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Deficiency or dysfunction of von Willebrand factor (VWF) is associated with a bleeding disorder known as von Willebrand disease (VWD). The clinical manifestations of VWD are heterogeneous, and are in part dictated by the structural or functional defects of VWF. The tools to control bleeding in VWD are dominated by VWF concentrates, desmopressin and antifibrinolytic therapy. In view of these treatments being considered as effective, it is surprising that quality‐of‐life studies consistently demonstrate a significant mental and physical burden in VWD patients, particularly in women. Apparently, the current weaponry to support the management of VWD is insufficient to fully address the needs of the patients. It is important therefore to continue to search for innovative treatment options which could better serve the VWD patients. In this short review, two of such options are discussed in more detail: emicizumab to correct for the deficiency of factor VIII (FVIII), and the pegylated aptamer BT200 to increase endogenous levels of the VWF/FVIII complex. [ABSTRACT FROM AUTHOR]

Deficiency or dysfunction of von Willebrand factor (VWF) is associated with a bleeding disorder known as von Willebrand disease (VWD). The clinical manifestations of VWD are heterogeneous, and are in part dictated by the structural or functional defects of VWF. The tools to control bleeding in VWD are dominated by VWF concentrates, desmopressin and antifibrinolytic therapy. In view of these treatments being considered as effective, it is surprising that quality‐of‐life studies consistently demonstrate a significant mental and physical burden in VWD patients, particularly in women. Apparently, the current weaponry to support the management of VWD is insufficient to fully address the needs of the patients. It is important therefore to continue to search for innovative treatment options which could better serve the VWD patients. In this short review, two of such options are discussed in more detail: emicizumab to correct for the deficiency of factor VIII (FVIII), and the pegylated aptamer BT200 to increase endogenous levels of the VWF/FVIII complex. [ABSTRACT FROM AUTHOR]

Aims Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. Methods and results VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvβ3 signalling abolished proliferation. However, VWF did not bind to αvβ3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvβ3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. Conclusion VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvβ3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation. [ABSTRACT FROM AUTHOR]

We have developed and tested a novel microfluidic device for blood oxygenation, which exhibits a large surface area of gas exchange and can support long-term sustainable endothelialization of blood microcapillaries, enhancing its hemocompatibility for clinical applications. The architecture of the parallel stacking of the trilayers is based on a central injection for blood and a lateral injection/output for gas which allows significant reduction in shear stress, promoting sustainable endothelialization since cells can be maintained viable for up to 2 weeks after initial seeding in the blood microchannel network. The circular design of curved blood capillaries allows covering a maximal surface area at 4 inch wafer scale, producing high oxygen uptake and carbon dioxide release in each single unit. Since the conventional bonding process based on oxygen plasma cannot be used for surface areas larger than several cm2, a new "wet bonding" process based on soft microprinting has been developed and patented. Using this new protocol, each 4 inch trilayer unit can be sealed without a collapsed membrane even at reduced 15 μm thickness and can support a high blood flow rate. The height of the blood channels has been optimized to reduce pressure drop and enhance gas exchange at a high volumetric blood flow rate up to 15 ml min−1. The simplicity of connecting different units in the stacked architecture is demonstrated for 3- or 5-unit stacked devices that exhibit remarkable performance with low primary volume, high oxygen uptake and carbon dioxide release and high flow rate of up to 80 ml min−1. [ABSTRACT FROM AUTHOR]

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a nextgeneration APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs. [ABSTRACT FROM AUTHOR]

Résumé: Le facteur VIII (FVIII) est une protéine de la cascade de coagulation qui exerce une activité de cofacteur non enzymatique vis-à-vis du facteur IXa (FIXa), l'enzyme qui va promouvoir l'activation du facteur X (FX). Un déficit fonctionnel en FVIII conduit à l'hémophilie A, une pathologie hémorragique grave dont la prévalence se situe autour de 1-2 cas pour 10 000 naissances de garçons. La prise en charge des complications hémorragiques associées à l'hémophilie A s'effectue par une thérapie de substitution visant à remplacer le FVIII manquant, préférentiellement par prophylaxie. En raison des limites inhérentes à cette approche (avec la nécessité d'infusions intraveineuses fréquentes et l'apparition d'anticorps inhibiteurs contre le FVIII thérapeutique), de nouvelles thérapies ont récemment été développées. L'emicizumab, un anticorps bispécifique, en est un représentant. Cet anticorps a pour fonction de mimer l'activité cofacteur du FVIII en permettant un rapprochement dans l'espace du FIXa et du FX. Dans cette revue, nous discuterons des aspects fonctionnels et cliniques de l'emicizumab, nous comparerons son mode d'action à celui du FVIII et les implications pour le suivi biologique des patients. De plus, nous présenterons les données cliniques obtenues dans les essais pivots HAVEN qui ont montré un bénéfice significatif de l'emicizumab avec une réduction importante des saignements spontanés chez les patients traités. Finalement, nous discuterons des effets secondaires associés à l'utilisation de l'emicizumab et nous conclurons sur son utilisation potentielle au-delà de l'hémophilie A. Coagulation factor VIII (FVIII) functions as a non-enzymatic cofactor for the enzyme factor IXa (FIXa) in the activation of factor X (FX). The functional deficiency of FVIII is associated with a severe bleeding disorder known as hemophilia A, affecting 1-2 per 10,000 male births. Bleeding complications have long been managed via FVIII substitution therapy, preferably on a prophylactic basis. Due to limitations of this approach (i.e., the need for frequent intravenous infusions, development of neutralizing antibodies), novel treatment options for hemophilia A have been evaluated, including the use of the bispecific antibody emicizumab. Emicizumab is designed to mirror FVIII cofactor function by promoting the spatial approximation of FIXa and FX. In this review, we will discuss functional and clinical aspects of emicizumab. We will compare its mode of action to that of FVIII and consider the implications for laboratory monitoring of this antibody. In addition, we highlight the clinical data obtained in the pivotal HAVEN-trials, which have shown a clear clinical benefit by strongly reducing the occurrence of spontaneous bleeding episodes. Finally, we discuss some of the adverse effects that have been reported and also the potential use of emicizumab beyond patients with severe hemophilia A. [ABSTRACT FROM AUTHOR]

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders. A platform for systemic therapeutic transgene expression independent of patient mutations needs a safe and highly transcribed locus. Here the authors ex vivo edit HPSCs using CRISPR-Cas9 to integrate transgenes under the α-globin promoter to achieve erythroid specific expression. [ABSTRACT FROM AUTHOR]

Novel therapies for hemophilia, including non‐factor replacement and in vivo gene therapy, are showing promising results in the clinic, including for patients having a history of inhibitor development. Here, we propose a novel therapeutic approach for hemophilia based on llama‐derived single‐domain antibody fragments (sdAbs) able to restore hemostasis by inhibiting the antithrombin (AT) anticoagulant pathway. We demonstrated that sdAbs engineered in multivalent conformations were able to block efficiently AT activity in vitro, restoring the thrombin generation potential in FVIII‐deficient plasma. When delivered as a protein to hemophilia A mice, a selected bi‐paratopic sdAb significantly reduced the blood loss in a model of acute bleeding injury. We then packaged this sdAb in a hepatotropic AAV8 vector and tested its safety and efficacy profile in hemophilic mouse models. We show that the long‐term expression of the bi‐paratopic sdAb in the liver is safe and poorly immunogenic, and results in sustained correction of the bleeding phenotype in hemophilia A and B mice, even in the presence of inhibitory antibodies to the therapeutic clotting factor. Synopsis: This study presents a novel therapeutic approach for hemophilia A and B, with and without inhibitors. By using single‐domain antibodies targeting the anticoagulant antithrombin, a sustained correction of the in vivo bleeding phenotype was achieved following protein or AAV8‐based gene therapy. Bi‐paratopic single domain antibodies (sdAbs) have been generated that neutralized the anticoagulant protein antithrombin.Neutralization of antithrombin normalized thrombin generation in hemophilic plasma, irrespective of the presence of inhibitory antibodies.Liver expression of a bivalent sdAb restored clotting in hemophilia A and in hemophilia B mice, even in the presence of anti‐FIX antibodies. [ABSTRACT FROM AUTHOR]

Background: Angiodysplasia is a vascular malformation associated with gastrointestinal bleeding, generally observed in the elderly. This condition is unexpectedly more frequent in patients with von Willebrand disease (VWD)-type 2A having low levels of VWF high-molecular-weight-multimers (HMWMs) and increased VWF-degradation fragments (1). Aim: To develop an innovative murine model of VWD-type 2A and study the role of degraded-VWF in vascular processes. Methods: Mice expressing human (h) VWF, carrying the type 2A (p.R1597W) variant or wild-type (as control) and human GPIba, have been generated (hVWF(p.R1597W) + / +/hGP1BA + / + and hVWF + / +/hGP1BA + / +). Haemoglobin (Hb), VWF:Ag, propeptide, multimer pattern and factor VIII activity were analyzed. Tail-clip and tail-vein-transection (TVT) bleeding assays were assessed. Results: Control hVWF + / +/hGP1BA + / + -mice expressed 15 ± 4% VWF:Ag, 44 ± 8% FVIII activity and normal VWF multimers. hVWF(p. R1597W) + / +/hGP1BA + / + -mice are viable and do not display spontaneous bleeding manifestations. These mice expressed 3 ± 1% VWF:Ag and 7 ± 1% FVIII activity combined with an abnormal multimer pattern, with only low multimers and few degradation bands visible. Despite the relatively low VWF:Ag levels, hVWF + / +/hGP1BA + / + -mice displayed normal haemostatic responses in both the severe- (tail-clip) and milder- (TVT) bleeding assays. In contrast, hVWF(p.R1597W) + / + / hGP1BA + / + -mice had a severe bleeding phenotype. Interestingly, in the TVT model, although the amount of blood shed was consistent with severe bleeding, 57% of type 2A mice were capable of forming an occlusive, although unstable clot within 15 min of the injury, differing from the bleeding profile of VWF-deficient mice. Conclusion: We developed a unique humanized mouse models for VWD-type 2A. Experiments are ongoing to study the vasculature of these mice. [ABSTRACT FROM AUTHOR]

Background & Aims In addition to their function in thrombosis and haemostasis, platelets play an important role in the stimulation of liver regeneration. It has been suggested that platelets deliver mitogenic cargo to the regenerating liver, and accumulation of platelets in the regenerating liver has been demonstrated. We studied kinetics of platelet influx in the regenerating liver and investigated the signal that initiates platelet influx. Methods We visualized platelets in the liver remnant after partial hepatectomy in mice using intravital microscopy and assessed liver regeneration by examination of liver/body weight ratio and the number of proliferating hepatocytes examined by immunohistochemistry. Results We demonstrated rapid but transient platelet influx into the liver remnant after a partial liver resection. Liver regeneration in thrombocytopenic mice was substantially impaired as evidenced by a reduced liver-to-body weight ratio and decreased numbers of proliferating hepatocytes at day 3 compared to mice with normal platelet counts. In contrast, liver regeneration was only mildly impaired when thrombocytopaenia was induced 2 hours after partial liver resection. Platelet influx into the liver remnant was virtually absent in the presence of an antibody to von Willebrand factor ( VWF) suggesting that VWF release from liver sinusoidal endothelial cells mediates platelet influx. Additionally, liver regeneration in mice deficient in VWF was markedly impaired. Conclusions A rapid but transient VWF-dependent platelet influx into the liver remnant drives platelet-mediated liver regeneration. [ABSTRACT FROM AUTHOR]

The development of therapeutic strategies for the treatment of hemophilia A has been a long-lasting issue since the initial identification of the disease in the early 19th century and has involved several major steps to reach contemporary treatment. The current replacement therapy involving intravenous infusion of plasmatic or recombinant factor VIII (FVIII) has been efficient to control bleeding episodes of patients with hemophilia A. Nonetheless, replacement therapy requires frequent infusions to maintain values of FVIII above the threshold of efficacy and represents a serious burden that significantly impacts the patient's quality of life. The recent half-life extension technologies have permitted the development of a first generation of longacting FVIII variants with improved circulating survival. These molecules, which are now in clinical phase III studies or have already received food and drug administration (FDA) approval for therapeutic use will allow a reduction in the frequency of infusion and lighten the treatment of hemophilia A. However, the half-life extension of FVIII was not as efficient as initially expected, particularly with regard to the results obtained with other therapeutic molecules. To overcomethese limitations inherent to the nature of FVIII biology, several bypassing therapies independent of the FVIII molecule are currently in development. These emerging approaches exploit the modulation of the coagulation/anticoagulation balance taking place in the hemostatic process to compensate for the FVIII deficiency. In this review, we recount the history of hemophilia A treatment up to the recent emerging therapies, with particular focus on infusion-based strategies. [ABSTRACT FROM AUTHOR]

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation. [ABSTRACT FROM AUTHOR]

Von Willebrand factor (VWF) is a large multimeric protein, the function of which has been demonstrated to be pivotal to the haemostatic system. Indeed, quantitative and/or qualitative abnormalities of VWF are associated with the bleeding disorder Von Willebrand disease (VWD). Moreover, increased plasma concentrations of VWF have been linked to an increased risk for thrombotic complications. In the previous decades, many studies have contributed to our understanding of how VWF is connected to the haemostatic system, particularly with regard to structure-function relationships. Interactive sites for important ligands of VWF (such as factor VIII, collagen, glycoprotein Ibα, integrin αIIbβ3 and protease ADAMTS13) have been identified, and mutagenesis studies have confirmed the physiological relevance of the interactions between VWF and these ligands. However, we have also become aware that VWF has a more versatile character than previously thought, given its potential role in various non-hemostatic processes, like intimal thickening, tumor cell apoptosis and inflammatory processes. In the presence review, a summary of our knowledge on VWF structure-function relationships is provided in the context of the "classical" haemostatic task of VWF and in perspective of pathological processes beyond haemostasis. [ABSTRACT FROM AUTHOR]

Up until recently, von Willebrand Factor (VWF) structure-function relationships have only been studied through in vitro approaches. A powerful technique known as hydrodynamic gene transfer, which allows transient expression of a transgene by mouse hepatocytes, has led to an important shift in VWF research. Indeed this approach has now enabled us to transiently express a number of VWF mutants in VWF- deficient mice in order to test the relative importance of specific residues in different aspects of VWF biology and functions in an in vivo setting. As a result, mice reproducing various types of von Willebrand disease have been generated, models that will be useful to test new therapies. This approach also allowed a more precise identification of the importance of VWF interaction with subendothelial collagens and with platelets receptors in hemostasis and thrombosis. The recent advances gathered from these studies as well as the pros and cons of the technique will be reviewed here. [ABSTRACT FROM AUTHOR]

Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s-1 through four independent channels with a height of 50 mm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (LagT), the rate of platelet accumulation (VPLT), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to VPLT and SC at all wall shear rates. A longer LagT for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s-1 and 300 s-1. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay. [ABSTRACT FROM AUTHOR]

Background: Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies. Methods: Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF), eptifibatide (Gp IIb/IIIa antagonist), and anti-Mac-1 Ab (anti-αMβ2 integrin Ab) were tested for the ability to inhibit phagocytosis. Results: None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+) all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01). Conclusions: Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver endothelial cells efficiently phagocytosed baboon platelets. Blocking vWF and integrin adhesion pathways prevented both aggregation and phagocytosis. [ABSTRACT FROM AUTHOR]

Factor X (FX), a plasma glycoprotein playing a central role in coagulation has a long circulatory half-life compared to closely related coagulation factors. The activation peptide of FX has been shown to influence its clearance with two N-glycans as key determinants of FX's relatively long survival. To decipher FX clearance mechanism, organ biodistribution and cellular interactions of human plasma FX (pd-FX), recombinant FX (rFX), N-deglycosylated FX (N-degly-FX) and recombinant FX mutated at both N-glycosylation sites (rFXN181A–N191A) were evaluated. Biodistribution analysis of 125I-labelled FX proteins after administration to mice revealed liver as major target organ for all FX variants. Liver tissue sections analysis showed an interaction of pd-FX and N-degly-FX to different cell types. These findings were confirmed in cell binding studies revealing that FX and FX without N-glycans interact with macrophages and hepatocytes, respectively. N-degly-FX appeared to be degraded in hepatocytes while interestingly pd-FX was not by macrophages. Furthermore, the chemical inactivation of macrophages by gadolinium chloride resulted in a significant decrease of circulating pd-FX into mice and not of N-degly-FX. Altogether our data lead to the conclusion that FX interaction with macrophages through its N-glycans protects it from a rapid clearance explaining its relatively long circulatory half-life. [ABSTRACT FROM AUTHOR]

Introduction: The percentage of time within the target INR range 2.0 to 3.0 (TTR) in patients treated with vitamin K antagonists varies considerably among efficacy-studies of novel anticoagulants. In order to properly asses the quality of anticoagulant control in upcoming cost-effectiveness studies and real life registries this systematic review reports a benchmark of TTR for different treatment durations in patients with venous thromboembolism and discusses ways to calculate TTR. Methods: Medline and Embase were searched for studies published between January 1990 and May 2012. Randomized controlled trials and cohort studies reporting the TTR in patients with objectively confirmed venous thromboembolism treated with vitamin K antagonists (VKA) were eligible. Duplicate reports, studies only reporting INR during initial treatment or with VKA treatment less than 3 months were excluded. Three authors assessed trials for inclusion and extracted data independently. Discrepancies were resolved by discussion between the reviewers. A meta-analysis was performed by calculating a weighted mean, based on the number of participants in each included study, for each time-period in which the TTR was measured since the confirmation of the diagnosis of VTE. Results: Forty studies were included (26064 patients). The weighted means of TTR were 54.0% in the first month since the start of treatment, 55.6% in months 1 to 3, 60.0% in months 2 to 3, 60.0% in the months1 to 6+ and 75.2% in months 4 to 12+. Five studies reported TTR in classes. The INR in these studies was ≥67% of time in therapeutic range in 72.0% of the patients. Conclusion: Reported quality of VKA treatment is highly dependent on the time-period since the start of treatment, with TTR ranging from approximately 56% in studies including the 1st month to 75% in studies excluding the first 3 months. [ABSTRACT FROM AUTHOR]

Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulone- phritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR TFLLR-NH2, and SLIGKV- NH2,, respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways. [ABSTRACT FROM AUTHOR]

Background: The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2. Methodology: In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor- I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. Conclusions: Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens. [ABSTRACT FROM AUTHOR]

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-OGly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some Oglycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments. [ABSTRACT FROM AUTHOR]

Hemostasis and thrombosis represent two sides of the same coin. Hemostasis maintains blood fluidity in the vascular system while allowing for rapid thrombus formation to prevent excessive hemorrhage after blood vessel injury. Thrombosis is a pathologic extension of the normal hemostatic mechanism, occurring when unwanted clot formation develops in certain pathological situations. The molecular mechanisms underlying both phenomena are fundamentally identical. One of the key players in both processes is the plasma glycoprotein von Willebrand factor, which perfectly illustrates this duality between hemostatic and thrombotic mechanisms. The purpose of this review is to discuss novel findings on the role of von Willebrand factor at this interface, and how some of these findings may help develop new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF. [ABSTRACT FROM AUTHOR]

LAIR-1 (Leukocyte Associated Ig-like Receptor -1) is a collagen receptor that functions as an inhibitory receptor on immune cells. It has a soluble family member, LAIR-2, that also binds collagen and can interfere with LAIR-1/collagen interactions. Collagen is a main initiator for platelet adhesion and aggregation. Here, we explored the potential of soluble LAIR proteins to inhibit thrombus formation in vitro. LAIR-2/Fc but not LAIR-1/Fc inhibited collagen-induced platelet aggregation. In addition, LAIR-2/Fc also interfered with platelet adhesion to collagen at low shear rate (300 s-1; IC50=18 μg/ml) and high shear rate (1500 s21; IC50=30 μg/ml). Additional experiments revealed that LAIR-2/Fc leaves interactions between collagen and α2β1 unaffected, but efficiently prevents binding of collagen to Glycoprotein VI and von Willebrand factor. Thus, LAIR-2/Fc has the capacity to interfere with platelet-collagen interactions mediated by Glycoprotein VI and the VWF/Glycoprotein Ib axis. [ABSTRACT FROM AUTHOR]

Background: The urgent medical need for innovative approaches to control influenza is emphasized by the widespread resistance of circulating subtype H1N1 viruses to the leading antiviral drug oseltamivir, the pandemic threat posed by the occurrences of human infections with highly pathogenic avian H5N1 viruses, and indeed the evolving swine-origin H1N1 influenza pandemic. A recently discovered class of human monoclonal antibodies with the ability to neutralize a broad spectrum of influenza viruses (including H1, H2, H5, H6 and H9 subtypes) has the potential to prevent and treat influenza in humans. Here we report the latest efficacy data for a representative antibody of this novel class. Methodology/Principal Findings: We evaluated the prophylactic and therapeutic efficacy of the human monoclonal antibody CR6261 against lethal challenge with the highly pathogenic avian H5N1 virus in ferrets, the optimal model of human influenza infection. Survival rates, clinically relevant disease signs such as changes in body weight and temperature, virus replication in lungs and upper respiratory tract, as well as macro- and microscopic pathology were investigated. Prophylactic administration of 30 and 10 mg/kg CR6261 prior to viral challenge completely prevented mortality, weight loss and reduced the amount of infectious virus in the lungs by more than 99.9%, abolished shedding of virus in pharyngeal secretions and largely prevented H5N1-induced lung pathology. When administered therapeutically 1 day after challenge, 30 mg/kg CR6261 prevented death in all animals and blunted disease, as evidenced by decreased weight loss and temperature rise, reduced lung viral loads and shedding, and less lung damage. Conclusions/Significance: These data demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant animal model of influenza and justify clinical development of this approach as intervention for both seasonal and pandemic influenza. [ABSTRACT FROM AUTHOR]

Background: Von Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life. Methodology: Standardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15-44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated. Principal Findings: FVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5±2.6 h versus 14.3±3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%). Conclusion: Our approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se. [ABSTRACT FROM AUTHOR]

Proteolytic processing of von Willebrand factor (VWF) by ADAMTS13 metalloproteinase is crucial for normal hemostasis. In vitro, cleavage of VWF by ADAMTS13 is slow even at high shear stress and is typically studied in the presence of denaturants. We now show that, under shear stress and at physiological pH and ionic strength, coagulation factor VIII (FVIII) accelerates, by a factor of ≈10, the rate of specific cleavage at the Tyr1605-Met1606 bond in VWF. Multimer analysis reveals that FVIII preferentially accelerates the cleavage of high-molecular-weight multimers. This rate enhancement is not observed with VWF predenatured with 1.5 M guanidine. The ability of FVIII to enhance VWF cleavage by ADAMTS13 is rapidly lost after pretreatment of FVIII with thrombin. A FVIII derivative lacking most of the B domain behaves equivalently to full-length FVIII. In contrast, a derivative lacking both the B domain and the acidic region a3 that contributes to the high-affinity interaction of FVIII with VWF exhibits a greatly reduced ability to enhance VWF cleavage. Our data suggest that FVIII plays a role in regulating proteolytic processing of VWF by ADAMTS13 under shear stress, which depends on the high-affinity interaction between FVIII and its carrier protein, VWF. [ABSTRACT FROM AUTHOR]

C4b-binding protein (C4BP) is a protein acting as a complement inhibitor and a carrier protein for anticoagulant protein S. Previously, we reported that the in vivo clearance of C4BP involves CD91, and that a CD91-interactive site overlaps the heparin-binding site within C4BP α-chains . Here, we investigated the C4BP-CD91 interaction in more detail. Binding of C4BP to CD91 was unaffected by protein S, which associates with C4BP β-chain. Second, mutagenesis of cationic residues within C4BP α-chains impaired CD91 binding, reducing the affinity of triple mutant C4BPα/R39Q-R64Q-R66Q by 20-fold (Kd= 10 nM versus 214 nM for wild-type and mutant C4BP, respectively). Accordingly, intracellular degradation of this mutant by CD91-expressing cells was reduced to levels of CD91-deficient cells. Moreover, C4BPα/R39Q-R64Q-R66Q displayed a 3-fold prolonged survival compared to normal C4BP in in vivo clearance experiments. Since these residues also contribute to heparin binding, we explored the role of heparin-sulfate proteoglycans (HSPG) in the endocytosis of C4BP. The absence of HSPG was associated with a near complete absence of cell binding and intracellular degradation of C4BP. Apparently, the cellular uptake of C4BP depends on both HSPG and CD91, involving interactions with positively charged residues within C4BP α-chain. [ABSTRACT FROM AUTHOR]

Background. Thrombocytopenia occurs early during malarial infection, but its underlying mechanism is unclear. Secretion of von Willebrand factor (vWF) occurs on endothelial cell activation, and it plays an important role in platelet agglutination. Methods. In 14 healthy human volunteers who were experimentally infected with Plasmodium falciparum, we studied vWF secretion and proteolysis as well as the relationship between changes in circulating platelet numbers and plasma levels of vWF and activated vWF. Results. Platelet numbers started to decrease between days 7 and 9 after infection, which corresponded to the earliest phase of blood-stage infection. With the decrease in platelet numbers, levels of vWF, vWF propeptide (markers of chronic and acute endothelial cell activation, respectively), and activated vWF (exposing the glycoprotein Iba platelet-binding domain) increased proportionally. A strong, reciprocal relationship was observed between platelet numbers and levels of both vWF and activated vWF. Activity of the vWF-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13)—a regulator of vWF activity—remained unchanged. Conclusions. P. falciparum induces systemic acute endothelial cell activation and release of activated vWF immediately after the onset of blood-stage infection. The resulting platelet agglutination may result in early thrombocytopenia and may play a role in the pathogenesis of malaria. [ABSTRACT FROM AUTHOR]

Protein A (Spa) is a surface-associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing VH3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A–vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig-binding region of protein A, Spa-EDABC, fused to glutathione- S-transferase (GST), bound recombinant vWF in a dose-dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length-Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D′-D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig-binding domain bound to the A1 domain in a similar manner to the full length-Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST-SpaD protein at sites known to be involved in IgG Fc or in VH3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized VH3 Fab had reduced binding to vWF A1 and D′-D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site. [ABSTRACT FROM AUTHOR]

Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy-chain region LysT13-Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X, phospholipids, and thrombin or factor Xa. Plasma-derived factor VIII is activated by thrombin with a second-order rate constant of 3.3±0.3×106 M-1 s-1, which proved to be slightly higher than for activation by factor Xa. The second-order rate constant of activation by thrombin of plasma-derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713-Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713-Arg740, des-(713–1637) factor VIII. This suggests that the region Lys713-Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy-chain cleavage of des-(713–1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713–1637)-factor VIII is clearly reduced. Furthermore, the second-order rate constant (0.2±0.1×106 M-1 s-1) of des-(713–1637)-factor VIII light-chain cleavage by thrombin was reduced tenfold compared with that of plasma-derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des-(713–1637)-factor VIII light chain cleavage by thrombin is similar to that of isolated light-chain, but isolated light-chain is cleaved by factor Xa 20-fold more efficiently than the light chain in des (713–1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light-chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy-chain sequence Lys713-Arg740 promotes both activation and light-chain proteolysis. [ABSTRACT FROM AUTHOR]

Background and Objectives: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary hemostasis but recent data suggest additional roles beyond hemostasis in angiogenesis and potentially in vascular smooth muscle cell (VSMC) proliferation. Our aim was to investigate how VWF can modulate VSMC proliferation and identified the underlying mechanisms and the in vivo pathophysiological relevance. Methods and Results: Cultured aortic VSMCs proliferation and migration were increased in the presence of VWF. VSMCs treatment with a siRNA targeting αv integrin or the RGT-peptide blocking αvβ3 signaling completely inhibited proliferation. VWF did not bind directly to αvβ3 on VSMCs. We identified that VWF A2 domain was able to bind VSMCs. Since the lowdensity lipoprotein-related receptor protein (LRP) family are known to act as co-receptors we hypothesized the involvement of a member in the signaling pathway. Using the universal LRP-inhibitor (RAP), we confirmed LRP-mediated VSMC proliferation. siRNA experiments and proximity ligation assay staining identified LRP4 as the VWF-counterreceptor on VSMCs and showed co-localization between αvβ3 and LRP4. Carotid ligations were applied to VWF +/+ and −/− mice and intimal hyperplasia (IH) was measured. Less VWF−/− mice developed IH compared to VWF +/+ mice. Finally the proliferative effect of VWF was confirmed in human atherosclerotic lesions from different vessels (aortas, carotids) showing a proximity between VWF and a-SM actin positive cells. Conclusions: VWF mediates VSMC proliferation through its A2 domain binding to the LRP4 receptor and integrin αvβ3signaling. The decreased IH following vascular injury suggests that targeting VWF-LRP4 interactions may contribute to limit remodeling. [ABSTRACT FROM AUTHOR]

Low-density lipoprotein receptor–related protein-1 (LRP) on brain capillaries clears amyloid β-peptide (Aβ) from brain. Here, we show that soluble circulating LRP (sLRP) provides key endogenous peripheral 'sink' activity for Aβ in humans. Recombinant LRP cluster IV (LRP-IV) bound Aβ in plasma in mice and Alzheimer's disease–affected humans with compromised sLRP-mediated Aβ binding, and reduced Aβ-related pathology and dysfunction in a mouse model of Alzheimer disease, suggesting that LRP-IV can effectively replace native sLRP and clear Aβ. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (<40 nm) in 31 ± 4% of PBMCs. In vitro perfusion assays revealed that leukocyte-rolling over E- and P-selectin was inhibited by sSiglec-5/Fc or sSiglec-5/C4BP, while adhesion onto VCAM1 was unaffected. When applied to healthy mice (0.8 mg/kg), sSiglec-5/C4BP significantly reduced the number of rolling leukocytes under basal conditions (10.9 ± 3.7 versus 23.5 ± 9.3 leukocytes/field/min for sSiglec-5/C4BP-treated and control mice, respectively; p = 0.0093). Moreover, leukocyte recruitment was inhibited over a 5-h observation period in an in vivo model of TNFalpha-induced inflammation following injection sSiglec-5/C4BP (0.8 mg/kg). Our data identify PSGL1 as a ligand for Siglec-5, and soluble Siglec-5 variants appear efficient in blocking PSGL1-mediated leukocyte rolling and the inflammatory response in general. [ABSTRACT FROM AUTHOR]