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Changes in the feeding pattern of a pig may indicate disease or welfare issues and can also be strongly related to productivity changes. Automatic measurements of the feeding patterns of group-housed growing-finishing pigs can enable the detection of such feeding related changes. In this paper, a high frequency (HF) Radio Frequency Identification (RFID) system was used to register pigs’ feeding patterns at a commercial-type feed trough. The raw RFID registrations had to be translated into feeding visits, as there were irregular time gaps between the registrations that were inherent to the system for this application. Data from two experiments were used: one used behavioural observations of six pigs during three days with the aim of finding the best method for visit construction (experiment 2, E2), whilst the other experiment used feeding pattern observations of 20 pigs on one day as an independent validation data-set (experiment 1, E1). Three methods for visit construction were proposed: (1) visit criteria (a bout criterion and a minimum duration criterion), (2) a digital filter (using a well-defined time delay and a threshold for feeding) and (3) erosion and dilation operations from mathematical morphology. The best results were obtained using a bout criterion equal to 10 s for two RFID tags per pig or 20 s for one RFID tag per pig. Removal of short visits was found to be unnecessary. This optimal method is equal to a closing operation (a dilation followed by an erosion). For the RFID system with the optimal bout criterion, using two tags per pig, average sensitivity was 83%, specificity 98%, accuracy 97% and precision was 75% for E2. Applying the same bout criterion on the independent test set (E1), resulted in an average sensitivity of 80%, a specificity of 99%, an accuracy of 98% and a precision of 78%. Performance of the method tended to be lower when using one tag per pig. Regression analysis revealed that the observed feeding duration was well-correlated with RFID-based feeding duration (R 2 = 0.86 for two tags per pig, E1 + E2). Other observed variables (number of feeding visits and inter-visit interval) were harder to predict by the RFID system. From these results, there was concluded that feeding patterns can be recorded accurately in an automatic way if the registrations from the RFID system are analysed using an appropriate bout criterion. [ABSTRACT FROM AUTHOR]

A 3D imaging technique using a high speed binocular stereovision system was developed in combination with corresponding image processing algorithms for accurate determination of the parameters of particles leaving the spinning disks of centrifugal fertilizer spreaders. Validation of the stereo-matching algorithm using a virtual 3D stereovision simulator indicated an error of less than 2 pixels for 90% of the particles. The setup was validated using the cylindrical spread pattern of an experimental spreader. A 2D correlation coefficient of 90% and a Relative Error of 27% was found between the experimental results and the (simulated) spread pattern obtained with the developed setup. In combination with a ballistic flight model, the developed image acquisition and processing algorithms can enable fast determination and evaluation of the spread pattern which can be used as a tool for spreader design and precise machine calibration. [ABSTRACT FROM AUTHOR]

Lameness in sows is an important welfare concern as well as a cause of considerable economic loss. In general, lameness in sows is evaluated by visual scoring of the gait. However, visual assessment is a subjective technique with low reproducibility. Therefore, the need arises for more accurate and objective methods to enable correct detection. A detection system, based on force stance variables derived from force plate analysis and visual stance variables derived from image processing was developed: SowSIS (sow stance information system). The transportable device and computer software was tested in a practical environment and was positively evaluated for its practical use and precise measurements. A preliminary study of the force stance variables was performed by comparing measurements of sound sows and sows with lameness in their hind leg. All lame sows were reluctant to bear weight on their lame leg which resulted in a lower weight exerted by the lame hind leg compared to the other legs. This shift in weight was mainly compensated by putting more weight on the contralateral sound hind leg. Sows showed more but shorter kicks (i.e. lifting leg of the ground) with their lame leg compared to the other legs. The results indicated that the developed system SowSIS has very promising potential to support future objective research in lameness in sows. [ABSTRACT FROM AUTHOR]

Patients with von Willebrand disease (VWD) are often heterozygous for a missense mutation in the von Willebrand factor (VWF) gene. Investigating the pathogenic features of VWF mutations in cells directly derived from patients has been challenging. Here, we have used blood outgrowth endothelial cells (BOECs) isolated from human peripheral blood to analyze the storage and secretion of VWF. BOECs showed full endothelial characteristics and responded to Weibel-Palade body (WPB) secretagogues except desmopressin. We examined BOECs derived from a single subject heterozygous for a type 2N mutation (p.Arg854Gln) and from 4 patients with type 1 VWD who were, respectively, heterozygous for p.Ser1285Pro, p.Leu1307Pro, p.Tyr1584Cys, and p.Cys2693Tyr. Compared with normal BOECs, BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr showed morphologically abnormal WPB and retention of VWF in the endoplasmic reticulum, whereas BOECs heterozygous for p.Arg854Gln or p.Tyr1584Cys showed normal WPB. The agonist-induced exocytosis of WPB from BOECs and formation of VWF strings on BOECs heterozygous for p.Ser12B5Pro, p.Leu1307Pro, or p.Cys2693Tyr, but not for p.Arg854Glnor p.Tyr1584Cys, were reduced. In conclusion, VWD phenotype can be recapitulated in BOECs, and thus BOECs provide a feasible bona tide cell model to study the pathogenic effects Of VWF mutations. [ABSTRACT FROM AUTHOR]

Cattle lameness causes considerable animal welfare problems and negatively affects the farm economy. Gait scoring techniques and claw health reports are commonly used for research and surveys, but few daily management solutions exist to monitor gait parameters from individual cows within a herd. A tool to automate and process the measurement of spatiotemporal kinematic and force variables was developed using a pressure sensitive walkway, commercial farm infrastructure and management tools. A fully automatic setup on ILVO’s experimental farm measures and analyses the gait of each cow that exits the milking parlour by registration of 20 basic kinematic gait variables. Based on this variable set, a wide range of typical gait parameters such as triple support time, abduction, etc. can be calculated. This paper presents some practical results and considerations related to this system of automated gait analysis. A first validation of the acquired variables shows that the Gaitwise system is capable of clustering observations in relation to the observer score with an overall sensitivity of 76–90% and specificity of 86–100%. Variables of asymmetry and speed seem most promising for further research on the detection of lameness. For future reference, the resulting cow gait variables will be stored for time series analysis to detect changes in individual cow walking behaviour in relation to any source (claw lesions, gestation stage, etc.). Such measurements could provide valuable information for management, veterinary check-ups and further research for automatic lameness detection in cattle. [ABSTRACT FROM AUTHOR]

Abstract: The low-density lipoprotein receptor-related protein (LRP) binds a range of proteins including receptor associated protein (RAP), activated α2-macroglobulin (α2M*), factor IXa (FIXa), and factor VIII (FVIII) light chain. The binding is mediated by the complement-type repeats, which are clustered in four distinct regions within LRP. Cluster II of 8 repeats (CR3–10) and cluster IV of 11 repeats (CR21–31) have been implicated in ligand-binding. Previous studies have aimed to identify the cluster II repeats involved in binding α2M* and RAP. We now evaluated the binding to RAP, α2M*, FIXa and FVIII light chain of triplicate repeat-fragments of not only clusters II but also of cluster IV. Employing surface plasmon resonance analysis, we found that most efficient ligand-binding was displayed by the repeats within region CR4–8 of cluster II and within region CR24–28 of cluster IV. Whereas the binding to RAP could be attributed to two consecutive repeats (CR5–6, CR26–27), combinations of three repeats showed most efficient binding to FIXa (CR6–8, CR26–28), FVIII light chain (CR5–7, CR6–8, CR24–26), and α2M* (CR4–6, CR24–26). The results imply that there is an internal functional duplication of complement-type repeats within LRP resulting in two clusters that bind the same ligands. [Copyright &y& Elsevier]

Low-density lipoprotein receptor-related protein (LRP) contributes to factor VIII (FVIII) catabolism in vivo. Besides LRP, FVIII also interacts with very low-density lipoprotein receptor (VLDLR) in vitro. We investigated the physiological role of VLDLR in FVIII catabolism, using knockout mouse models for VLDLR and LRP, alone and in combination. VLDLR−/− mice displayed normal plasma FVIII, whereas VLDLR−/− LRP− double-knockout mice had slightly increased FVIII compared with LRP-deficient mice. Remarkably, VLDLR deficiency slightly accelerated FVIII clearance. Adenovirus-mediated overexpression of VLDLR did not lower plasma FVIII in LRP-deficient mice. We conclude that VLDLR does not act in concert with LRP in FVIII clearance in vivo. [ABSTRACT FROM AUTHOR]

Studies the activation of blood coagulation factor IX to activated factor IX. Development of enzymatic activity; Exposure of the binding sites; Use of an affinity-purified soluble form of lipoprotein receptor-related protein.

Presents the use of Complementary Metal Oxide Semiconductors (CMOS) for integration of a Radio Frequency (RF) transceiver front-end. Attempts of producing single-chip CMOS RF circuits for cellular applications; Discussion of fundamental pitfalls and limitations of RF CMOS; Emphasis on optimizing transceiver as a single block.

Background: Activated factor IX (FIXa) is an inefficient enzyme that needs activated factor VIII (FVIII) for full activity. Recently, we identified a network of FVIII-driven changes in FIXa employing hydrogen-deuterium eXchange mass spectrometry (HDX-MS). Some changes also occurred in active-site inhibited FIXa, but others were not cofactor-driven, in particular those within the 220-loop (in chymotrypsin numbering)., Objective: The aim of this work is to better understand the zymogen-to-enzyme transition in FIX, with specific focus on substrate-driven changes at the catalytic site., Methods: Footprinting mass spectrometry by HDX and Tandem-Mass Tags (TMT) labelling were used to explore changes occurring upon the conversion from FIX into FIXa. Mutagenesis and kinetic studies served to assess the role of the 220-loop., Results: HDX-MS displayed remarkably few differences between FIX and FIXa. In comparison with FIX, FIXa did exhibit decreased deuterium uptake at the N-terminus region. This was more prominent when the FIXa active site was occupied by an irreversible inhibitor. TMT-labelling showed that the N-terminus is largely protected from labelling, and that inhibitor binding increases protection to a minor extent. Occupation of the active site also reduced deuterium uptake within the 220-loop backbone. Mutagenesis within the 220-loop revealed that a putative H-bond network contributes to FIXa activity. TMT labeling of the N-terminus suggested that these 220-loop variants are more zymogen-like than wild-type FIXa., Conclusion: In the absence of cofactor and substrate, FIXa is predominantly zymogen-like. Stabilization in its enzyme-like form involves, apart from FVIII-binding, also interplay between the 220-loop, N-terminus, and the substrate binding site., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system., (© 2020 by The American Society of Hematology.)

In the complex with von Willebrand factor (VWF) factor VIII (FVIII) is protected from rapid clearance from circulation. Although it has been established that the FVIII binding site resides in the N-terminal D'-D3 domains of VWF, detailed information about the amino acid regions that contribute to FVIII binding is still lacking. In the present study, hydrogen-deuterium exchange mass spectrometry was employed to gain insight into the FVIII binding region on VWF. To this end, time-dependent deuterium incorporation was assessed in D'-D3 and the FVIII-D'-D3 complex. Data showed reduced deuterium incorporation in the D' region Arg782-Cys799 in the FVIII-D'-D3 complex compared to D'-D3. This implies that this region interacts with FVIII. Site-directed mutagenesis of the six charged amino acids in Arg782-Cys799 into alanine residues followed by surface plasmon resonance analysis and solid phase binding studies revealed that replacement of Asp796 affected FVIII binding. A marked decrease in FVIII binding was observed for the D'-D3 Glu787Ala variant. The same was observed for D'-D3 variants in which Asp796 and Glu787 were replaced by Asn796 and Gln787. Site-directed mutagenesis of Leu786, which together with Glu787 and Cys789 forms a short helical region in the crystal structure of D'-D3, also had a marked impact on FVIII binding. The combined results show that the amino acid region Arg782-Cys799 is part of a FVIII binding surface. Our study provides new insight into FVIII-VWF complex formation and defects therein that may be associated with bleeding caused by markedly reduced levels of FVIII., (Copyright© 2020 Ferrata Storti Foundation.)

Background: The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surface-exposed elements involved in VWF and FIXa interaction., Objective: The aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding., Methods: Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays., Results: Five surface-exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding., Conclusion: The surface-exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle., (© 2019 International Society on Thrombosis and Haemostasis.)

It has been proposed that differences may exist between umbilical cord blood (CB) platelets and adult peripheral blood (APB) platelets, including altered protein levels of the main platelet integrins. We have now compared the protein expression profiles of CB and APB platelets employing a label-free comparative proteomics approach. Aggregation studies showed that CB platelets effectively aggregate in the presence of thromboxane A2 analogue, collagen, and peptide agonists of the proteinase-activated receptors 1 and 4. In agreement with previous studies, higher concentrations of the agonists were required to initiate aggregation in the CB platelets. Mass spectrometry analysis revealed no significant difference in the expression levels of critical platelet receptors like glycoprotein (GP)Ib, GPV, GPIX, and integrin αIIbβ3. This was confirmed using flow cytometry-based approaches. Gene ontology enrichment analysis revealed that elevated proteins in CB platelets were in particular enriched in proteins contributing to mitochondrial energy metabolism processes. The reduced proteins were enriched in proteins involved in, among others, platelet degranulation and activation. In conclusion, this study reveals that the CB and APB platelets are distinct. In particular, changes were observed for proteins that belong to metabolic and energy generation processes and not for the critical adhesive platelet integrins and glycoproteins.

Objective: Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of PARs (protease-activated receptors). The predominant thrombin receptor is PAR1, and in endothelial cells (ECs), thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear whether thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N terminus have remained an unresolved issue., Approach and Results: Here, we used a quantitative phosphoproteomics approach to show that classical and peptide activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in the presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs., Conclusions: Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrate that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-tethered ligand peptide induce similar phosphoregulation, and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating that it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology., (© 2017 American Heart Association, Inc.)

Genetics play a significant role in venous thromboembolism (VTE), yet current clinical laboratory-based testing identifies a known heritable thrombophilia (factor V Leiden, prothrombin gene mutation G20210A, or a deficiency of protein C, protein S, or antithrombin) in only a minority of VTE patients. We hypothesized that a substantial number of VTE patients could have lesser-known thrombophilia mutations. To test this hypothesis, we performed whole-exome sequencing (WES) in 64 patients with VTE, focusing our analysis on a novel 55-gene extended thrombophilia panel that we compiled. Our extended thrombophilia panel identified a probable disease-causing genetic variant or variant of unknown significance in 39 of 64 study patients (60.9%), compared with 6 of 237 control patients without VTE (2.5%) ( P < .0001). Clinical laboratory-based thrombophilia testing identified a heritable thrombophilia in only 14 of 54 study patients (25.9%). The majority of WES variants were either associated with thrombosis based on prior reports in the literature or predicted to affect protein structure based on protein modeling performed as part of this study. Variants were found in major thrombophilia genes, various SERPIN genes, and highly conserved areas of other genes with established or potential roles in coagulation or fibrinolysis. Ten patients (15.6%) had >1 variant. Sanger sequencing performed in family members of 4 study patients with and without VTE showed generally concordant results with thrombotic history. WES and extended thrombophilia testing are promising tools for improving our understanding of VTE pathogenesis and identifying inherited thrombophilias., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Factor VIII C-domains are believed to have specific functions in cofactor activity and in interactions with von Willebrand factor. We have previously shown that factor VIII is co-targeted with von Willebrand factor to the Weibel-Palade bodies in blood outgrowth endothelial cells, even when factor VIII carries mutations in the light chain that are associated with defective von Willebrand factor binding. In this study, we addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and cofactor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, factor VIII or YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells for in vitro characterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, factor V did not target to Weibel-Palade bodies. The chimeras showed reduced Weibel-Palade body targeting, suggesting that this requires the factor VIII C1-C2 region. The factor VIII/V-C1 chimera did not bind von Willebrand factor and had reduced affinity for activated factor IX, whereas the factor VIII/V-C2 chimera showed a minor reduction in von Willebrand factor binding and normal interaction with activated factor IX. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand factor and activated factor IX. Foot-printing analysis of the chimeras revealed increased exposure of lysine residues in the A1/C2- and C1/C2-domain interface, suggesting increased C2-domain mobility and disruption of the natural C-domain tandem pair orientation. Apparently, this affects intracellular trafficking, but not extracellular function., (Copyright© Ferrata Storti Foundation.)

Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function.

Patients with von Willebrand disease (VWD) are often heterozygous for a missense mutation in the von Willebrand factor (VWF) gene. Investigating the pathogenic features of VWF mutations in cells directly derived from patients has been challenging. Here, we have used blood outgrowth endothelial cells (BOECs) isolated from human peripheral blood to analyze the storage and secretion of VWF. BOECs showed full endothelial characteristics and responded to Weibel-Palade body (WPB) secretagogues except desmopressin. We examined BOECs derived from a single subject heterozygous for a type 2N mutation (p.Arg854Gln) and from 4 patients with type 1 VWD who were, respectively, heterozygous for p.Ser1285Pro, p.Leu1307Pro, p.Tyr1584Cys, and p.Cys2693Tyr. Compared with normal BOECs, BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr showed morphologically abnormal WPB and retention of VWF in the endoplasmic reticulum, whereas BOECs heterozygous for p.Arg854Gln or p.Tyr1584Cys showed normal WPB. The agonist-induced exocytosis of WPB from BOECs and formation of VWF strings on BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr, but not for p.Arg854Gln or p.Tyr1584Cys, were reduced. In conclusion, VWD phenotype can be recapitulated in BOECs, and thus BOECs provide a feasible bona fide cell model to study the pathogenic effects of VWF mutations.

Purpose: To determine the optimal timing for imaging brain tumours and other brain lesions with 18F-labelled fluoromethylcholine (18F-FCho) PET., Materials and Methods: Dynamic PET imaging with 18F-FCho (acquisition time of 28 min) was performed in 24 patients with space-occupying lesions in the brain. On the coregistered PET and MRI, lesion-to-normal tissue uptake ratios (LNRs) were calculated. Time-activity curves (TACs) were generated on the basis of the LNRs. Changes in LNR over time were calculated on the basis of the linear part of the TAC (last 22 min of the acquisition)., Results: TACs for 18F-FCho in gliomas of different grading showed that, after a rapid uptake phase, the mean increase in LNR was 1.07 ± 0.93 for glioblastomas, -0.52 ± 1.56 for anaplastic astrocytomas, 0.04 ± 0.13 for grade 2 oligoastrocytomas and 0.37 in a case of a pilocytic astrocytoma. The average increase in LNR was 0.46 for a brain metastasis, 0.41 ± 0.69 for radiation-induced mass lesions and 1.07 for a tumefactive demyelinating lesion. In contrast, TACs for 18F-FCho in meningiomas showed that, after a rapid uptake phase, the average change in LNR was -5.25 ± 4.19 for typical meningiomas and -3.04 in a case of a mixed angiomatous and clear cell meningioma., Conclusion: On the basis of the TACs, PET imaging with 18F-FCho starting within minutes after the administration of the tracer is preferred for the detection of brain tumours and other brain lesions. If discrimination between meningioma and other brain tumours is of concern, both 'early' and 'late' PET imaging could be helpful.

Purpose: To evaluate the distribution of 18F-labelled fluoromethylcholine (FCho) in normal structures and tumors of the head region using positron emission tomography (PET) and magnetic resonance imaging., Materials and Methods: We retrospectively reviewed the positron emission tomography, magnetic resonance imaging, and the coregistered images obtained in 17 patients with suspected high-grade gliomas. The accumulation of 18F-FCho in the normal structures and in brain lesions was visually and semiquantitatively assessed. A 4-point grading system was used for the visual analysis. A standardized uptake value (SUV) was used to quantify uptake., Results: In the normal brain parenchyma, 18F-FCho uptake was faint (SUVmean, 0.15 ± 0.03 (SD)). Uptake was generally moderate in the choroid plexus (SUVmean, 0.82 ± 0.16), cavernous sinus (SUVmean, 0.87 ± 0.19), extraocular eye muscles (SUVmean, 1.10 ± 0.27), masticatory muscles (SUVmean, 0.99 ± 0.22), and bone marrow (SUVmean, 1.06 ± 0.26), whereas uptake was usually moderately intense in the pituitary gland (SUVmean, 1.90 ± 0.21). Uptake was variable in the lacrimal glands and the mucosa of the nasal cavity (for SUVmean of subgroups see text). Intense uptake was observed in the parotid glands (SUVmean, 3.27 ± 0.73). (Moderately) intense 18F-FCho uptake was observed in glioblastomas (range SUVmax, 2.26-6.37) and typical meningiomas (range SUVmax, 3.75-5.81). Uptake was globally faint in grade II and III gliomas (range SUVmax, 0.33-0.78). 18F-FCho uptake was also demonstrated in benign lesions, such as a tumefactive demyelinating brain lesion., Conclusions: 18F-FCho uptake was faint in the normal brain parenchyma and usually moderate in the choroid plexus, cavernous sinus, extraocular eye muscles, masticatory muscles, and bone marrow. Uptake in the pituitary gland was generally moderately intense, whereas uptake in the lacrimal glands and the mucosa of the nasal cavity was variable. Parotid glands had intense uptake. Also, uptake in glioblastomas and meningiomas was usually (moderately) intense, whereas uptake in grade II and III gliomas was globally faint. However, 18F-FCho uptake was not tumor specific.

Purpose: [(18)F]Fluoromethylcholine ([(18)F]FCho) is a radiotracer generally used for tumour visualization in patients. Due to high levels of dimethylaminoethanol (DMAE) remaining in [(18)F]FCho solutions synthesized by currently available methods, tumour visualization might be compromised., Methods: An improved purification method involving an optimized purification step for reducing the levels of DMAE was conceived. The physiological explanation for the interference of residual DMAE in [(18)F]FCho pharmacokinetics was further elaborated in a xenograft mouse model., Results: The use of a series of polymer solid-phase extraction cartridges (Oasis HLB/WCX), instead of the commonly used combination of tC18 and Accell CM cartridges, reduced DMAE levels from 402.2±49.6 ppm to 3.0±0.5 ppm. Subsequent in vitro tests proved that (1) [(18)F]FCho uptake was reduced in the presence of DMAE at concentrations above 0.5 µM and (2) DMAE is a competitive inhibitor of [(18)F]FCho transport. In vivo experiments in xenograft mouse models corroborated reduced tumour uptake at DMAE plasma levels of about 2.5 µM as found in patients injected with contaminated [(18)F]FCho., Conclusion: Residual DMAE, even at levels below choline plasma concentrations found during fasting, compromises [(18)F]FCho uptake in vivo and care should be taken to avoid its interference in molecular imaging with [(18)F]FCho.

Purpose: To give an up-to-date overview of the potential clinical utility of (18)F-labelled choline derivatives for tumour imaging with positron emission tomography., Methods: A PubMed search for (18)F-labelled choline analogues was performed. Review articles and reference lists were used to supplement the search findings., Results: (18)F-labelled choline analogues have been investigated as oncological PET probes for many types of cancer on the basis of enhanced cell proliferation. To date, studies have focused on the evaluation of prostate cancer. Available studies have provided preliminary results for detecting local and metastatic disease. Experience with (18)F-fluorocholine PET in other tumour types, including brain and liver tumours, is still limited. In the brain, excellent discrimination between tumour and normal tissue can be achieved due to the low physiological uptake of (18)F-fluorocholine. In the liver, in which there is a moderate to high degree of physiological uptake in normal tissue, malignancy discrimination may be more challenging., Conclusion: PET/CT with (18)F-fluorocholine can be used to detect (recurrent) local prostate cancer, but seems to have limited value for T (tumour) and N (nodal) staging. In patients presenting with recurrent biochemical prostate cancer, it is a suitable single-step examination with the ability to exclude distant metastases when local salvage treatment is intended. In the brain, high-grade gliomas, metastases and benign lesions can be distinguished on the basis of (18)F-fluorocholine uptake. Moreover, PET imaging is able to differentiate between radiation-induced injury and tumour recurrence. In the liver, (18)F-fluorocholine PET/CT seems promising for the detection of hepatocellular carcinoma.

Background: The increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest of scientific research in this field. Therefore, a vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma (HCC) in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for HCC research, in which tumours develop in a natural background of fibrosis and (2) to assess the time-dependent angiogenic changes in the pathogenesis of HCC., Methods: Weekly intraperitoneal injections with the hepatocarcinogenic compound N-nitrosodiethylamine was applied as induction method and samples were taken at several time points to assess the angiogenic changes during the progression of HCC., Results: The N-nitrosodiethylamine-induced mouse model provides well vascularised orthotopic tumours after 25 weeks. It is a representative model for human HCC and can serve as an excellent platform for the development of new therapeutic targets.

Objective: The aim of this study was to investigate whether normoglycemic glucose concentrations interfere with cerebral F-18 FDG uptake., Methods: The analysis was based on 2 sets of paired PET scans in 94 patients who were in complete metabolic remission after the successful completion of treatment for lymphoma. For these 188 PET scans, 2 subgroups were defined according to the plasma glucose level at the time of scanning. Group 1 contained the PET images that were associated with the lower of both normoglycemic plasma glucose levels, whereas group 2 contained the PET images that were associated with the higher of both plasma glucose levels. SUVs (standard uptake values) in the cerebellum between both groups were compared using paired sample T test. Subsequently, SUVs were normalized to a standard glucose concentration and normalized SUVs were again compared. Further, we calculated the coefficient of variation of SUVs in group 1 and 2 both before and after the normalization step., Results: Mean plasma glucose level was 86 mg/dL (SD of 9 mg/dL) in group 1 and 97 mg/dL (SD of 10 mg/dL) in group 2. Mean SUV was 3.8 (SD of 1.1) for group 1 and 3.5 (SD of 1.1) for group 2. Mean SUV in group 1 was slightly but statistically significantly higher than the mean SUV in group 2 (p < 0.01). Mean normalized SUV was 3.6 (SD of 1.1) in group 1 and 3.7 (SD of 1.3) in group 2. A paired comparison between normalized SUVs in both groups indicated that there was no statistically significant difference (p < 0.31). The coefficient of variation for the SUVs in group 1 and 2 before normalization was 29 and 30%, respectively. The coefficient of variation for the normalized SUVs in group 1 and 2 was 30 and 34%, respectively., Conclusions: Our results indicated that plasma glucose levels that are within the normoglycemic range have a small but systematic effect on F-18 FDG uptake in the brain (following an inverse relationship). Normalizing plasma glucose levels to a standard glucose concentration successfully reduced the intra-subject variability of SUV measures. Inter-subject variability, however, remained high suggesting that other factors have an influence as well.

Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33(scFv), and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 --> Ala/Ala (fVIII(YFP 2092/93)) and 2 single mutants Lys2092 --> Ala and Phe2093 --> Ala. KM33(scFv) inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIII(YFP 2092/93) had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33(scFv) was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33(scFv) inhibited binding to platelets and fVIII(YFP 2092/93) had reduced binding to A23187-stimulated platelets. fVIII(YFP 2092) exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIII(YFP 2093) showed a reduction of activity with Ptd-L-Ser less than 12%. fVIII(YFP 2092/93) had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain.

von Willebrand factor (VWF) serves as adhesive surface for platelets to adhere to the vessel wall. We have recently found that beta2-glycoprotein I is able to inhibit platelet binding to VWF, indicating a role in the pathophysiology of arterial thrombosis. In the present study, we investigated whether differences in beta2-glycoprotein I plasma levels influence the risk of myocardial infarction. We have measured beta2-glycoprotein I and VWF antigen levels in 539 men with a first myocardial infarction and in 611 control subjects. Although we did not find a profound effect of beta2-glycoprotein I plasma levels on myocardial infarction in the overall population, we found a dose-dependent protective effect of increasing beta2-glycoprotein I plasma levels on myocardial infarction in men 60 years and older. In this age group, we found an odds ratio of 0.41 (95% confidence interval, 0.22-0.74) for high beta2-glycoprotein I levels compared with low levels. High plasma levels of beta2-glycoprotein I remained protective for myocardial infarction despite high levels of VWF. To conclude, high circulating levels of beta2-glycoprotein I appeared to be associated with a reduced risk of myocardial infarction in elderly men. In vivo experiments are needed to investigate the exact contribution of beta2-glycoprotein I on the pathophysiology of myocardial infarction.

Background: Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion., Design and Methods: Human blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human B-domain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy., Results: Blood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6+/-1.0 pmol/1 x 10(6) cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6- to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15-22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies., Conclusions: We conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A.

Weibel-Palade bodies (WPBs) are the endothelial storage organelles that are formed upon von Willebrand factor (VWF) expression. Apart from VWF, WPBs contain a variety of hemostatic and inflammatory proteins. Some of these are thought to be targeted to WPBs by directly interacting with VWF in the secretory pathway. Previous studies have demonstrated that coexpression of factor VIII (FVIII) with VWF results in costorage of both proteins. However, whether cotrafficking is driven by intracellular FVIII-VWF assembly has remained unclear. We now have addressed this issue using recombinant VWF type 2N variants that are known to display reduced FVIII binding in the circulation. Binding studies using purified fluorescent FVIII and VWF type 2N variants revealed FVIII binding defects varying from moderate (Arg854Gln, Cys1060Arg) to severe (Arg763Gly, Thr791Met, Arg816Trp). Upon expression in HEK293 cells, all VWF variants induced formation of WPB-like organelles that were able to recruit P-selectin, as well as FVIII. WPBs containing FVIII did not display their typical elongated shape, suggesting that FVIII affects the organization of VWF tubules therein. The finding that VWF type 2N variants are still capable of cotargeting FVIII to storage granules implies that trafficking of WPB cargo proteins does not necessarily require high-affinity assembly with VWF.

The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein-coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca(2+)- or cAMP-raising agonists.

The discovery that antiphospholipid antibodies recognize plasma proteins that bind to phospholipids rather than recognizing phospholipids themselves has been a major advance in research into antiphospholipid syndrome (APS). It is now established that beta2-glycoprotein I (beta2 GPI) is the most important antigen for antiphospholipid antibodies. However, the possible pathologic mechanism is still much debated. This is mainly because not all patients with anti-beta2 GPI antibodies show clinical symptoms that are related to APS. Several reports indicate that anti-beta2 GPI antibodies with lupus anticoagulant (LA) activity are clinically of much importance. Most patients with LA caused by anti-beta2 GPI antibodies suffer from thrombosis as a result of recognition of the first domain of beta2 GPI by these antibodies. In the search for a pathologic mechanism that might explain the high occurrence of thrombosis in patients with anti-domain I antibodies (LA-causing anti-beta2 GPI antibodies), it was found that these antibodies show increased resistance to the anticoagulant activity of annexin A5. We have shown that the same population of antibodies also displays increased resistance to activated protein C. Owing to the diversity of clinical symptoms related to APS, it is likely that other pathologic mechanisms also contribute to the occurrence of APS-related symptoms.

The low-density lipoprotein receptor-related protein (LRP) binds a range of proteins including receptor associated protein (RAP), activated alpha2-macroglobulin (alpha2M*), factor IXa (FIXa), and factor VIII (FVIII) light chain. The binding is mediated by the complement-type repeats, which are clustered in four distinct regions within LRP. Cluster II of 8 repeats (CR3-10) and cluster IV of 11 repeats (CR21-31) have been implicated in ligand-binding. Previous studies have aimed to identify the cluster II repeats involved in binding alpha2M* and RAP. We now evaluated the binding to RAP, alpha2M*, FIXa and FVIII light chain of triplicate repeat-fragments of not only clusters II but also of cluster IV. Employing surface plasmon resonance analysis, we found that most efficient ligand-binding was displayed by the repeats within region CR4-8 of cluster II and within region CR24-28 of cluster IV. Whereas the binding to RAP could be attributed to two consecutive repeats (CR5-6, CR26-27), combinations of three repeats showed most efficient binding to FIXa (CR6-8, CR26-28), FVIII light chain (CR5-7, CR6-8, CR24-26), and alpha2M* (CR4-6, CR24-26). The results imply that there is an internal functional duplication of complement-type repeats within LRP resulting in two clusters that bind the same ligands.

The light chain of activated factor IX (FIXa) is involved in a number of functional properties, including FIXa enzymatic activity. This suggests the existence of a functional link between the FIXa light chain and the catalytic domain. The FIXa structure includes a few putative interactions between EGF2 and the protease domain. The role thereof has been addressed in this study. Recombinant FIX variants FIX-N92A, FIX-N92H, FIX-Y295A, and FIX-F299A were produced in 293 cells. After activation, the purified mutants were analyzed for a variety of functional parameters. None of these substitutions had a major effect on the interaction with antithrombin or the cleavage of the chromogenic substrate CH(3)SO(2)-d-CHG-Gly-Arg-p-nitroanilide. All FIXa mutants, however, exhibited a reduced level of factor X (FX) activation. Defective proteolytic activity occurred both in the absence and in the presence of activated factor VIII (FVIIIa). All mutants also exhibited a reduced level of FX activation in the absence of phospholipids. This suggests that putative interdomain contacts involving residues Asn(92), Tyr(295), and Phe(299) affect reactivity toward FX. Detailed kinetic studies in the presence of phospholipids and FVIIIa revealed substrate inhibition, particularly for mutants FIXa-N92A and FIXa-N92H. Surface plasmon resonance demonstrated that the same replacements weaken the association with the isolated factor VIII (FVIII) A2 domain and the FVIII light chain. This implies a defect in the formation of the FX-activating complex that is membrane-independent. We conclude that contacts between EGF2 and the protease domain of FIXa are crucial for FIXa enzymatic activity and for the assembly of the FX-activating complex.

Low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) are members of the LDLR family of endocytic receptors. LRP recognizes a wide spectrum of structurally and functionally unrelated ligands, including coagulation factor VIII (FVIII). In contrast, the ligand specificity of LDLR is restricted to apolipoproteins E and B-100. Ligand binding to the LDLR family is inhibited by receptor-associated protein (RAP). We have previously reported that, apart from LRP, other RAP-sensitive mechanisms contribute to the regulation of FVIII in vivo. In the present study, we showed that the extracellular ligand-binding domain of LDLR interacts with FVIII in vitro and that binding was inhibited by RAP. The physiologic relevance of the FVIII-LDLR interaction was addressed using mouse models of LDLR or hepatic LRP deficiency. In the absence of hepatic LRP, LDLR played a dominant role in the regulation and clearance of FVIII in vivo. Furthermore, FVIII clearance was accelerated after adenovirus-mediated gene transfer of LDLR. The role of LDLR in FVIII catabolism was not secondary to increased plasma lipoproteins or to changes in lipoprotein profiles. We propose that LDLR acts in concert with LRP in regulating plasma levels of FVIII in vivo. This represents a previously unrecognized link between LDLR and hemostasis.