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New therapeutic agents are needed to overcome the toxicity and suboptimal efficacy observed in current treatment of glomerulonephritis (GN). BaxB01 is a fully human monoclonal antibody targeting a disease-related immunologically distinct isoform of Macrophage migration Inhibitory Factor (MIF), designated oxidized MIF (oxMIF) and locally expressed in inflammatory conditions. We report the pharmacokinetic profile of BaxB01, and its dose and exposure-related disease-modifying activity in experimentally induced rat GN. BaxB01 bound to rat oxMIF with high affinity and reduced rat macrophage migration in vitro. After intravenous administration in rats, BaxB01 demonstrated favorable pharmacokinetics, with a half-life of up to nine days. Disease modification was dose-related (≥ 10mg/kg) as demonstrated by significantly reduced proteinuria and diminished histopathological glomerular crescent formation. Importantly, a single dose was sufficient to establish an exposure-related, anti-inflammatory milieu via amelioration of glomerular cellular inflammation. Pharmacodynamic modeling corroborated these findings, consistently predicting plasma exposures that were effective in attenuating both anti-inflammatory activity and reducing loss of kidney function. This pharmacologic benefit on glomerular function and structure was sustained during established disease, while correlation analyses confirmed a link between the antibody's anti-inflammatory activity and reduced crescent formation in individual rats. Finally, safety assessment in rats showed that the experimental therapeutic was well tolerated without signs of systemic toxicity or negative impact on kidney function. These data define therapeutically relevant exposures correlated with mechanism-based activity in GN, while toxicological evaluation suggests a large therapeutic index and provides evidence for achieving safe and effective exposure to a MIF isoform-directed therapeutic in nephritis-associated disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large-scale manufacturing and raises concerns for in vivo applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell-derived polymorphic class-I major histocompatibility complexes (MHC-I) are incorporated into the LV surface and trigger allogeneic T-cell responses. By disrupting the beta-2 microglobulin gene in producer cells, we obtained MHC-free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single-copy inducible vector components, which can be reproducibly converted into high-yield LV producers upon site-specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement-mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen-free LV with higher purity and increased complement resistance that are better suited for in vivo gene therapy., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

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 -1 , 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.

Von Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions. Re-compaction of Von Willebrand factor is accelerated by intramolecular interactions and increases GPIbα dissociation rate. This mechanism enables VWF to be locally activated by hydrodynamic force in hemorrhage and rapidly deactivated downstream, providing a paradigm for hierarchical mechano-regulation of receptor-ligand binding.Von Willebrand factor (VWF) is a blood protein involved in clotting and is proposed to be activated by flow, but the mechanism is unknown. Here the authors show that VWF is first converted from a compact to linear form by flow, and is subsequently activated to bind GPIbα in a tension-dependent manner.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, which was shown to be upregulated in cancers and to exhibit tumor promoting properties. Unlike other cytokines, MIF is ubiquitously present in the circulation and tissue of healthy subjects. We recently described a previously unrecognized, disease-related isoform of MIF, designated oxMIF, which is present in the circulation of patients with different inflammatory diseases. In this article, we report that oxMIF is also linked to different solid tumors as it is specifically expressed in tumor tissue from patients with colorectal, pancreatic, ovarian and lung cancer. Furthermore, oxMIF can be specifically targeted by a subset of phage display-derived fully human, monoclonal anti-MIF antibodies (mAbs) that were shown to neutralize pro-tumorigenic activities of MIF in vivo. We further demonstrate that anti-oxMIF mAbs sensitize human cancer cell lines (LNCaP, PC3, A2780 and A2780ADR) to the action of cytotoxic drugs (mitoxantrone, cisplatin and doxorubicin) in vitro and in an A2780 xenograft mouse model of ovarian cancer. We conclude that oxMIF is the disease related isoform of MIF in solid tumors and a potential new diagnostic marker and drug target in cancer.

Purpose: Recent findings demonstrated anti-PEG antibody formation in some healthy individuals and patients who have not received PEGylated biotherapeutics. Some of these findings evoked criticism because of shortcomings in the antibody assays used. To better understand this topic, we established robust antibody analytics and screened two cohorts of healthy individuals and one cohort of hemophilia patients for the expression of anti-PEG antibodies., Methods: A flow cytometry approach and a fully validated ELISA platform were established to detect specific anti-PEG antibodies. Immunohistochemistry was used to test for potential binding of anti-PEG antibodies to human tissues., Results: IgM and/or IgG anti-PEG antibodies are expressed by some healthy individuals and by some patients with hemophilia who have not received PEGylated biotherapeutics. These antibodies can be either transient or persistent and recognize PEGs of different sizes with or without terminal methoxy groups. Age and location of healthy individuals influence the prevalence of IgG but not of IgM antibodies. Anti-PEG antibodies do not cross-react with human tissues supporting the safety of the antibodies., Conclusion: We confirm that some healthy individuals and some patients with hemophilia express specific antibodies against PEG which are not associated with any pathology and do not bind to human tissues.

Neutralizing autoantibodies against factor VIII (FVIII), also called FVIII inhibitors, are the cause of acquired hemophilia A (AHA). They are quantified in the Bethesda assay or Nijmegen-modified Bethesda assay by their ability to neutralize FVIII in normal human plasma. However, FVIII inhibitors do not represent the whole spectrum of anti-FVIII autoantibodies. Here, we studied isotypes, immunoglobulin G subclasses, and apparent affinities of anti-FVIII autoantibodies to assess their prognostic value for the outcome in AHA. We analyzed baseline samples from patients enrolled in the prospective GTH-AH 01/2010 study. Our data suggest that anti-FVIII immunoglobulin A (IgA) autoantibodies are predictors of poor outcome in AHA. Anti-FVIII IgA-positive patients achieved partial remission similar to anti-FVIII IgA-negative patients but had a higher risk of subsequent recurrence. Consequently, IgA-positive patients achieved complete remission less frequently (adjusted hazard ratio [aHR], 0.35; 95% confidence interval [CI], 0.18-0.68; P < .01) and had a higher risk of death (aHR, 2.62; 95% CI, 1.11-6.22; P < .05). Anti-FVIII IgA was the strongest negative predictor of recurrence-free survival after achieving partial remission and remained significant after adjustment for baseline demographic and clinical characteristics. In conclusion, anti-FVIII IgA represents a potential novel biomarker that could be useful to predict prognosis and tailor immunosuppressive treatment of AHA., (© 2016 by The American Society of Hematology.)

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., (© 2016 by The American Society of Hematology.)

Tissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 µM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2-1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1-150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25-0.5 nM TFPI) inhibit FXa with a t1/2 between 3-15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.

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., (© 2015 by The American Society of Hematology.)

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., (© 2015 American Heart Association, Inc.)

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.

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., (© 2015 AABB.)

Unlabelled: BACKGROUND &, Methods: Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS &, Discussion: The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures., Conclusions: From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

Purpose: Sub-visible particles were shown to facilitate unwanted immunogenicity of protein therapeutics. To understand the root cause of this phenomenon, a comprehensive analysis of these particles is required. We aimed at establishing a flow-cytometry-based technology to analyze the amount, size distribution and nature of sub-visible particles in protein solutions., Methods: We adjusted the settings of a BD FACS Canto II by tuning the forward scatter and the side scatter detectors and by using size calibration beads to facilitate the analysis of particles with sizes below 1 μM. We applied a combination of Bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and DCVJ (9-(2,2-dicyanovinyl)julolidine) to identify specific characteristics of sub-visible particles., Results: The FACS technology allows the analysis of particles between 0.75 and 10 μm in size, requiring relatively small sample volumes. Protein containing particles can be distinguished from non-protein particles and cross-β-sheet structures contained in protein particles can be identified., Conclusions: The FACS technology provides robust and reproducible results with respect to number, size distribution and specific characteristics of sub-visible particles between 0.75 and 10 μm in size. Our data for number and size distribution of particles is in good agreement with results obtained with the state-of-the-art technology micro-flow imaging.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy., (Copyright © 2015 by The American Association of Immunologists, Inc.)

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.

Recently, lower thrombin generation has been associated with excess bleeding post-cardiopulmonary bypass (CPB). Therefore, treatment to correct thrombin generation is a potentially important aspect of management of bleeding in this group of patients. The objective of the present study was to investigate the effects of fresh frozen plasma (FFP), recombinant factor VIIa (rFVIIa), prothrombin complex concentrate (PCC) and tissue factor pathway inhibitor (TFPI) inhibition on thrombin generation when added ex vivo to the plasma of patients who had undergone cardiac surgery requiring CPB. Patients undergoing elective cardiac surgery were recruited. Blood samples were collected before administration of heparin and 30 min after its reversal. Thrombin generation was measured in the presence and absence of different concentrations of FFP, rFVIIa, PCC and an anti-TFPI antibody. A total of 102 patients were recruited. Thrombin generation following CPB was lower compared with pre-CPB (median endogenous thrombin potential pre-CPB 339 nmol/l per min, post-CPB 155 nmol/l per min, P < 0.0001; median peak thrombin pre-CPB 35 nmol/l, post-CPB 11 nmol/l, P < 0.0001). Coagulation factors and anticoagulants decreased, apart from total TFPI, which increased (55-111 ng/ml, P < 0.0001), and VWF (144-170 IU/dl, P < 0.0001). Thrombin generation was corrected to pre-CPB levels by the equivalent of 15 ml/kg FFP, 45 μg/kg rFVIIa and 25 U/kg of PCC. Inhibition of TFPI resulted in an enhancement of thrombin generation significantly beyond pre-CPB levels. This study shows that FFP, rFVIIa, PCC and inhibition of TFPI correct thrombin generation in the plasma of patients who have undergone surgery requiring CPB. Inhibition of TFPI may be a further potential therapeutic strategy for managing bleeding in this group of patients.

Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

Recently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIII-specific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses., (© 2015 by The American Society of Hematology.)

Fucoidans are complex sulfated polysaccharides extracted from brown algae. Depending on the concentration, they have been shown to stimulate and inhibit blood coagulation in vitro. Promotion of coagulation is mediated by blocking tissue factor pathway inhibitor (TFPI). We screened fucoidan extracts from four brown algae species in vitro with respect to their potential to improve coagulation in bleeding disorders. The fucoidans' pro- and anticoagulant activities were assessed by global hemostatic and standard clotting assays. Results showed that fucoidans improved coagulation parameters. Some fucoidans also activated the contact pathway of coagulation, an undesired property reported for sulfated glycosaminoglycans. Chemical evaluation of fucoidans' complex and variable structure included molecular weight (Mw), polydispersity (polyD), structural heterogeneity, and organic and inorganic impurities. Herewith, we describe a screening strategy that facilitates the identification of crude fucoidan extracts with desired biological and structural properties for improvement of compromised coagulation like in hemophilia., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

Fucoidan is a highly complex sulfated polysaccharide commonly extracted from brown seaweed. In addition to their many biological activities, fucoidans have recently been demonstrated to inhibit or increase coagulation at different concentration ranges. Their structural features, i.e. molecular weight (Mw), Mw distribution, degree of sulfation, monosaccharide composition, and different linkages, are known to affect these activities. Therefore, structure-activity relationship (SAR) analysis of fucoidan is crucial for its potential use as a procoagulant. In this study, Fucus vesiculosus (F.v.) fucoidan was fractionated by charge and size as well as over- and desulfated to different degrees to yield preparations with various structural properties. The fractions' pro- and anticoagulant activities were assessed by calibrated automated thrombography (CAT) and activated partial thromboplastin time(aPTT) assays. Binding to and inhibition of the anticoagulant protein tissue factor pathway inhibitor (TFPI) and the ability to activate coagulation via the contact pathway were also investigated. This paper discusses the impact of charge density, size, and sugar composition on fucoidan's pro- and anticoagulant activities. Fucoidan requires a minimal charge density of 0.5 sulfates per sugar unit and a size of 70 sugar units to demonstrate desired procoagulant activities for improvement of haemostasis in factor VIII/factor IX-deficient plasma.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nM). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nM) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nM). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

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.

Purpose: Human factor VIIa (FVIIa) is commonly used as bypassing therapy to treat bleeding episodes in hemophilia patients with neutralizing antibodies to factors VIII (FVIII) or IX (FIX). There is a need for a suitable animal model to assess the immunogenicity of new FVIIa products during preclinical development. The aim of this study was the design of a novel transgenic mouse model with immune tolerance to human FVIIa., Methods: The model was generated by transgenic expression of human F7 cDNA. FVIIa-specific immune responses after treatment with human FVIIa were assessed by analyzing circulating antibodies, antibody producing plasma cells and CD4(+) T cells., Results: In contrast to wild-type mice, human FVII transgenic mice did not develop antibodies when treated with human FVIIa. The immune tolerance was specific and could be broken by application of human FVIIa together with a strong stimulus of the innate immune system. Break of tolerance was associated with increased numbers of pro-inflammatory FVIIa-specific CD4(+) T cells., Conclusions: The new mouse model is suitable to study the influence of the innate immune system on maintenance and break of immune tolerance against FVIIa and could be used to assess the immunogenicity of new FVIIa products during pre-clinical development.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biologic properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix, and promoting angiogenesis. Recently described fully human anti-MIF antibodies were tested in vitro and in vivo for their ability to influence growth rate and invasion of the human PC3 prostate cancer cell line. In vitro, the selected candidate antibodies BaxG03, BaxB01, and BaxM159 reduced cell growth and viability by inhibiting MIF-induced phosphorylation of the central kinases p44/42 mitogen-activated protein kinase [extracellular signal-regulated kinase-1 and -2 (ERK1/2)] and protein kinase B (AKT). Incubation of cells in the presence of the antibodies also promoted activation of caspase-3/7. The antibodies furthermore inhibited MIF-promoted invasion and chemotaxis as transmigration through Matrigel along a MIF gradient was impaired. In vivo, pharmacokinetic parameters (half-life, volume of distribution, and bioavailability) of the antibodies were determined and a proof-of-concept was obtained in a PC3-xenograft mouse model. Treatment with human anti-MIF antibodies blunted xenograft tumor growth in a dose-dependent manner. We therefore conclude that the anti-MIF antibodies described neutralize some of the key tumor-promoting activities of MIF and thus limit tumor growth in vivo.

Background: Tissue factor pathway inhibitor (TFPI) is a multidomain protein that negatively regulates the coagulation cascade. TFPI inhibits the tissue factor (TF)-activated factor VII-activated FX (FXa) complex during TF-mediated coagulation initiation. The aptamer BAX 499 binds specifically to TFPI and inhibits its function, mediating a procoagulant effect in both in vitro and in vivo models of hemophilia., Objectives: This study sought to identify the regions of TFPI that are critical for BAX 499 binding, and to determine how binding mediates aptamer inhibition of TFPI., Methods and Results: In vitro biochemical methods were used to evaluate the BAX 499 interaction with and inhibition of TFPI. Binding experiments indicated that the full-length TFPI protein is required for tight aptamer binding. Binding-competition experiments implicated the Kunitz 1, Kunitz 3 and C-terminal domains of TFPI in aptamer binding, a finding that is supported by hydrogen-deuterium exchange experiments, and indicated that aptamer and FXa can bind simultaneously to TFPI. In enzymatic assays, BAX 499 inhibited TFPI in a manner that is distinct from domain-specific antibodies, and aptamer inhibitory activity is reduced in the presence of the TFPI cofactor protein S., Conclusions: These studies demonstrate that BAX 499 binds to TFPI via multiple domains of the protein in a manner that is distinct from other TFPI inhibitors, mediating a mechanism of inhibition that does not involve direct competition with FXa. With this unique inhibitory mechanism, BAX 499 provides a useful tool for studying TFPI biology in health and disease., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Tissue factor pathway inhibitor (TFPI) is a multi-Kunitz domain protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa., Objectives: To investigate the role of the three Kunitz domains (KDs) of TFPI in FVIIa inhibition using full-length TFPI (TFPIfl ) and truncated TFPI constructs., Methods: Inhibition of FVIIa with/without relipidated tissue factor (TF) or soluble TF (sTF) by TFPIfl /TFPI constructs was quantified with a FVIIa-specific chromogenic substrate., Results and Conclusions: TFPIfl inhibited TF-FVIIa via a monophasic reaction, which is rather slow at low TFPIfl concentrations (t½  ≈ 5 min at 2 nm TFPI) and has a Ki of 4.6 nm. In the presence of sTF and without TF, TFPIfl was a poor FVIIa inhibitor, with Ki values of 122 nm and 1118 nm, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPIfl . TFPI constructs without the KD3-c-terminus (TFPI1-150 and KD1-KD2) were 7-10-fold less effective than TFPIfl in inhibiting TF-FVIIa and sTF-FVIIa, indicating that the KD3-C-terminus significantly contributes to direct inhibition of FVIIa by TFPI. Compared with KD1-KD2, KD1 was a poor TF-FVIIa inhibitor (Ki =434 nm), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF-FVIIa inhibition by TFPIfl (Ki =0.7 nm). In the presence of FXa, a tight quaternary TF-FVIIa-TFPI-FXa complex is formed with TFPIfl , TFPI1-150 and KD1-KD2, with Ki values of < 0.15 nm, 0.5 nm and 0.8 nm, respectively, indicating the KD3-C-terminus is not a prerequisite for quaternary complex formation. Phospholipids and the Gla-domain of FXa are required for quaternary complex formation., (© 2013 International Society on Thrombosis and Haemostasis.)

Tissue factor pathway inhibitor (TFPI) is a major inhibitor of coagulation. We therefore hypothesised that high plasmatic TFPI levels are associated with impaired ex vivo clotting in a model of acquired haemophilia. Blood samples were collected in a prospective clinical study from 30 healthy volunteers. Coagulation in normal or factor VIII (FVIII)-inhibited human blood or plasma was measured by the calibrated automated thrombogram (CAT) and rotational thromboelastometry (ROTEM). Both methods are global haemostatic assays that provide insight into the whole coagulation process. Monoclonal mouse antibodies raised against either the C-terminus or the Kunitz domain 2 of TFPI were used to determine full-length (fl-) and total TFPI by an enzyme-immunoassay. Clotting times and parameters of thrombin generation correlated with TFPI levels. Subjects with low fl-TFPI levels had significantly shorter clotting times and a higher endogenous thrombin potential (ETP) compared to those with high fl-TFPI levels (p≤0.005 for all). An even stronger effect was seen in FVIII-inhibited blood/plasma: ROTEM clotting time was 26% shorter (p=0.01) and the ETP assessed by CAT was >2-fold higher in subjects with low fl-TFPI levels (p≤0.0001). Plasmatic TFPI is a major determinant of coagulation in global haemostatic tests particularly when FVIII is missing. Thus, inhibition of TFPI might be a promising novel treatment approach, especially in haemophilia patients with FVIII inhibitors.

Neutralizing antibodies against factor VIII (FVIII) remain the major complication in the replacement therapy of hemophilia A patients. To better understand the evolution of these antibodies it is important to generate comprehensive datasets which include both neutralizing and nonneutralizing antibodies, their isotypes, and IgG subclasses. We developed sensitive ELISAs to analyze FVIII-binding antibodies in different cohorts of hemophilia A patients and in healthy individuals. Our data reveal the prevalence of FVIII-binding antibodies among healthy individuals (n = 600) to be as high as 19%, with a prevalence of antibody titers > or =1:80 of 2%. The prevalence of FVIII-binding antibodies was 34% (5% for titers > or =1:80) in patients without FVIII inhibitors (n = 77), 39% (4% for titers > 1:80) in patients after successful immune tolerance induction therapy (n = 23), and 100% (n = 20, all titers > or =1:80) in patients with FVIII inhibitors. We found significant differences for IgG subclasses of FVIII-binding antibodies between the different study cohorts. IgG4 and IgG1 were the most abundant IgG subclasses in patients with FVIII inhibitors. Strikingly, IgG4 was completely absent in patients without FVIII inhibitors and in healthy subjects. These findings point toward a distinct immune regulatory pathway responsible for the development of FVIII-specific IgG4 associated with FVIII inhibitors.

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.

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.

PEGylation is the most successful approach, to date, to prolong the in vivo survival of recombinant proteins. The conjugation of the polymer to glycoproteins results in challenging analysis, and furthermore, requires a wide variety of analytical tools for the determination of the extent of PEGylation. Herein, we present microchip capillary gel electrophoresis (MCGE) with a non-commercial high-molecular-weight protein assay for the analysis of the PEGylation degree with a focus on multiple PEGylation. To show the potential of the modified MCGE system, high-mass PEGylated glycoproteins (e.g. coagulation factor VIII) were analyzed. For the von Willebrand factor, the influence of glycans and the hydrodynamic radius on migration time and molecular weight determination is shown. The modified MCGE assay system is a powerful tool for the rapid assessment of the degree of PEGylation, demonstrating conjugate quality or reaction control of PEGylated proteins. This is the main advantage over time-consuming conventional SDS-PAGE. Furthermore, electrophoretic separation, staining, destaining, and fluorescence detection in one step combined with automated data analysis show that the MCGE system is a promising technique for high-throughput monitoring. The MCGE system can be used for rapid structure confirmation ("MCGE fingerprinting") of multiply PEGylated glycoproteins beyond the 230 kDa molecular mass range., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a β-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this β-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2011 International Society on Thrombosis and Haemostasis.)

Background: von Willebrand factor (VWF) released from endothelial cells is rich in ultra-large (UL) multimers that are intrinsically active in binding platelets, whereas plasma-type VWF multimers require shear stress to be activated. This functional difference may be attributed to thiols exposed on the surface of plasma-type VWF multimers, but not on ULVWF multimers. Shear stress induces the exposed thiols to form disulfide bonds between laterally apposed plasma-type VWF multimers, leading to enhanced VWF binding to platelets., Objectives: We tested a hypothesis that ADAMTS-13 has a disulfide bond reducing activity that regulates shear-induced thiol-disulfide exchange of VWF., Methods: Thiol blocking agents and active thiol bead capturing were used to identify and locate this activity, along with truncated ADAMTS-13 mutants., Results: ADAMTS-13 contains a disulfide bond reducing activity that primarily targets disulfide bonds in plasma-type VWF multimers induced by high shear stress or formed with thiol beads, but not disulfide bonds in native multimeric structures. Cysteine thiols targeted by this activity are in the VWF C-domain and are known to participate in shear-induced thiol-disulfide exchange. ADAMTS-13 contains cysteine thiols that remain exposed after being subjected to hydrodynamic forces. Blocking these active thiols eliminates this reducing activity and moderately decreases ADAMTS-13 activity in cleaving ULVWF strings anchored to endothelial cells under flow conditions, but not under static conditions. This activity is located in this C-terminal region of ADAMTS-13., Conclusions: This novel disulfide-bond-reducing activity of ADAMTS-13 may prevent covalent lateral association and increased platelet adherence of plasma-type VWF multimers induced by high fluid shear stress., (© 2010 International Society on Thrombosis and Haemostasis.)

Stroke is a leading cause of death and disability. The only therapy available is recombinant tissue plasminogen activator, but side effects limit its use. Platelets play a crucial role during stroke, and the inflammatory reaction promotes neurodegeneration. von Willebrand factor (VWF), an adhesion molecule for platelets, is elevated in patients with acute stroke. The activity of VWF is modulated by ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) that cleaves VWF to smaller less-active forms. We recently documented that ADAMTS13 negatively regulates both thrombosis and inflammation. We report that deficiency or reduction of VWF reduces infarct volume up to 2-fold after focal cerebral ischemia in mice, thus showing the importance of VWF in stroke injury. In contrast, ADAMTS13 deficiency results in larger infarctions, but only in mice that have VWF. Importantly, infusion of a high dose of recombinant human ADAMTS13 into a wild-type mouse immediately before reperfusion reduces infarct volume and improves functional outcome without producing cerebral hemorrhage. Furthermore, recombinant ADAMTS13 did not enhance bleeding in a hemorrhagic stroke model. Our findings show the importance of VWF in regulating infarction and suggest that recombinant ADAMTS13 could be considered as a new therapeutic agent for prevention and/or treatment of stroke.

Background: Activated coagulation factor IX (FIXa) has low catalytic activity towards its physiologic substrate FX when activated FVIII (FVIIIa) is absent. One reason for this is that the FIX surface loop 99 stabilizes FIXa in a conformation that limits access of FX to the active site., Objectives: To investigate the effect of mutations in loop 99 and in the active site on FIXa activity with and without FVIIIa., Methods: Five full-length FIX mutants with amino acid exchanges in the catalytic domain of FIX were constructed and characterized by measuring their activity in FX activation in model systems and in plasma., Results and Conclusions: The mutants showed no or marginally improved catalytic properties in FX activation by the intrinsic tenase complex (FIXa-FVIIIa-Ca(2+)-phospholipid). The combination of mutations Y94F and K98T hardly affected FX activation in the presence of FVIIIa, but yielded a FIX molecule that, in FIX-depleted plasma, had approximately 2.5-fold higher clotting activity and approximately 3.5-fold higher activity in a thrombin generation assay than plasma-derived FIX (pdFIX). Two FIXa mutants had considerably increased activities towards FX in the absence of FVIIIa. FIXa-Y94F/K98T/Y177F/I213V/E219G (FIXa-L) and FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIXa-M) activated FX with catalytic efficiencies (k(cat)/K(m)) that, as compared with activated pdFIX, were increased 17-fold and six-fold, respectively. However, in plasma, their zymogen forms performed similarly to pdFIX. This indicates that the introduced mutations not only affected the activity of FIXa but may have also influenced the lifetime of the activated mutant molecules in plasma by modifying their activation and/or inhibition rates.

Background: ADAMTS13-neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP)., Objective: To analyze the IgG subclass distribution of anti-ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis., Methodology: An enzyme-linked immunosorbent assay-based method was used to quantify the relative amounts of IgG subclasses of anti-ADAMTS13 antibodies in acquired TTP plasma., Results: IgG(4) (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG(1) (52%), IgG(2) (50%), and IgG(3) (33%). IgG(4) was found either alone (17/52) or with other IgG subclasses (35/52). IgG(4) was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG(4) and IgG(1) antibodies (P < 0.01). Patients with high IgG(4) levels and undetectable IgG(1) are more prone to relapse than patients with low IgG(4) levels and detectable IgG(1)., Conclusions: All IgG subclasses of anti-ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG(4), followed by IgG(1), antibodies dominating the anti-ADAMTS13 immune response. Levels of IgG(4) could be useful for the identification of patients at risk of disease recurrence.

Background: von Willebrand factor (VWF) is composed of a series of multimers, the sizes of which are regulated by the plasma metalloprotease ADAMTS13., Objective: Proteolysis of human recombinant VWF (rVWF) by ADAMTS13 present in the plasma of different species typically used as preclinical animal models was investigated to evaluate the efficacy and safety of rVWF., Methods: Degradation of rVWF was studied in vitro under moderate denaturing conditions and was monitored by multimer analysis, residual collagen binding, and immunoblot analysis. In vivo cleavage was determined by administration of rVWF to cynomolgus monkeys, rabbits and VWF-deficient mice and subsequent analysis of plasma samples by immunoblot. Plasma ADAMTS13 levels were determined with a synthetic human VWF peptide (FRETS-VWF73)., Results: From the animals tested, only rabbit plasma was as efficient as human plasma in proteolysing rVWF in vitro. Mouse plasma virtually failed to cleave rVWF. Administration of human rVWF resulted in ADAMTS13-specific cleavage products in rabbits and, to a lesser extent, in cynomolgus monkeys at various doses of rVWF. Virtually no cleavage occurred in mice. ADAMTS13 activity levels in rabbit and monkey plasma were similar to those in human plasma and were not significantly altered upon infusion of rVWF up to very high doses, indicating that rVWF did not lead to an exhaustion of endogenous ADAMTS13 in both species., Conclusions: The differences in susceptibility to cleavage of rVWF by different species need to be considered when interpreting the physiology of human rVWF from results of tests in animal models.

ADAMTS13, a metalloprotease primarily synthesized in liver and endothelial cells, cleaves von Willebrand factor (VWF) at the central A2 domain, thereby reducing the sizes of circulating VWF multimers. Genetic or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). To date, plasma infusion or exchange is the only proven effective therapy for TTP. In search for a better therapy, an autologous transplantation of hematopoietic progenitor cells transduced ex vivo with a self-inactivating lentiviral vector encoding a full-length murine Adamts13 and an enhanced green fluorescent protein (GFP) reporter gene was performed in Adamts13(-/-) mice after irradiation. All recipient mice showed detectable ADAMTS13 antigen and proteolytic activity in plasma despite only low levels of bone marrow chimerism. The levels of plasma ADAMTS13 were sufficient to eliminate the ultralarge VWF multimers and offered systemic protection against ferric chloride-induced arterial thrombosis. The data suggest that hematopoietic progenitor cells can be genetically modified ex vivo and transplanted in an autologous model to provide adequate levels of functional ADAMTS13 metalloprotease. This success may provide the basis for development of a novel therapeutic strategy to cure hereditary TTP in humans.

Background: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies., Objective: We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies., Methods and Results: Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies., Conclusions: These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.

von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprotease with thrombospondin type I repeats-13 (ADAMTS13) activity is decreased in both acute and chronic inflammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and inflammation. Using intravital microscopy, we show that ADAMTS13 deficiency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in inflamed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13(-/-) mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13(-/-) compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13(-/-) but not in WT mice. Furthermore, in inflammatory models, ADAMTS13 deficiency resulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our findings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during inflammation.

Background: Factor VIIIa (FVIIIa) binds to activated FIX and enhances the activation of FX by several orders of magnitude. Deficiency of FVIII causes the bleeding disorder hemophilia A and is treated by i.v. infusion of FVIII concentrates., Objectives: To explore whether or not FVIII activity can be supplied by alternative molecules, e.g. molecules with FIXa-binding activity., Methods: Conventional hybdridoma technology was used to discover antibodies exhibiting FVIII-like activity., Results: We identified a series of antibodies specific for human FIX that mimicked the stimulatory effect of FVIIIa on FIXa. Upon binding to human FIXa, these antibodies enhanced the protease activity of FIXa towards its natural substrate FX about tenfold. A similar enhancement was also achieved with 5 pm FVIIIa (i.e. 16 mU mL(-1) or 1.6% activated FVIII). Procoagulant activity of these anti-FIXa antibodies was observed in model systems containing purified proteins as well as in plasma., Conclusion: Our findings show that FVIII can, at least partially, be replaced by an unrelated molecule. Procoagulant antibodies might potentially aid the development of an FVIII substitute for hemophilia A treatment.

To study both the pathophysiologic and the prognostic value of ADAMTS13 in thrombotic microangiopathies (TMAs), we enrolled a cohort of 35 adult patients combining a first acute episode of TMA, an undetectable (below 5%) ADAMTS13 activity in plasma, and no clinical background such as sepsis, cancer, HIV, and transplantation. All patients were treated by steroids and plasma exchange, and an 18-month follow-up was scheduled. Remission was obtained in 32 patients (91.4%), and 3 patients died (8.6%) after the first attack. At presentation, ADAMTS13 antigen was decreased in 32 patients (91.4%), an ADAMTS13 inhibitor was detectable in 31 patients (89%), and an anti-ADAMTS13 IgG/IgM/IgA was present in 33 patients (94%). The 3 decedent patients were characterized by the association of several anti-ADAMTS13 Ig isotypes, including very high IgA titers, while mortality was independent of the ADAMTS13 inhibitor titer. In survivors, ADAMTS13 activity in remission increased to levels above 15% in 19 patients (59%) but remained undetectable in 13 patients (41%). Six patients relapsed either once or twice (19%) during the follow-up. High levels of inhibitory anti-ADAMTS13 IgG at presentation were associated with the persistence of an undetectable ADAMTS13 activity in remission, the latter being predictive for relapses within an 18-month delay.