Vienna, Austria (A.S., B.P., F.S., H.R.).

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.
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http://dx.doi.org/10.1161/ATVBAHA.115.306014DOI Listing
November 2015

Ca(2+) concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy.

Anal Bioanal Chem 2015 Aug 23;407(20):6051-6. Epub 2015 May 23.

Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-IAC, 1060, Vienna, Austria.

FVIII is a multi-domain protein organized in a heavy and a light chain, and a B-domain whose biological function is still a matter of debate. The 3D structure of a B-domain-deleted FVIII variant has been determined by X-ray crystallography, leaving unexplained the functional nature of the flexible B-domain which could play an important role in the structure-function relationship since it is removed during the activation process. To obtain clues on the function of the B-domain, the morphology of full-length FVIII and its isolated domains was determined in the absence or presence of Ca(2+). Recombinant full-length FVIII, the purified heavy chain, light chain and B-domain as well as B-domain-deleted rFVIII were analysed in buffers of different Ca(2+) concentrations by atomic force microscopy. In the absence of Ca(2+), FVIII appeared as a globular molecule, whereas at high amounts of Ca(2+) up to 50-nm long tail structures emerged. These tails could be identified as unravelled B-domains, as images of isolated B-domains showed the same morphology and heavy chains which include the B-domain were also rich of tails, whereas the isolated light chains and B-domain-deleted FVIII lacked any deviation from a globular shape. The images further suggested that the B-domain interacts with the light chain particularly at low Ca(2+) concentrations. Our results show a Ca(2+)-regulated conformational change of the B-domain in the context of full-length rFVIII. As the B-domain tightly associated with the core of the FVIII molecule under low Ca(2+)-concentrations, a stabilizing function on FVIII under non-activating conditions may be proposed.
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http://dx.doi.org/10.1007/s00216-015-8778-zDOI Listing
August 2015

Genetic variants in the ADAMTS13 and SUPT3H genes are associated with ADAMTS13 activity.

Blood 2015 Jun 1;125(25):3949-55. Epub 2015 May 1.

Department of Hematology.

A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) cleaves von Willebrand factor, reducing its prothrombotic activity. The genetic determinants of ADAMTS13 activity remain unclear. We performed a genome-wide association study of ADAMTS13 activity in the Rotterdam Study, a population-based cohort study. We used imputed genotypes of common variants in a discovery sample of 3443 individuals and replication sample of 2025 individuals. We examined rare exonic variant associations in ADAMTS13 in 1609 individuals using an exome array. rs41314453 in ADAMTS13 was associated with ADAMTS13 activity in both our discovery (β, -20.2%; P = 1.3 × 10(-33)) and replication sample (P = 3.3 × 10(-34)), and explained 3.6% to 6.5% of the variance. In the combined analysis of our discovery and replication samples, there were 2 further independent associations at the ADAMTS13 locus: rs3118667 (β, 3.0; P = 9.6 × 10(-21)) and rs139911703 (β, -11.6; P = 3.6 × 10(-8)). In addition, rs10456544 in SUPT3H was associated with a 4.2 increase in ADAMTS13 activity (P = 1.13.6 × 10(-8)). Finally, we found 3 independent associations with rare coding variants in ADAMTS13: rs148312697 (β, -32.2%; P = 3.7 × 10(-6)), rs142572218 (β, -46.0%; P = 3.9 × 10(-5)), and rs36222275 (β, -13.9%; P = 2.9 × 10(-3)). In conclusion, we identified rs41314453 as the main genetic determinant of ADAMTS13 activity, and we present preliminary findings for further associations at the ADAMTS13 and SUPT3H loci.
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http://dx.doi.org/10.1182/blood-2015-02-629865DOI Listing
June 2015

Nonacog gamma, a novel recombinant factor IX with low factor IXa content for treatment and prophylaxis of bleeding episodes.

Expert Rev Clin Pharmacol 2015 Mar 8;8(2):163-77. Epub 2015 Feb 8.

Baxter Innovations GmbH, Vienna, Austria.

Nonacog gamma is a new recombinant factor IX to treat factor IX deficiency. It is indicated for control of bleeding episodes, perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia B. Nonacog gamma was first approved in the USA in June 2013 under the trade name RIXUBIS followed by market approvals in Australia and the EU in 2014, and marketing authorization decision is pending in Japan. Nonacog gamma is derived from a recombinant Chinese hamster ovary cell line using a state of the art biotechnological manufacturing process. Recombinant factor IX is produced by Baxter's protein-free fermentation technology, which was first developed for ADVATE. The product is purified and formulated in the absence of any human or animal-derived protein. Nonacog gamma was characterized both in comprehensive in vitro and in vivo non-clinical studies as well as in an extensive clinical trial program.
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http://dx.doi.org/10.1586/17512433.2015.1011126DOI Listing
March 2015

Visualization of a protein-protein interaction at a single-molecule level by atomic force microscopy.

Anal Bioanal Chem 2014 Feb 21;406(5):1411-21. Epub 2013 Dec 21.

Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-IAC, 1060, Wien, Austria.

Atomic force microscopy is unmatched in terms of high-resolution imaging under ambient conditions. Over the years, substantial progress has been made using this technique to improve our understanding of biological systems on the nanometer scale, such as visualization of single biomolecules. For monitoring also the interaction between biomolecules, in situ high-speed imaging is making enormous progress. Here, we describe an alternative ex situ imaging method where identical molecules are recorded before and after reaction with a binding partner. Relocation of the identical molecules on the mica surface was thereby achieved by using a nanoscale scratch as marker. The method was successfully applied to study the complex formation between von Willebrand factor (VWF) and factor VIII (FVIII), two essential haemostatic components of human blood. FVIII binding was discernible by an appearance of globular domains appended to the N-terminal large globular domains of VWF. The specificity of the approach could be demonstrated by incubating VWF with FVIII in the presence of a high salt buffer which inhibits the interaction between these two proteins. The results obtained indicate that proteins can maintain their reactivity for subsequent interactions with other molecules when gently immobilized on a solid substrate and subjected to intermittent drying steps. The technique described opens up a new analytical perspective for studying protein-protein interactions as it circumvents some of the obstacles encountered by in situ imaging and other ex situ techniques.
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http://dx.doi.org/10.1007/s00216-013-7563-0DOI Listing
February 2014

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

Haematologica 2014 Apr 15;99(4):779-87. Epub 2013 Nov 15.

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.
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http://dx.doi.org/10.3324/haematol.2013.094151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971089PMC
April 2014

Preclinical safety and efficacy of a new recombinant FIX drug product for treatment of hemophilia B.

Int J Hematol 2013 Nov 24;98(5):525-32. Epub 2013 Sep 24.

Baxter Innovations GmbH, Wagramer Straße 17-19, 1221, Vienna, Austria.

Baxter has developed a new recombinant factor IX (rFIX) drug product (BAX326) for treating patients with hemophilia B, or congenital FIX deficiency. An extensive preclinical program evaluated the pharmacokinetics, efficacy, and safety of BAX326 in different species. The efficacy of BAX326 was tested in three mouse models of primary pharmacodynamics: tail-tip bleeding, carotid occlusion, and thrombelastography. The pharmacokinetics was evaluated after a single intravenous bolus injection in mice, rats, and macaques. Toxicity was assessed in rats and macaques, safety pharmacology in rabbits and macaques, and immunogenicity in mice. BAX326 was shown to be efficacious in all three primary pharmacodynamic studies (P ≤ 0.0076). Hemostatic efficacy was dose related and similar for the three lots tested. Pharmacokinetic results showed that rFIX activity and rFIX antigen concentrations declined in a bi-phasic manner, similar to a previously licensed rFIX product. BAX326 was well tolerated in rabbits and macaques at all dose levels; no thrombogenic events and no adverse clinical, respiratory, or cardiovascular effects occurred. BAX326 was also shown to have a similar immunogenicity profile to the comparator rFIX product in mice. These results demonstrate that BAX326 has a favorable preclinical safety and efficacy profile, predictive of a comparable effect to that of the previously licensed rFIX in humans.
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http://dx.doi.org/10.1007/s12185-013-1448-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091414PMC
November 2013

Protective anti-inflammatory effect of ADAMTS13 on myocardial ischemia/reperfusion injury in mice.

Blood 2012 Dec 22;120(26):5217-23. Epub 2012 Aug 22.

Immune Disease Institute, Boston, MA 02115, USA.

Coronary heart disease is a major cause of death in the western world. Although essential for successful recovery, reperfusion of ischemic myocardium is inevitably associated with reperfusion injury. To investigate a potential protective role of ADAMTS13, a protease cleaving von Willebrand factor multimers, during myocardial ischemia/reperfusion, we used a mouse model of acute myocardial infarction. We found that Adamts13(-/-) mice developed larger myocardial infarctions than wild-type control mice, whereas treatment of wild-type mice with recombinant human ADAMTS13 (rhADAMTS13) led to smaller infarctions. The protective effect of ADAMTS13 was further confirmed by a significant reduction of cardiac troponin-I release and less myocardial apoptosis in mice that received rhADAMTS13 compared with controls. Platelets adherent to the blood vessel wall were observed in few areas in the heart samples from mice treated with vehicle and were not detected in samples from mice treated with rhADAMTS13. However, we observed a 9-fold reduction in number of neutrophils infiltrating ischemic myocardium in mice that were treated with rhADAMTS13, suggesting a potent anti-inflammatory effect of ADAMTS13 during heart injury. Our data show that ADAMTS13 reduces myocardial ischemia/reperfusion injury in mice and indicate that rhADAMTS13 could be of therapeutic value to limit myocardial ischemia/reperfusion injury.
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http://dx.doi.org/10.1182/blood-2012-06-439935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537313PMC
December 2012

Development and validation of a predictive model for death in acquired severe ADAMTS13 deficiency-associated idiopathic thrombotic thrombocytopenic purpura: the French TMA Reference Center experience.

Haematologica 2012 Aug 11;97(8):1181-6. Epub 2012 May 11.

Service de Médecine Interne, Hôpital Charles Nicolle, Rouen, France.

Background: Acquired thrombotic thrombocytopenic purpura is still associated with a 10-20% death rate. It has still not been possible to clearly identify early prognostic factors of death. This study involved thrombotic thrombocytopenic purpura patients with acquired severe (<10% of normal activity) ADAMTS13 deficiency and aimed to identify prognostic factors associated with 30-day death.

Design And Methods: The study involved a prospective cohort of patients and was carried out between October 2000 and August 2010. A validation cohort of patients was set up from September 2010 to August 2011. Altogether, 281 (analysis cohort) and 66 (validation cohort) consecutive adult thrombotic thrombocytopenic purpura patients with acquired severe ADAMTS13 deficiency were enrolled. The study evaluated 30-day mortality after treatment initiation according to characteristics at inclusion.

Results: Non-survivors (11%) were older (P=10(-6)) and more frequently presented arterial hypertension (P=5.10(-4)) and ischemic heart disease (P=0.013). Prognosis was increasingly poor with age (P=0.004). On presentation, cerebral manifestations were more frequent in non-survivors (P=0.018) and serum creatinine level was higher (P=0.008). The most significant independent variables determining death were age, severe cerebral involvement and LDH level 10 N or over. A 3-level risk score for early death was defined and confirmed in the validation cohort using these variables, with higher values corresponding to increased risk of early death.

Conclusions: A risk score for early death was defined in patients with thrombotic thrombocytopenic purpura and validated on an independent cohort. This score should help to stratify early treatment and identify patients with a worse prognosis.
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http://dx.doi.org/10.3324/haematol.2011.049676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409815PMC
August 2012

A new mouse model mimicking thrombotic thrombocytopenic purpura: correction of symptoms by recombinant human ADAMTS13.

Blood 2012 Jun 23;119(25):6128-35. Epub 2012 Apr 23.

Department of Preclinical Pharmacology & Toxicology, Baxter Innovations GmbH, Industriestrasse 67, Vienna, Austria.

Deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a VWF-cleaving protease, is the key factor in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy. It is well established that ADAMTS13 deficiency results in elevated plasma levels of ultra-large VWF multimers (ULVWF), which are prone to induce platelet aggregation; however, the actual trigger of TTP development remains uncertain. Here we describe a new animal model in which some TTP-like symptoms can be triggered in ADAMTS13 knockout mice by challenge with 2000 units/kg body weight of recombinant human VWF containing ULVWF multimers. Animals rapidly showed clinical symptoms and developed severe thrombocytopenia. Schistocytosis, a decrease in hematocrit, and elevated serum lactate dehydrogenase levels were observed. The heart was identified as the most sensitive target organ with rapid onset of extensive platelet aggregation in the ventricles and myocardial necrosis. Prophylactic administration of 200 units/kg recombinant human ADAMTS13 protected ADAMTS13 knockout mice from developing TTP. Therapeutic administration of 320 units/kg rhADAMTS13 reduced the incidence and severity of TTP findings in a treatment interval-dependent manner. We therefore consider this newly established mouse model of thrombotic microangiopathy highly predictive for investigating the efficacy of new treatments for TTP.
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http://dx.doi.org/10.1182/blood-2011-09-380535DOI Listing
June 2012

Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2-D DIGE.

Electrophoresis 2011 Jun 26;32(11):1292-301. Epub 2011 Apr 26.

Baxter Innovations GmbH, Vienna, Austria.

2-D DIGE is a method that circumvents the gel-to-gel variability inherent in conventional 2-DE and is particularly useful for studying proteome changes in diverse applications such as developmental biology and tissue proteomics. We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The factor VIII heterodimer is composed of heterogeneous, heavily glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments whose identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII's glycans, due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate various PEGylated rFVIII conjugates. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool for quality control of very complex pharmaceutically active ingredients.
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http://dx.doi.org/10.1002/elps.201000627DOI Listing
June 2011

Identification of PEX33, a novel component of the peroxisomal docking complex in the filamentous fungus Neurospora crassa.

Eur J Cell Biol 2010 Dec 21;89(12):955-64. Epub 2010 Aug 21.

Institut für Physiologische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Germany.

The docking complex of peroxisomal matrix protein import is composed of PEX13 and PEX14 in all species analyzed so far, whereas only yeast appears to possess an additional component, PEX17. In this report we isolated PEX14 complexes of Neurospora crassa. Among the complex constituents, one protein designated as PEX33 possessed homology to PEX14 but only in a short N-terminal domain. The PEX14/PEX33 interaction was verified by means of two-hybrid analysis. Moreover, PEX33 was shown to interact with itself and the PTS1-receptor PEX5. Localization studies demonstrated that PEX33 constitutes a glyoxysomal protein. Growth tests of the pex33 deletion strain revealed a defect of this strain in the biogenesis of glyoxysomes and Woronin bodies. As the function of PEX33 was not redundant to that of PEX14, it is a genuine novel peroxin. Based on our experimental data, the function of PEX33 seems to resemble that of yeast PEX17 despite clear structural differences.
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http://dx.doi.org/10.1016/j.ejcb.2010.07.003DOI Listing
December 2010

A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa.

Proteomics 2010 Sep;10(18):3222-34

Department of Systems Biochemistry, Institute of Physiological Chemistry, Ruhr-University of Bochum, Bochum, Germany.

Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI-MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl-CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.
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http://dx.doi.org/10.1002/pmic.201000095DOI Listing
September 2010

De novo synthesis of peroxisomes upon mitochondrial targeting of Pex3p.

Eur J Cell Biol 2010 Dec 24;89(12):947-54. Epub 2010 Jul 24.

Institut für Physiologische Chemie, Abteilung für Systembiochemie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany.

Peroxisomes can form either by growth and division of pre-existing peroxisomes or by de novo synthesis from the endoplasmic reticulum. Pex3p is the key component for both pathways and its targeting to the ER is thought to initiate the de novo formation of peroxisomes. Here, we addressed the question whether Pex3p also can induce peroxisome formation from mitochondrial membranes. Pex3p was targeted to mitochondria by fusion with the mitochondrial targeting signal of Tom20p. The Tom20p-Pex3p-fusion protein was expressed in Pex3p-deficient cells, which are characterized by the lack of peroxisomal membranes. De novo formation of import-competent peroxisomes was observed upon expression of the mitochondrial Pex3p in the mutant cells. This de novo synthesis is independent of the GTPases Vps1p and Dnm1p, two proteins required for peroxisome fission. We conclude that natural or artificial targeting of Pex3p to any endomembrane may initiate peroxisome formation and that also Pex3p-containing mitochondria can serve as source for the de novo synthesis of peroxisomes.
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http://dx.doi.org/10.1016/j.ejcb.2010.06.012DOI Listing
December 2010

Structure and function of a recombinant von Willebrand factor drug candidate.

Semin Thromb Hemost 2010 Jul 15;36(5):510-21. Epub 2010 Jul 15.

Baxter BioScience, Vienna, Austria.

The complex structure, large size, and multiple posttranslational modifications of von Willebrand factor (VWF) presented a technological challenge for the production of recombinant VWF (rVWF). Nonetheless, we developed an rVWF product for treating von Willebrand disease, whereupon rVWF is coexpressed with recombinant factor VIII (rFVIII) in Chinese hamster ovary cells used to produce rFVIII for the treatment of hemophilia A. Here we describe the characterization of the structure and function of the rVWF drug product, with a focus on its in vitro platelet aggregation and matrix protein binding functions. Electron microscopy and multimer analysis revealed a highly organized structure for the rVWF protein, with a homogeneous multimer distribution including ultrahigh molecular weight multimers. The specific activity for binding to collagen and platelets mediated by ristocetin is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. The affinity and binding capacity of rVWF to FVIII is comparable to VWF in plasma. rVWF effectively binds to platelets and promotes platelet adhesion under shear stress similar to VWF in human plasma.
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http://dx.doi.org/10.1055/s-0030-1255445DOI Listing
July 2010