Peptidylarginine deiminase 4 and ADAMTS13 activity in Staphylococcus aureus bacteraemia.

Staphylococcus aureus infection is associated with increased levels of neutrophil extracellular traps (NETs) and von Willebrand factor (VWF), and with reduced activity of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). Peptidylarginine deiminase 4 (PAD4) contributes to NET formation and inactivates ADAMTS13 in vitro. The role of PADs in the dynamics of NETs, VWF and ADAMTS13 has not yet been studied. We thus aimed to assess the longitudinal evolution of NETs, PADs, VWF and ADAMTS13 activity in S. aureus infection. Plasma samples from S. aureus bacteraemia patients were longitudinally collected and analysed for NETs, PAD4/PAD2, VWF and ADAMTS13 activity. Correlation analyses with clinical data were performed. Recombinant PAD4 and S. aureus were assessed in vitro for their potential to modulate ADAMTS13 activity. Sixty-seven patients were included. Plasma levels of NETs, VWF, PAD4 and PAD2 were increased and ADAMTS13 activity was decreased. Levels of PADs were negatively correlated with ADAMTS13 activity. NETs were positively correlated with PADs, and negatively with ADAMTS13 activity. In vitro, recombinant PAD4 but not S. aureus reduced ADAMTS13 activity in plasma. Levels of PAD4 and PAD2 correlate with reduced ADAMTS13 activity, with neutrophils as the likely source of PAD activity in S. aureus bacteraemia. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Management of Bleeding and Hemolysis During Percutaneous Microaxial Flow Pump Support: A Practical Approach.

Percutaneous ventricular assist devices (pVADs) are increasingly being used because of improved experience and availability. The Impella (Abiomed), a percutaneous microaxial, continuous-flow, short-term ventricular assist device, requires meticulous postimplantation management to avoid the 2 most frequent complications, namely, bleeding and hemolysis. A standardized approach to the prevention, detection, and treatment of these complications is mandatory to improve outcomes. The risk for hemolysis is mostly influenced by pump instability, resulting from patient- or device-related factors. Upfront echocardiographic assessment, frequent monitoring, and prompt intervention are essential. The precarious hemostatic balance during pVAD support results from the combination of a procoagulant state, due to critical illness and contact pathway activation, together with a variety of factors aggravating bleeding risk. Preventive strategies and appropriate management, adapted to the impact of the bleeding, are crucial. This review offers a guide to physicians to tackle these device-related complications in this critically ill pVAD-supported patient population.

Emicizumab for acquired haemophilia A: A case series.

BACKGROUND: Emicizumab is approved to prevent bleeding in patients with congenital haemophilia A with or without inhibitors. However, no randomized trials addressed the efficacy of emicizumab in acquired haemophilia A (AHA). AIMS: To report the clinical and biochemical response of emicizumab in AHA. METHODS: This single-centre retrospective study included seven adults with AHA between November 2020 and May 2022. We collected patient characteristics, laboratory coagulation parameters, the use of haemostatic agents, bleeds and thrombotic events. Treatment was monitored using chromogenic FVIII assays. The assay with human reagents assesses both the emicizumab FVIII-like-activity and native patient FVIII-activity. The assay with bovine reagents only measures the patients' native FVIII-activity as emicizumab does not bind to bovine reagents. RESULTS: Patients presented with spontaneous hematoma (n = 7), intramuscular bleeding (n = 2), haematuria (n = 2) and/or gastro-intestinal bleeding (n = 2). Six patients had major bleedings. At diagnosis, APTT was prolonged (91 seconds, IQR 73-103), FVIII activity was 0% (IQR 0-1) and FVIII inhibitor 182 BU/mL (IQR 104-228). Emicizumab was administered weekly (3 mg/kg) for 4 weeks, and thereafter every 2 weeks until regression of the inhibitor. Three patients received activated FVIIa (cumulative dose of 1.7 mg/kg, IQR 1.2-2.2). All bleedings were controlled after treatment initiation, without further bleeds. After starting emicizumab, FVIII-like activity reached ≥5% at 12 days (IQR 7-14), whereas recovery of the intrinsic FVIII-activity ≥5% occurred at 128 days (IQR 88-173), coinciding with the disappearance of the FVIII inhibitor. There were no safety issues. CONCLUSION: In this AHA case series, no new clinically relevant bleeds were observed after initiation of emicizumab in conjunction with standard immunosuppressive therapy.

Impairment of Angiogenesis-Driven Clot Resolution Is a Key Event in the Progression to Chronic Thromboembolic Pulmonary Hypertension: Validation in a Novel Rabbit Model.

BACKGROUND: Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening condition and rare complication of acute pulmonary embolism. Mechanisms underlying impaired clot resolution and in sustained fibrothrombotic obstruction of the pulmonary arterial bed remain poorly understood. Since defective angiogenesis correlated to defective clot resolution based on observations in surgical material from patients with CTEPH, we aimed to validate its crucial pathogenic role by intrathrombus inhibition of angiogenesis in a novel CTEPH rabbit model. METHODS: We aimed to compare whether intrathrombus administration of an antifibrinolytic agent, tranexamic acid, or an inhibitor of angiogenesis, SU5416, would contribute to CTEPH progression. Both products were administered on a weekly basis by autologous clot embolization in rabbits. Right ventricular pressure was monitored by telemetry, right ventricular function by transthoracic echocardiography, and a complete pulmonary hemodynamic evaluation was obtained through right heart catheterization. Markers of inflammation, endothelial dysfunction, heart failure, and fibrinolysis were measured in plasma. Pulmonary vessel remodeling was analyzed by immunohistochemistry. RESULTS: Impairing intrathrombus angiogenesis by repeatedly embolizing autologous blood clots containing SU5416 resulted in elevated mean pulmonary arterial pressure (38 mm Hg), increased indexed pulmonary vascular resistance, and enhanced right ventricular hypertrophy (80%, 1.9-fold, 36%, respectively, compared with rabbits embolized with clots containing an antifibrinolytic agent). This was caused by both obstruction of large pulmonary arteries with fibrothrombotic material and muscularization of pulmonary microvessels, and accompanied by inflammatory cell infiltration and increased circulating endothelin-1. CONCLUSIONS: The key role of angiogenesis-driven clot resolution was validated in a reliable small-animal model reproducing the major pathophysiological hallmarks of CTEPH.

Clinical application of multigene panel testing for bleeding, thrombotic, and platelet disorders: a 3-year Belgian experience.

BACKGROUND: The international study ThromboGenomics has evaluated the diagnostic rate using a targeted multigene panel test for the screening of inherited bleeding, thrombotic and platelet disorders. OBJECTIVES: We retrospectively analyzed the results of the implementation of genetic testing for inherited bleeding, thrombotic and platelet disorders in Belgian clinical practice and evaluated possible reclassification of reported variants. PATIENTS/METHODS: We implemented a Thrombosis-Hemostasis multigene panel test using whole exome sequencing to diagnose 487 patients recruited by 27 different Belgian hospitals with the implementation of stringent laboratory accreditation standards and by studying up to 100 diagnostic-grade genes. RESULTS: This Thrombosis-Hemostasis multigene panel test was able to detect at least one genetic variant in 58% of the 487 patients of which 50% were (likely) pathogenic variants and the others were variants of unknown significance. Polygenic variants were detected in 65 patients (13%). A multi-step workflow for results discussion by multidisciplinary team meetings and patients' recalls for segregation studies and additional laboratory testing was set up. Variants were also submitted to the GoldVariants database from the International Society on Thrombosis and Haemostasis (ISTH). The aim of these approaches is to optimize variant interpretation and to (re)classify variants of unknown significance as (likely) pathogenic or (likely) benign. CONCLUSIONS: The growing implementation of multigene panel tests in clinical diagnostics comes with difficulties in interpreting genetic results. Additional efforts are needed to continuously optimize the diagnostic outcome.

Pro-haemostatic effect of DDAVP is partially derived through non-classical (CD14dim /CD16++ ) monocytes residing the spleen.

The splenic endothelial Weibel-palade bodies are one of the most important candidate organelles to release von Willebrand factor upon stimulation with desmopressin. However, the presence of functional desmopressin-specific receptor has not yet been demonstrated on endothelial cells. Experimental evidences are in favour of an indirect pro-haemostatic effect of desmopressin, but the exact mediator and its cellular origin are largely elusive. Here, we report partially hampered desmopressin response in a splenectomised severe haemophilia A/Beta Thalassemia patient without any genetic variant relevant to his incomplete desmopressin response. To further investigate the role of the spleen in this phenomenon, the release of VWF from desmopressin-treated human splenic endothelial cells was assessed in vitro. As a result, desmopressin induced the release of VWF from endothelial cells when the cells were co-cultured with non-classical (CD14dim /CD16++ ), but not other subtypes of monocytes or PBMCs. This in vitro study which resembles close proximity of endothelial cells of sinusoids to monocyte reservoir reside in parenchyma of subcapsular red pulp of the spleen sheds a light upon the role of this highly vascularized VWF-producing organ in driving indirect effect of desmopressin.

Dysregulation of the kallikrein-kinin system in bronchoalveolar lavage fluid of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a critical component of the kallikrein-kinin system. Its dysregulation may lead to increased vascular permeability and release of inflammatory chemokines. Interactions between the kallikrein-kinin and the coagulation system might further contribute to thromboembolic complications in COVID-19. METHODS: In this observational study, we measured plasma and tissue kallikrein hydrolytic activity, levels of kinin peptides, and myeloperoxidase (MPO)-DNA complexes as a biomarker for neutrophil extracellular traps (NETs), in bronchoalveolar lavage (BAL) fluid from patients with and without COVID-19. FINDINGS: In BAL fluid from patients with severe COVID-19 (n = 21, of which 19 were mechanically ventilated), we observed higher tissue kallikrein activity (18·2 pM [1·2-1535·0], median [range], n = 9 vs 3·8 [0·0-22·0], n = 11; p = 0·030), higher levels of the kinin peptide bradykinin-(1-5) (89·6 [0·0-2425·0], n = 21 vs 0·0 [0·0-374·0], n = 19, p = 0·001), and higher levels of MPO-DNA complexes (699·0 ng/mL [66·0-142621·0], n = 21 vs 70·5 [9·9-960·0], n = 19, p < 0·001) compared to patients without COVID-19. INTERPRETATION: Our observations support the hypothesis that dysregulation of the kallikrein-kinin system might occur in mechanically ventilated patients with severe pulmonary disease, which might help to explain the clinical presentation of patients with severe COVID-19 developing pulmonary oedema and thromboembolic complications. Therefore, targeting the kallikrein-kinin system should be further explored as a potential treatment option for patients with severe COVID-19. FUNDING: Research Foundation-Flanders (G0G4720N, 1843418N), KU Leuven COVID research fund.

Apixaban in a porcine model of mechanical valve thrombosis in pulmonary position-a pilot study.

OBJECTIVES: The newest mechanical valves have low thrombogenicity, making them candidates for anticoagulation with a direct oral anticoagulant. While these drugs hold great promise to replace warfarin, clinical trials have been disappointing so far. We aimed to evaluate apixaban in a porcine model of mechanical valve thrombosis with On-X® (CryoLife) aortic valves implanted in pulmonary position. METHODS: On-X® valves were implanted in pulmonary valve position in 9 Yucatan pigs. Animals received prophylactic enoxaparin 40 mg for 1 week. Pigs in the low-dose group received 5 mg apixaban twice daily for 10 weeks. The intermediary-dose group received 5 mg twice daily for 6 weeks and then 10 mg twice daily afterwards. The high-dose group received 15 mg twice daily for 10 weeks. After sacrifice, valves were macroscopically evaluated and thrombus weight was documented. RESULTS: The median weight of the 9 animals was 64.3 kg, range 52.5-70.9. In the low-dose group (2 animals), both valves showed manifest, chronic thrombosis with blocked hinges. In the intermediary-dose group, a normal functioning valve without thrombosis was seen in 2/4 animals. In the high-dose group (3 pigs), there was no valve thrombosis. No bleeding events occurred. In all animals, apixaban plasma levels were low compared to clinical target levels. CONCLUSIONS: The pulmonary position seems to be an aggressive model for mechanical valve thrombosis in pigs. Apixaban has the potential to prevent valve thrombosis, even in these thrombogenic conditions. Detailed pharmacokinetic studies are needed to determine the ideal apixaban dosage for future experiments and to enable extrapolation to the clinical situation.

Coagulation Factors Accumulate During Normothermic Liver Machine Perfusion Regardless of Donor Type and Severity of Ischemic Injury.

BACKGROUND: Coagulation factors may inform on liver function during normothermic machine perfusion (NMP). We investigated whether graft ischemic injury impairs the accumulation of anticoagulation factors during NMP of porcine and human livers. METHODS: Dynamics of FV, FVII, FVIII, FIX, and FX during NMP and their correlation with graft injury was investigated in porcine livers with minimal (no warm ischemia, n = 5) or severe injury (60 min warm ischemia, n = 5). Next, FV, FVIII, FIX, fibrinogen, and antithrombin were measured in 35 matched human liver NMPs from the COPE trial. Correlation of these factors with outcomes was explored. Livers were categorized in to 4 groups depending on donor type and posttransplant peak aspartate aminotransferase (AST) as surrogate of minimal (peak < 500 IU/L) or moderate injury (peak > 1000 IU/L). RESULTS: Factor concentrations increased significantly during NMP regardless of severity of injury. In porcine livers, factor concentrations were 2- to 6-fold lower in severely injured grafts (all P < 0.05). All factors negatively correlated with AST (coefficient range: from -0.50 to -0.93; all P < 0.05) and lactate (range: from -0.51 to -0.67; all P < 0.05). In human livers, no difference in factor accumulation rates and no correlation with other markers were observed. One graft with primary nonfunction had low rate of factor accumulation. CONCLUSIONS: Anticoagulation factors accumulate during NMP regardless of donor type and severity of injury. In pigs, severe ischemic injury resulted in significantly lower factor concentrations. In human livers with life-sustaining function, they do not correlate with hepatic injury. Whether low concentrations predict nonfunction in high-risk livers with severe injury requires further investigation.

Factor VIII-Fc Activates Natural Killer Cells via Fc-Mediated Interactions With CD16.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level <1%) hemophilia A (HA) patients and are a serious hindrance to effective management of HA. Consequently, strategies that can either prevent anti-FVIII inhibitors from developing or "tolerize" individuals who develop such antibodies represent a clinically important unmet need. One intervention for patients with high-titer inhibitors is immune tolerance induction (ITI) therapy. Although ITI therapy is the only clinically proven strategy to eradicate anti-FVIII inhibitors, mechanisms of inhibitor reduction remain unknown. Factor VIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor used in replacement therapy for HA. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasma half-life via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Venous Thromboembolism in Patients Discharged after COVID-19 Hospitalization.

BACKGROUND: Venous thromboembolism (VTE) is a frequent complication of COVID-19, so that the importance of adequate in-hospital thromboprophylaxis in patients hospitalized with COVID-19 is well established. However, the incidence of VTE after discharge and whether postdischarge thromboprophylaxis is beneficial and safe are unclear. In this prospective observational single-center study, we report the incidence of VTE 6 weeks after hospitalization and the use of postdischarge thromboprophylaxis. METHODS: Patients hospitalized with confirmed COVID-19 were invited to a multidisciplinary follow-up clinic 6 weeks after discharge. D-dimer and C-reactive protein were measured, and all patients were screened for deep vein thrombosis with venous duplex-ultrasound. Additionally, selected high-risk patients received computed tomography pulmonary angiogram or ventilation-perfusion (V/Q) scan to screen for incidental pulmonary embolism. RESULTS: Of 485 consecutive patients hospitalized from March through June 2020, 146 patients were analyzed, of which 39% had been admitted to the intensive care unit (ICU). Postdischarge thromboprophylaxis was prescribed in 28% of patients, but was used more frequently after ICU stay (61%) and in patients with higher maximal D-dimer and C-reactive protein levels during hospitalization. Six weeks after discharge, elevated D-dimer values were present in 32% of ward and 42% of ICU patients. Only one asymptomatic deep vein thrombosis (0.7%) and one symptomatic pulmonary embolism (0.7%) were diagnosed with systematic screening. No bleedings were reported. CONCLUSION: In patients who had been hospitalized with COVID-19, systematic screening for VTE 6 weeks after discharge revealed a low incidence of VTE. A strategy of selectively providing postdischarge thromboprophylaxis in high-risk patients seems safe and potentially effective.

Optimization of the detection of inhibitory autoantibodies against the VWF-cleaving protease ADAMTS13 with an automated chemiluminescent ADAMTS13 activity immunoassay.

INTRODUCTION: Acquired thrombotic thrombocytopenic purpura is a rare disease associated with the production of autoantibodies against the VWF-cleaving protease ADAMTS13. The detection of these antibodies is made difficult by the instability of ADAMTS13 in citrated plasma and the time-consuming ADAMTS13 assays. The aim of our study was to evaluate the optimal conditions for detecting anti-ADAMTS13 inhibitory antibodies with the novel automated chemiluminescent immunoassay HemosILR AcuStar ADAMTS13 Activity assay. METHODS: The parallelism between the AcuStar ADAMTS13 calibration curve and ADAMTS13 concentrations in serially diluted citrated plasma was evaluated after 2 hours incubation at 25°C, 37°C, or 37°C after addition of Ca2+ to preserve the activity of the metalloprotease. Using Bethesda assays based on the 3 incubation procedures and the HemosILR AcuStar ADAMTS13 Activity assay, the inhibitor titers were determined in patients' samples with ADAMTS13 antibodies and compared with those determined using the TechnozymR ADAMTS13 activity ELISA. RESULTS: The criterion of parallelism was respected for the 3 incubation methods over the range of ADAMTS13 concentrations relevant for the detection of ADAMTS13 inhibitor antibodies in a Bethesda assay. In agreement with this observation, all the incubation methods permitted the accurate detection and quantification of inhibitory anti-ADAMTS13 antibodies in the samples from patients with acquired thrombotic thrombocytopenic purpura. CONCLUSION: Incubation of plasma samples with normal plasma at 25°C, 37°C, or 37°C after addition of Ca2+ can be used in a Bethesda assay for quantifying the inhibitory activity of antibodies interfering with ADAMTS13 in the chemiluminescent HemosILR AcuStar ADAMTS13 Activity assay.

Apixaban in patients on haemodialysis: a single-dose pharmacokinetics study.

BACKGROUND: Apixaban, a direct oral anticoagulant inhibiting factor Xa, has been proven to reduce the risk of atrial fibrillation-related stroke and thromboembolism in patients with mild to moderate renal insufficiency. Patients on renal replacement therapy, however, were excluded from randomized controlled trials. Therefore, uncertainty remains concerning benefits, dosing and timing of intake in haemodialysis population. METHODS: We conducted a Phase II pharmacokinetics study in which 24 patients on maintenance haemodialysis were given a single dose (2.5 mg or 5 mg) of apixaban, either 30 min before or immediately after dialysis on the mid-week dialysis day. RESULTS: Apixaban 5 mg resulted in higher area under the curve (AUC0-48) in comparison with 2.5 mg, although significance could only be reached for dosing pre-dialysis (2.5 mg versus 5 mg, P = 0.008). In line, peak concentrations (Cmax) after dosing pre-dialysis were significantly higher in the 5 mg than in the 2.5 mg groups (P = 0.02). In addition, dialysis resulted in significant reduction of drug exposure. AUC0-48 pre-dialysis were on average 48% (2.5 mg) and 26% (5 mg) lower than the AUC0-48 post-dialysis, in line with Cmax. As a result, a dose of 2.5 mg post-dialysis and a dose of 5 mg pre-dialysis resulted in similar AUC0-48. In contrast, significant differences were found between the 5 mg group post-dialysis and the 2.5 mg group pre-dialysis (P = 0.02). CONCLUSIONS: Our data suggest that exposure to apixaban in patients on maintenance haemodialysis is dependent not only on drug dose but also on timing of intake relative to the haemodialysis procedure.

Von Willebrand factor and ADAMTS13 impact on the outcome of Staphylococcus aureus sepsis.

BACKGROUND: Previous clinical evidence correlates levels of von Willebrand factor (VWF) and its cleaving protease ADAMTS13 with outcome in septic patients. No previous studies addressed if VWF and ADAMTS13 affected the outcome of Staphylococcus aureus sepsis. OBJECTIVES: We studied the role of VWF and ADAMTS13 in S. aureus sepsis both in patients and in mice. METHODS: VWF levels and ADAMTS13 activity levels were measured in plasma samples from 89 S. aureus bacteremia patients by chemiluminescent assays and were correlated with clinical sepsis outcome parameters. In wild-type mice and mice deficient in VWF and ADAMTS13, we investigated the outcome of S. aureus sepsis and quantified bacterial clearance and organ microthrombi. RESULTS: In patients with S. aureus bloodstream infections, high VWF levels and low ADAMTS13 activity levels correlated with disease severity and with parameters of inflammation and disseminated intravascular coagulation. In septic mice, VWF deficiency attenuated mortality, whereas ADAMTS13 deficiency increased mortality. Bacterial clearance was enhanced in VWF-deficient mice. The differences in mortality for the studied genotypes were associated with differential loads of organ microthrombi in both liver and kidneys. CONCLUSIONS: In conclusion, this study reports the consistent relation of VWF, ADAMTS13 and their ratio to disease severity in patients and mice with S. aureus sepsis. Targeting VWF multimers and/or the relative ADAMTS13 deficiency that occurs in sepsis should be explored as a potential new therapeutic target in S. aureus endovascular infections.

Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves.

INTRODUCTION: The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs. AIM: The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels. METHODS: We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism. RESULTS: Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25 IU/dL in spiked samples and in detection of FVIII activity (≥1 IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results. CONCLUSION: Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments.

Pure ultra-fine carbon particles do not exert pro-coagulation and inflammatory effects on microvascular endothelial cells.

Pro-thrombotic and inflammatory changes play an important role in cardiovascular morbidity and mortality, resulting from short-term exposure to fine particulate air-pollution. Part of those effects has been attributed to the ultra-fine particles (UFPs) that pass through the lung and directly contact blood-exposed and circulating cells. Despite UFP-induced platelet activation, it is unclear whether the penetrated particles exert any direct effect on endothelial cells. While exposure levels are boosting as a result of world-wide increases in economic development and desertification, which create more air-polluted regions, as well as increase in demands for synthetic UFPs in medicine and various industries, further studies on the health effects of these particles are required. In this study, human pulmonary and cardiac microvascular endothelial cells (MECs) have been exposed to 0.1, 1, 10, and 100 µg/ml suspensions of either a natural (carbon black) or a synthetic (multi-walled carbon nano-tubes) type of UFPs, in vitro. As a result, no changes in the levels of coagulation factor VIII, Von Willebrand factor, Interleukin 8, and P-selectin measured in the cells' supernatant were observed prior to and 6, 12, and 24 h after exposure. In parallel, the spatio-temporal effect of UFPs on cardiac MECs was evaluated by Transmission Electron Microscopy. Despite phagocytic uptake of pure UFPs observed on cellular sections of the treated cells, Weibel-Palade bodies remained intact in shape and similar in number when compared with the untreated cells. Our work shows that carbon itself is a non-toxic carrier for endothelial cells.