Quantitative analysis of heme and hemoglobin for the detection of intravascular hemolysis.

BACKGROUND: Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin. RESULTS: Two commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02-45 µM) and hemoglobin (∼0.002-17 µM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme. SIGNIFICANCE: Our results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.

Prediction of diagnosis and diastolic filling pressure by AI-enhanced cardiac MRI: a modelling study of hospital data.

BACKGROUND: With increasing numbers of patients and novel drugs for distinct causes of systolic and diastolic heart failure, automated assessment of cardiac function is important. We aimed to provide a non-invasive method to predict diagnosis of patients undergoing cardiac MRI (cMRI) and to obtain left ventricular end-diastolic pressure (LVEDP). METHODS: For this modelling study, patients who had undergone cardiac catheterisation at University Hospital Heidelberg (Heidelberg, Germany) between July 15, 2004 and March 16, 2023, were identified, as were individual left ventricular pressure measurements. We used existing patient data from routine cardiac diagnostics. From this initial group, we extracted patients who had been diagnosed with ischaemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, or amyloidosis, as well as control individuals with no structural phenotype. Data were pseudonymised and only processed within the university hospital's AI infrastructure. We used the data to build different models to predict either demographic (ie, AI-age and AI-sex), diagnostic (ie, AI-coronary artery disease and AI-cardiomyopathy [AI-CMP]), or functional parameters (ie, AI-LVEDP). We randomly divided our datasets via computer into training, validation, and test datasets. AI-CMP was not compared with other models, but was validated in a prospective setting. Benchmarking was also done. FINDINGS: 66 936 patients who had undergone cardiac catheterisation at University Hospital Heidelberg were identified, with more than 183 772 individual left ventricular pressure measurements. We extracted 4390 patients from this initial group, of whom 1131 (25·8%) had been diagnosed with ischaemic cardiomyopathy, 1064 (24·2%) had been diagnosed with dilated cardiomyopathy, 816 (18·6%) had been diagnosed with hypertrophic cardiomyopathy, 202 (4·6%) had been diagnosed with amyloidosis, and 1177 (26·7%) were control individuals with no structural phenotype. The core cohort only included patients with cardiac catherisation and cMRI within 30 days, and emergency cases were excluded. AI-sex was able to predict patient sex with areas under the receiver operating characteristic curves (AUCs) of 0·78 (95% CI 0·77-0·78) and AI-age was able to predict patient age with a mean absolute error of 7·86 years (7·77-7·95), with a Pearson correlation of 0·57 (95% CI 0·56-0·57). The AUCs for the classification tasks ranged between 0·82 (95% CI 0·79-0·84) for ischaemic cardiomyopathy and 0·92 (0·91-0·94) for hypertrophic cardiomyopathy. INTERPRETATION: Our AI models could be easily integrated into clinical practice and provide added value to the information content of cMRI, allowing for disease classification and prediction of diastolic function. FUNDING: Informatics for Life initiative of the Klaus-Tschira Foundation, German Center for Cardiovascular Research, eCardiology section of the German Cardiac Society, and AI Health Innovation Cluster Heidelberg.

Comprehensive domain-specific analysis and immunoglobulin G profiling of anti-factor VIII antibodies using a bead-based multiplex immunoassay.

BACKGROUND: Antibodies against factor (F)VIII are a major complication in the treatment of patients with severe hemophilia A. The Nijmegen-Bethesda assay (NBA) is the gold standard for detection of neutralizing antibodies (inhibitors), whereas both inhibitors and nonneutralizing antibodies can be detected by immunoassays such as enzyme-linked immunosorbent assay (ELISA) and multiplex bead-based assays. OBJECTIVES: Evaluation of an in-house Luminex bead-based assay (LumiTope) compared with a commercially available ELISA and NBA. METHODS: The LumiTope method comprised full-length and B-domain-deleted FVIII as well as 9 purified FVIII single or multidomains. The respective proteins were coupled to magnetic beads to detect domain-specific immunoglobulin (IgG; IgG1-4) anti-FVIII antibodies in a large cohort of patients with hemophilia A with and without inhibitors. RESULTS: Overall, LumiTope assay had a high sensitivity (94.9%) and specificity (91.2%), particularly in patients with low-titer inhibitors compared with ELISA (sensitivity of 72.2% vs 27.7%). IgG4 was the most abundant IgG subclass in NBA-positive patients. NBA-positive and -negative patients showed different domain profiles. Patients with genetic variants in the heavy chain predominantly exhibited antibodies specific to this chain, while those with a light-chain variant showed a more diverse distribution of antibody specificities. Patients with an intron 22 inversion resembled those with a light-chain defect, with a majority of antibodies targeting the light chain. CONCLUSION: LumiTope assay provides a sensitive and specific method for not only detection but also domain specification of anti-FVIII-antibodies. Implementation of bead-based assays could improve antibody detection, profiling, and comparability of results and complement NBA.

Fibrinolysis biomarker, thrombin, and activated protein C level alterations after coagulation activation depend on type of thrombophilia and clinical phenotype.

Background: Recently, we have shown alterations in the anticoagulant response to recombinant activated factor VII (rFVIIa)-induced coagulation activation in patients with thrombophilia. Objectives: This study aimed to extend this in vivo model to fibrinolysis biomarkers. Methods: This interventional in vivo study included 56 patients with thrombophilia and previous venous thromboembolism (VTE+), 38 without VTE (VTE-), and 35 healthy controls. Plasma levels of D-dimer, plasmin-α2-antiplasmin (PAP) complex, and plasminogen activator inhibitor-1 (PAI-1) were monitored for over 8 hours after rFVIIa infusion (15 µg/kg) along with thrombin markers and activated protein C (APC). Results: Throughout cohorts, median PAP increased by 40% to 52% (P < 3.9 × 10-10) and PAI-1 decreased by 59% to 79% (P < 3.5 × 10-8). In contrast to thrombin-antithrombin (TAT) complex, which also increased temporarily (44% to 115%, P < 3.6 × 10-6), changes in PAP and PAI-1 did not reverse during the observation period. The area under the measurement-time curves (AUCs) of PAP and TAT, which are measures of plasmin and thrombin formation, respectively, were each greater in the VTE+ cohort than in healthy controls (median PAP-AUC = 0.48 vs 0.27 ng·h/L [P = .003], TAT-AUC = 0.12 vs 0.03 nmol·h/L [P = 2.5 × 10-4]) and were correlated with one another (r = 0.554). As evidenced by the respective AUCs, asymptomatic factor (F)V Leiden carriers showed less PAP formation (0.22 vs 0.41 ng·h/L, P = 9 × 10-4), more pronounced PAI-1 decline (0.10 vs 0.18 ng·h/L, P = .01), and increased APC formation (28.7 vs 15.4 pmol·h/L, P = .02) than those within the VTE+ group (n = 19 each). Conclusion: rFVIIa-induced thrombin formation is associated with fibrinolysis parameter changes outlasting the concomitant anticoagulant response. Both correlate with thrombosis history in FV Leiden and might help explain its variable clinical expressivity.

Two-center validation of assays for the detection of binding and neutralizing anti-factor VIII antibodies.

INTRODUCTION: Patients with hemophilia A treated with coagulation Factor VIII (FVIII) products are at risk for developing anti-FVIII antibodies. The ABIRISK Consortium aimed to provide knowledge on the formation and detection of anti-drug antibodies against biopharmaceutical products, including FVIII. Accordingly, standardized and validated assays for the detection of binding (total) and neutralizing antibodies are needed. AIM: Two-center validation of an ELISA for the detection of total FVIII-binding IgG-antibodies and Nijmegen-Bethesda assays for the quantification of FVIII-neutralizing antibodies according to consensus validation guidelines. METHODS: Validation of assays at both sites was done according to published recommendations and included preanalytics, the determination of key assay parameters, including cut-points, assay sensitivity, precision, and FVIII interference. RESULTS: The validated assays reproducibly detected FVIII-binding and -neutralizing antibodies with comparable performance in both laboratories. Floating screening cut-points were established for both assays. Determined mass-based sensitivity of both assays (all values ≤66 ng/mL) complied with the minimum sensitivity for the detection of anti-drug antibodies as recommended by the FDA (<100 ng/mL). Intra- and inter-assay coefficients of variation did not exceed 25%. Assay validation further revealed that pre-analytical heat treatment led to potentially false-positive ELISA results, while up to 0.15 IU/mL, residual FVIII showed no significant impact. Overall, good agreement of results was found for patient samples analyzed at both study sites. CONCLUSION: Comprehensive validation of different anti-FVIII-antibody assays in two laboratories gave novel insights into the impact of pre-analytical sample treatment as well as the comparability of test results generated by the use of methodically different assays.

Contiguous Silver(I)-Mediated Base Pairs of Imidazophenanthroline and Canonical Nucleobases in DNA Duplexes: Formation of Classical Duplexes versus Homodimer Formation.

Metal-mediated base pairs represent a topical alternative to canonical hydrogen-bonded base pairs. In this context, the ligand 1H-imidazo[4,5-f][1,10]phenanthroline (P) was introduced as an artificial nucleobase in a glycol nucleic acid-based nucleoside analogue into a DNA oligonucleotide in a way that the oligonucleotide contains a central block of six contiguous P residues. The ability to engage in Ag+-mediated base pairing was evaluated with respect to the four canonical nucleosides in positions complementary to P. Highly stabilizing Ag+-mediated base pairs were formed with cytosine and guanine (i.e., P-Ag+-C and P-Ag+-G base pairs), whereas the analogous base pairs with thymine and adenine were much less stable and hence formed incompletely. Surprisingly, the intermediate formation of a homodimeric duplex of the P-containing oligonucleotide was observed in all cases, albeit to a different extent. The homodimer is composed of P-Ag+-P base pairs and 18 overhanging mismatched canonical nucleobases. It demonstrates the obstacles present when designing metal-mediated base pairs as metal complexation may take place irrespective of the surrounding natural base pairs. Homodimer formation was found to be particularly prominent when the designated metal-mediated base pairs are of low stability, suggesting that homodimers and regular duplexes are formed in a competing manner.

6-Pyrazolylpurine and its deaza derivatives as nucleobases for silver(I)-mediated base pairing with pyrimidines.

The artificial nucleobase 6-pyrazolylpurine (6PP) and its deaza derivatives 1-deaza-6-pyrazolylpurine (1D6PP), 7-deaza-6-pyrazolylpurine (7D6PP), and 1,7-dideaza-6-pyrazolylpurine (1,7D6PP) were investigated with respect to their ability to differentiate between the canonical nucleobases cytosine and thymine by means of silver(I)-mediated base pairing. As shown by temperature-dependent UV spectroscopy and by circular dichroism spectroscopy, 6PP and (to a lesser extent) 7D6PP form stable silver(I)-mediated base pairs with cytosine, but not with thymine. 1D6PP and 1,7D6PP do not engage in the formation of stabilizing silver(I)-mediated base pairs with cytosine or thymine. The different behavior of 1D6PP, 7D6PP, and 1,7D6PP indicates that silver(I) binding occurs via the N1 position of the purine derivative, i.e. via the Watson-Crick face. The data show that 6PP is capable of differentiating between cytosine and thymine, which is potentially relevant in the context of detecting single-nucleotide polymorphisms.

Functional determination of emicizumab in presence of factor VIII activity.

BACKGROUND: Accurate measurement of emicizumab in the presence of factor (F) VIII is required in patients with severe hemophilia A treated with emicizumab, as well as additional need for FVIII substitution or emicizumab prophylaxis in patients with acquired or moderate to mild hemophilia A. However, the presence of FVIII potentially biases the results. OBJECTIVES: To assess the impact of plasma FVIII activity on determined emicizumab levels and evaluate different strategies for correction for or preanalytical inhibition of FVIII. METHODS: Evaluated strategies comprised of the following: (1) calculation of actual emicizumab plasma levels based on measured FVIII activities and FVIII-affected emicizumab values, (2) preanalytical heat treatment (56 °C for 40 minutes), and (3) neutralization of FVIII activity using FVIII inhibitors. Emicizumab levels and FVIII activities were measured using a modified FVIII one-stage clotting assay and a chromogenic FVIII assay based on bovine factors, respectively. RESULTS: Spiking experiments revealed a consistent linear association between FVIII activities and determined (FVIII-affected) emicizumab results at different emicizumab input levels (∼0.12 µg/mL per IU/dL of FVIII). This principally allowed for mathematical correction of measured emicizumab levels in the presence of FVIII. While a 40% to 50% activity loss of intrinsic plasma emicizumab through heat treatment was observed in patient samples, emicizumab spiked into FVIII-deficient plasma was not or only marginally affected. Application of inhibitor-based FVIII neutralization led to good agreement of results when compared with direct quantification of emicizumab by liquid chromatography-tandem mass spectrometry. CONCLUSION: Inhibitor-based FVIII neutralization appears to be a feasible strategy for accurate measurement of plasma emicizumab levels in the presence of FVIII activity.

Open-Source Implementations of the Reactive Asset Administration Shell: A Survey.

The use of open-source software is crucial for the digitalization of manufacturing, including the implementation of Digital Twins as envisioned in Industry 4.0. This research paper provides a comprehensive comparison of free and open-source implementations of the reactive Asset Administration Shell (AAS) for creating Digital Twins. A structured search on GitHub and Google Scholar was conducted, leading to the selection of four implementations for detailed analysis. Objective evaluation criteria were defined, and a testing framework was created to test support for the most common AAS model elements and API calls. The results show that all implementations support at least a minimal set of required features while none implement the specification in all details, which highlights the challenges of implementing the AAS specification and the incompatibility between different implementations. This paper is therefore the first attempt at a comprehensive comparison of AAS implementations and identifies potential areas for improvement in future implementations. It also provides valuable insights for software developers and researchers in the field of AAS-based Digital Twins.

First Contact, First Learnings - Nursing Staff Approaching Robotics in Health Care.

Systems for service and assistance robotics become relevant in nursing care. Workshops with target user groups can support the reflection and identification of scenarios for the use of robotic systems.

Sila[1]ruthenocenophanes: Long Sought-After Family Members.

During the last three decades, silicon-bridged [1]ferrocenophanes have been developed into an important class of monomers with the resulting polymers being the subject of hundreds of publications. For unknown reasons, not a single example of a silicon-bridged [1]ruthenocenophane is described in the literature. We set out to address this mystery and could synthesize the first sila[1]ruthenocenophanes. Their molecular structures as well as their heat release in the melt, render them as highly strained sandwich compounds that are polymerizable. Why did it take so long to prepare the first sila[1]ruthenocenophanes? This question is addressed by a detailed analysis of the kinetics of the salt-metathesis approach, which reveals a striking difference between ferrocene and ruthenocene.

Site-specific covalent metalation of DNA oligonucleotides with phosphorescent platinum(ii) complexes.

Phosphorescent Pt(II) complexes, composed of a tridentate N^N^C donor ligand and a monodentate ancillary ligand, were covalently attached to DNA oligonucleotides. Three modes of attachment were investigated: positioning the tridentate ligand as an artificial nucleobase via a 2'-deoxyribose or a propane-1,2-diol moiety and orienting it towards the major groove by appending it to a uridine C5 position. The photophysical properties of the complexes depend on the mode of attachment and on the identity of the monodentate ligand (iodido vs. cyanido ligand). Significant duplex stabilization was observed for all cyanido complexes when they are attached to the DNA backbone. The luminescence strongly depends on whether a single or two adjacent complexes are introduced, with the latter showing an additional emission band indicative of excimer formation. The doubly platinated oligonucleotides could be useful as ratiometric or lifetime-based oxygen sensors, as the green photoluminescence intensities and average lifetimes of the monomeric species are drastically boosted upon deoxygenation, whereas the red-shifted excimer phosphorescence is nearly insensitive to the presence of triplet dioxygen in solution.

Antidrug antibodies against the polyethylene glycol moiety inhibit the procoagulant activity of therapeutic polyethylene glycolated factor VIII.

BACKGROUND: The standard therapy for patients with hemophilia A (HA) is the replacement with factor VIII (FVIII) therapeutics. To overcome the limitation of short half-life of wild-type FVIII protein, polyethylene glycol (PEG) can be coupled to therapeutic FVIII to improve pharmacokinetics. OBJECTIVES: We aimed to characterize antibodies developed against a FVIII therapeutic PEGylated with a 40-kDa PEG (40PEG-BDDFVIII) in 2 patients with mild HA. METHODS: An inhouse bead-based immunoassay was developed to characterize and confirm the specificity of the detected antibodies. The neutralizing nature of the antibodies toward PEGylated therapeutics was determined by a modified Nijmegen-Bethesda assay. RESULTS: Two out of 46 patients treated with 40PEG-BDDFVIII developed inhibitory antibodies toward the drug. Switching to a non-PEGylated FVIII successfully increased the FVIII activity in both patients. In patient 1, antibodies were raised against FVIII and PEG. Anti-FVIII antibodies were of the immunoglobulin (Ig)G isotype, whereas anti-PEG antibodies were of IgG, IgM, and IgA isotypes. In patient 2, antibodies of IgG and IgA isotypes were directed only against the PEG moiety. Competitive assays confirmed the specificity of the antibodies against PEG. The applied Nijmegen-Bethesda assay revealed that patients' anti-PEG antibodies and AGP3, an antibody against the backbone of PEG, can inhibit all currently available PEGylated therapeutics but to different degrees. No inhibitory FVIII antibodies were detected. CONCLUSION: Antibodies against the PEG moiety of 40PEG-BDDFVIII abolished the efficacy of the drug. This is the first report on real-world experiences with the development of neutralizing anti-PEG antibodies after treatment with PEGylated FVIII therapeutics in mild HA.

Unexpectedly Large Structural Variations in Benzyl-Substituted Sila[1]ferrocenophanes.

A set of four planar-chiral sila[1]ferrocenophanes equipped with a benzyl group in the α-position, either on one or both Cp rings, and substituted on the bridging silicon atom, either by methyl or phenyl groups, were prepared. While NMR, UV/Vis, and DSC measurements did not show anything uncommon, single crystal X-ray analyses revealed unexpectedly large variations of the dihedral angles between both Cp rings (α tilt angle). While DFT calculations predicted α between 19.6 and 20.8°, measured values were found between 16.6(2) and 21.45(14)°. However, experimentally determined conformers differ significantly from those calculated for the gas phase. For the sila[1]ferrocenophane with the largest mismatch between the experimental and predicted α angle, it was shown that the orientation of benzyl groups have a significant influence on the ring-tilted structure. Packing of molecules in the crystal lattice forces benzyl groups into unusual orientations, resulting in a significantly reduced α angle through steric repulsions.

Efficiency of CMV serodiagnosis during pregnancy in daily laboratory routine.

BACKGROUND: Maternal acute primary cytomegalovirus (CMV) infection during the first trimester may cause severe long-term sequelae in newborns. For risk assessment, serological screening is routinely performed in pregnant women based on IgM, IgG and avidity tests using whole-virus antigen. A recent study evaluated the diagnostic value of recombinant protein-based ELISAs as second-line tests in pregnancy CMV screening, including anti-p52 IgM and anti-gB IgG as markers defining the early and late phase of infection, respectively. In the present study, these recombinant ELISAs were used as first-line screening tests in daily laboratory routine and compared to lysate-based assays with respect to [i] the number of conclusive results obtained with the initial sample and [ii] the underlying workload. METHODS: 553 unselected routine serum samples from pregnant women were tested for anti-CMV IgM and IgG antibodies using lysate-based ELISAs and avidity testing. Anti-CMV IgM antibodies against recombinant p52 and anti-CMV IgG antibodies against recombinant glycoprotein B (gB) were also determined by ELISA. All assays were performed and interpreted according to the manufacturer's instructions. RESULTS: For lysate-based IgM, IgG and avidity testing, 84.6 % of samples yielded conclusive results in a total of 1156 tests, while 15.4 % needed follow-up testing of a consecutive sample. Anti-p52 CMV IgM and anti-gB CMV IgG testing produced conclusive results for 92.8 % of samples in a total of 1026 tests, while 7.2 % samples required follow-up testing. CONCLUSIONS: The first-line use of ELISAs measuring anti-p52 CMV IgM and anti-gB CMV IgG antibodies to test for maternal CMV infection increases the number of conclusive results derived from an initial serum sample while requiring a considerably lower number of tests compared to the lysate-based approach. For day-to-day routines in a diagnostic laboratory, this high efficiency of the recombinant testing approach has significant practical relevance.

Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency.

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.

Chiral-at-Metal Silver-Mediated Base Pairs: Metal-Centred Chirality versus DNA Helical Chirality.

When covalently incorporating ligands capable of forming chiral metal complexes into a DNA oligonucleotide duplex, an enantiospecific formation of metal-mediated base pairs is possible. We have been investigating the chirality of the silver-mediated base pair P-AgI -P (P, 1H-imidazo[4,5-f][1,10]phenanthroline) depending on the number of consecutive P : P pairs within a series of duplexes. Towards this end, both enantiomers of the nucleoside analogue 3-(1H-imidazo[4,5-f][1,10]phenanthrolin-1-yl)propane-1,2-diol comprising an acyclic backbone were introduced into DNA duplexes, resulting in diastereomeric metal-mediated base pairs. The same chiral-at-metal complex is formed inside the duplex for up to five neighbouring P-AgI -P pairs, irrespective of whether (S)-P or (R)-P is used. With six silver-mediated base pairs, the chirality of the metal complex is inverted for (S)-P but not for (R)-P. This indicates an intricate balance of what determines the configuration of the metal complex, the intrinsically preferred metal-centred chirality or the DNA helical chirality.

Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach.

BACKGROUND: The endothelial cell-dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation. METHODS: Endothelial colony-forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC (activated PC) formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of TM (thrombomodulin) expression, ECFCs were stimulated with IL-1ß (interleukin 1ß). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII). RESULTS: The median peak APC concentration was 1.12 nmol/L in experiments with IL-1ß stimulated ECFCs and 3.66 nmol/L without IL-1ß. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031, P=0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo. CONCLUSIONS: Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.

Markers of Hereditary Thrombophilia with Unclear Significance.

Thrombophilia leads to an increased risk of venous thromboembolism. Widely accepted risk factors for thrombophilia comprise deficiencies of protein C, protein S, and antithrombin, as well as the factor V "Leiden" mutation, the prothrombin G20210A mutation, dysfibrinogenemia, and, albeit less conclusive, increased levels of factor VIII. Besides these established markers of thrombophilia, risk factors of unclear significance have been described in the literature. These inherited risk factors include deficiencies or loss-of-activity of the activity of ADAMTS13, heparin cofactor II, plasminogen, tissue factor pathway inhibitor (TFPI), thrombomodulin, protein Z (PZ), as well as PZ-dependent protease inhibitor. On the other hand, thrombophilia has been linked to the gain-of-activity, or elevated levels, of α2-antiplasmin, angiotensin-converting enzyme, coagulation factors IX (FIX) and XI (FXI), fibrinogen, homocysteine, lipoprotein(a), plasminogen activator inhibitor-1 (PAI-1), and thrombin-activatable fibrinolysis inhibitor (TAFI). With respect to the molecular interactions that may influence the thrombotic risk, more complex mechanisms have been described for endothelial protein C receptor (EPCR) and factor XIII (FXIII) Val34Leu. With focus on the risk for venous thrombosis, the present review aims to give an overview on the current knowledge on the significance of the aforementioned markers for thrombophilia screening. According to the current knowledge, there appears to be weak evidence for a potential impact of EPCR, FIX, FXI, FXIII Val34Leu, fibrinogen, homocysteine, PAI-1, PZ, TAFI, and TFPI on the thrombotic risk.