Assessment of inter-operator variability in peripheral monocyte subset gating strategy using flow cytometry in patients with suspected acute stroke.

BACKGROUND: Innovative tools to reliably identify patients with acute stroke are needed. Peripheral monocyte subsets, that is, classical-Mon1, intermediate-Mon2, and non-classical-Mon3, with their activation marker expression analyzed using flow-cytometry (FCM) could be interesting cell biomarker candidates. AIM: To assess the inter-operator variability in a new peripheral monocyte subset gating strategy using FCM in patients with suspected acute stroke. METHODS: In BOOST-study ("Biomarkers-algOrithm-for-strOke-diagnoSis-and Treatment-resistance-prediction," NCT04726839), patients ≥18 years with symptoms suggesting acute stroke within the last 24 h were included. Blood was collected upon admission to emergency unit. FCM analysis was performed using the FACS-CANTO-II® flow-cytometer and Flow-Jo™-software. Analyzed markers were CD45/CD91/CD14/CD16 (monocyte backbone) and CD62L/CD11b/HLA-DR/CD86/CCR2/ICAM-1/CX3CR1/TF (activation markers). Inter-operator agreement (starting from raw-data files) was quantified by the measure distribution and, for each patient, the coefficient of variation (CV). RESULTS: Three operators analyzed 20 patient blood samples. Median inter-operator CVs were below the pre-specified tolerance limits (10% [for Mon1 counts], 20% [Mon2, Mon3 counts], 15% [activation marker median-fluorescence-intensities]). We observed a slight, but systematic, inter-operator effect. Overall, absolute inter-operator differences in fractions of monocyte subsets were <0.03. CONCLUSION: Our gating strategy allowed monocyte subset gating with an acceptable inter-operator variability. Although low, the inter-operator effect should be considered in monocyte data analysis of BOOST-patients.

Importance of cytochrome 3A4 and 2D6-mediated drug-drug interactions in oxycodone consumption among older adults hospitalized for hip fracture: a cross-sectional study.

Hip fracture is a common injury and represents a major health problem with an increasing incidence. In older adults, opioids such as oxycodone are often preferred to other analgesics such as tramadol because of a lower risk of delirium. Different parameters, such as inhibition of cytochrome P450 (CYP450) 2D6 and/or 3A4, can potentially lead to pharmacokinetic variations of oxycodone representing a risk of adverse drugs effects or lack of drug response. There is a risk of drug-drug interactions involving CYP450 in older adults due to the high prevalence of polypharmacy. This study sought to identify patient characteristics that influence oxycodone administration. A single-center observational study included 355 patients with a hip fracture hospitalized in a geriatric postoperative unit. Composite endpoint based on form, duration, and timing to intake separated patients into three groups: "no oxycodone", "low oxycodone ", and "high oxycodone ". CYP450 interactions were studied based on a composite variable defining the most involved CYP450 pathways between CYP2D6 and CYP3A4. CYP450 interactions with CYP2D6 pathway involved were associated with the risk of "high oxycodone" [odds ratio adjusted on age and the type of hip fracture (OR*) 4.52, 95% confidence interval (CI) 1.39-16.83, p = 0.02)], as well as serum albumin levels (OR* 1.09, 95% CI 1.02-1.17, p = 0.01). Cognitive impairment was negatively associated with the risk of "high oxycodone" (OR* 0.38, 95% CI 0.18-0.77, p = 0.02). This study showed an association between CYP2D6 interactions and higher oxycodone consumption indirectly reflecting the existence of uncontrolled postoperative pain.

Case series of massive direct oral anticoagulant ingestion-Treatment and pharmacokinetics data.

BACKGROUND: Direct oral anticoagulants (DOAC) are widely used due to favourable benefit/risk ratio. However, consequences of massive ingestion have been poorly investigated. OBJECTIVES: We aimed to report outcome and pharmacokinetic parameters in patients who massively ingested DOACs. METHODS: We conducted a 5-year cohort study including consecutive massive DOAC ingestion patients admitted to two critical care departments. Patients were managed in accordance with standards of care. We collected the main history, clinical, laboratory, management and outcome data. The time-course of plasma DOAC concentrations measured using specific assays was modelled. RESULTS: Twelve patients (3F/9M; age, 55 years [41-63], median [25th-75th percentiles]) were included. Ingestions involved rivaroxaban (n = 7), apixaban (n = 3) and dabigatran (n = 2), with presumed doses of 9.4-fold [5.0-22.0] the full daily dose. Six patients received activated charcoal but no antidote nor blood-derived product. No bleeding was observed. One patient died due to refractory cardiogenic shock related to bisoprolol co-intoxication. Highest observed peak plasma concentrations were 1720 ng/ml (rivaroxaban), 750 ng/ml (apixaban) and 644 ng/ml (dabigatran). Times to reach DOAC concentration below 50 ng/ml were ~20-45 h (rivaroxaban), ~125 h (apixaban) and ~30-50 h (dabigatran). Elimination half-lives were 2.5-25.5 h (rivaroxaban), 22.0 and 36.5 h (apixaban), and 5.8 and 15.5 h (dabigatran), with substantial interindividual variability and prolongation in case of cardiovascular failure related to co-intoxicants. Charcoal administration, even if delayed, may have contributed to limit toxicity, possibly by reducing absorption and/or enteroenteric recycling. CONCLUSION: No bleeding was observed in this series of massive DOAC ingestions despite elevated plasma concentrations. No patient required specific haemostatic agents. Charcoal administration should be considered to limit toxicity.

White blood count, D-dimers, and ferritin levels as predictive factors of pulmonary embolism suspected upon admission in noncritically ill COVID-19 patients: The French multicenter CLOTVID retrospective study.

BACKGROUND: A high prevalence of pulmonary embolism (PE) has been described during COVID-19. Our aim was to identify predictive factors of PE in non-ICU hospitalized COVID-19 patients. METHODS: Data and outcomes were collected upon admission during a French multicenter retrospective study, including patients hospitalized for COVID-19, with a CT pulmonary angiography (CTPA) performed in the emergency department for suspected PE. Predictive factors significantly associated with PE were identified through a multivariate regression model. RESULTS: A total of 88 patients (median [IQR] age of 68 years [60-78]) were analyzed. Based on CTPA, 47 (53.4%) patients were diagnosed with PE, and 41 were not. D-dimer ≥3000 ng/mL (OR 8.2 [95% CI] 1.3-74.2, sensitivity (Se) 0.84, specificity (Sp) 0.78, P = .03), white blood count (WBC) ≥12.0 G/L (29.5 [2.3-1221.2], Se 0.47, Sp 0.92, P = .02), and ferritin ≥480 µg/L (17.0 [1.7-553.3], Se 0.96, Sp 0.44, P = .03) were independently associated with the PE diagnosis. The presence of the double criterion D-dimer ≥3000 ng/mL and WBC ≥12.0 G/L was greatly associated with PE (OR 21.4 [4.0-397.9], P = .004). CONCLUSION: The white blood count, the D-dimer and ferritin levels could be used as an indication for CTPA to confirm PE on admission in non-ICU COVID-19 patients.

Bleeding and Thrombosis: Insights into Pathophysiology of Bothrops Venom-Related Hemostasis Disorders.

Toxins from Bothrops venoms targeting hemostasis are responsible for a broad range of clinical and biological syndromes including local and systemic bleeding, incoagulability, thrombotic microangiopathy and macrothrombosis. Beyond hemostais disorders, toxins are also involved in the pathogenesis of edema and in most complications such as hypovolemia, cardiovascular collapse, acute kidney injury, myonecrosis, compartmental syndrome and superinfection. These toxins can be classified as enzymatic proteins (snake venom metalloproteinases, snake venom serine proteases, phospholipases A2 and L-amino acid oxidases) and non-enzymatic proteins (desintegrins and C-type lectin proteins). Bleeding is due to a multifocal toxicity targeting vessels, platelets and coagulation factors. Vessel damage due to the degradation of basement membrane and the subsequent disruption of endothelial cell integrity under hydrostatic pressure and tangential shear stress is primarily responsible for bleeding. Hemorrhage is promoted by thrombocytopenia, platelet hypoaggregation, consumption coagulopathy and fibrin(ogen)olysis. Onset of thrombotic microangiopathy is probably due to the switch of endothelium to a prothrombotic phenotype with overexpression of tissue factor and other pro-aggregating biomarkers in association with activation of platelets and coagulation. Thrombosis involving large-caliber vessels in B. lanceolatus envenomation remains a unique entity, which exact pathophysiology remains poorly understood.

International Council for Standardization in Haematology Recommendations for Hemostasis Critical Values, Tests, and Reporting.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), the aim of which is to provide hemostasis-related guidance documents for clinical laboratories. The current ICSH document was developed by an ad hoc committee, comprising an international collection of both clinical and laboratory experts. The purpose of this ICSH document is to provide laboratory guidance for (1) identifying hemostasis (coagulation) tests that have potential patient risk based on analysis, test result, and patient presentations, (2) critical result thresholds, (3) acceptable reporting and documenting mechanisms, and (4) developing laboratory policies. The basis for these recommendations was derived from published data, expert opinion, and good laboratory practice. The committee realizes that regional and local regulations, institutional stakeholders (e.g., physicians, laboratory personnel, hospital managers), and patient types (e.g., adults, pediatric, surgical) will be additional confounders for a given laboratory in generating a critical test list, critical value thresholds, and policy. Nevertheless, we expect this guidance document will be helpful as a framework for local practice.

Modified ROTEM for the detection of rivaroxaban and apixaban anticoagulant activity in whole blood: A diagnostic test study.

BACKGROUND: Rapid detection of the anticoagulant effect of oral factor Xa (FXa) inhibitors may be essential in several emergency clinical situations. Specific assays quantifying the drugs are performed in plasma and require a turnaround time that is too long to be useful in emergency situations. Rotational thromboelastometry (ROTEM) is a whole blood coagulation assay of blood viscoelasticity and could be of interest for FXa inhibitor detection in emergency. However, conventional ROTEM reagents only detect high amounts of inhibitors. OBJECTIVE: The aim of this study was first to assess the effect of whole blood components on the viscoelastic measurement of the effects of FXa inhibitors, and second to evaluate whether a modified ROTEM, triggered with a low amount of tissue factor and a saturating amount of phospholipid vesicles, can reliably detect low levels of FXa inhibitor activity in whole blood. DESIGN: Diagnostic test study. SETTINGS: A university research laboratory. From November 2014 to April 2016. PATIENTS: Sixty-six patients: 30 treated with rivaroxaban, 17 with apixaban and 19 without treatment. INTERVENTION: ROTEM was triggered with 2.5 pmol l of tissue factor and 10 µmol l of phospholipid vesicles. MAIN OUTCOME MEASURES: Modified ROTEM parameters were measured in different experimental conditions: platelet-poor plasma (PPP), platelet-rich plasma, PPP supplemented with fibrinogen and reconstituted whole blood with various haematocrit levels adjusted between 30 and 60%. Modified ROTEM was further validated using whole blood from patients who were either treated or not treated with FXa inhibitors. RESULTS: Modified ROTEM allowed detection of as little as 25 ng ml FXa inhibitors in PPP, with at least a 1.4-fold increase of the clotting time (P ≤ 0.02). Neither changes of fibrinogen concentration nor variations of platelet count or haematocrit precluded FXa inhibitor detection. A lengthened modified ROTEM clotting time of more than 197 s allowed detection of FXa inhibitor concentrations above 30 ng ml in whole blood with 90% sensitivity and 85% specificity. CONCLUSION: Modified ROTEM may be applicable in emergency situations for the detection of FXa inhibitors in whole blood.

Thrombin generation test: A reliable tool to evaluate the pharmacodynamics of vitamin K antagonist rodenticides in rats.

Vitamin K antagonist rodenticide pharmacodynamics (PD) is studied in rodents with traditional laboratory tests. We wondered if thrombin generation test (TGT) could add value. Difethialone (10 mg/kg) was administered per os to 97 OFA-Sprague Dawley rats. PD was studied over a 72 h-period using the Calibrated Automated Thrombogram on platelet poor plasma before and after intoxication (3 female and 3 male rats for each 13 time points) and TGT parameters were compared with the prothrombin time (PT) and vitamin K dependent factor activities previously reported. Following intoxication, preliminary tests evidenced rapid and full inhibition of thrombin generation triggered with 5 or 20 pM human recombinant tissue factor. To study the evolution of TGT parameters following difethialone intake, we adapted the test by complementing intoxicated rat samples with pooled normal rat plasma (3/1, v/v). Adapted TGT confirmed the known higher procoagulant basal level in females compared to males through higher endogenous thrombin potential (ETP) and peak height (PH) (p < 0.0001 and p = 0.0003, respectively). An exponential model fitted well the PH and ETP decay after intoxication. In contrast to PT, the decreases were observed immediately following VKA intake and had comparable time to halving values: 10.5 h (95% CI [8.2; 13.6]) for ETP and 10.4 h (95% CI [7.8; 14.1]) for PH. The decrease of FVII and FX preceded that of PH, ETP and FII while FIX decreased later on, contributing to the severe hypo-coagulability. We demonstrated that TGT performed in samples of intoxicated rats complemented with normal plasma is a reliable tool for evaluation of VKA rodenticide PD in rats.

[Von Willebrand disease in the elderly]. / Maladie de Willebrand du sujet âgé.

Von Willebrand disease in the elderly. Von Willebrand disease (VWD) is a rare inherited haemorrhagic disorder, the prevalence of symptomatic individuals is around 1/10 000. Von Willebrand factor level increases with advanced age, explaining a lower frequency and a lower severity of cutaneous haemorrhagic symptoms with aging. The management of comorbidities in VWD patients is multidisciplinary, on a case by case basis, taking into account scientific society guidelines and haemostasis expert recommendations. The haemorrhagic risk should be systematically evaluated before an invasive procedure or the start of treatment with anticoagulant or antiplatelet drugs, or before the use of some cancer chemotherapy.

Maladie de Willebrand du sujet âgé. La maladie de Willebrand est une maladie hémorragique rare héréditaire (prévalence des formes symptomatiques : 1/10 000). Le facteur Willebrand augmente physiologiquement avec l'âge, d'où une diminution de la fréquence et de la sévérité de la symptomatologie hémorragique. La prise en charge des comorbidités des patients âgés doit rester multidisciplinaire et se faire au cas par cas, en adaptant les recommandations des sociétés savantes et des spécialistes de l'hémostase. Le risque hémorragique est à évaluer avant toute procédure invasive ou tout traitement pouvant majorer ce risque (anticoagulants, antiagrégants plaquettaires, certaines chimiothérapies anticancéreuses).

Vitamin K antagonists in children with heart disease: height and VKORC1 genotype are the main determinants of the warfarin dose requirement.

Managing vitamin K antagonist (VKA) therapy is challenging in children because of a narrow therapeutic range and wide inter- and intra-individual variability in dose response. Only a few small studies have investigated the effect of nongenetic and genetic factors on the dose response to VKAs in children. In a cohort study including 118 children (median age 9 years; range, 3 months-18 years) mostly with cardiac disease, we evaluated by multivariate analysis the relative contribution of nongenetic factors and VKORC1/CYP2C9/CYP4F2 genotypes on warfarin (n = 83) or fluindione (n = 35) maintenance dose and the influence of these factors on the time spent within/above/below the range. The results showed that height, target international normalized ratio and VKORC1 and CYP2C9 genotypes were the main determinants of warfarin dose requirement, accounting for 48.1%, 4.4%, 18.2%, and 2.0% of variability, respectively, and explaining 69.7% of the variability. Our model predicted the warfarin dose within 7 mg/wk in 86.7% of patients. None of the covariates was associated with the time spent above or below the international normalized ratio range. Whether this model predicts accurately the effective maintenance dose is currently being investigated.

Comparisons between diluted thrombin time, ecarin chromogenic assays, and UPLC-MS for plasma level dabigatran quantification: Results from DRIVING study.

INTRODUCTION: The knowledge of dabigatran levels is helpful for decision-making in specific situations such as urgent surgery or when the question of reversal arises (uncontrolled bleeding, eligibility for thrombolysis). However, a limited number of observational studies are available regarding comparisons between quantification methods. The objective of the study was to compare dabigatran plasma levels using three assays including the reference method (high-performance liquid chromatography coupled with mass spectrometry), focusing on the agreement around the 30-50 ng/mL clinically relevant thresholds. METHODS: Sixty healthy volunteers from DRIVING trial (NCT01627665) were given a single 300-mg dabigatran etexilate dose. Serial blood samplings were performed at pre-defined time points (0 to 24 h). We analyzed plasma samples using ultra-performance-liquid chromatography coupled with tandem mass spectrometry (UPLC-MS) (dabigatran reference method); ii/diluted thrombin time (dTT) (Hemoclot-DTI-Hyphen-Biomed); iii/ecarin-based chromogenic assay (ECA-II-Stago). RESULTS: Nine hundred sixty samples were analyzed using the three assays (2759 values). dTT and ECA-II values were highly correlated with those of UPLC-MS (Deming regression). Most values >50 ng/mL were higher using dTT and ECA-II compared to UPLC-MS: biases were constant, +14% and +16% with dTT and ECA-II, respectively (Bland-Altman plots), suggesting that active metabolites accounted for ~15% of thrombin inhibition. Regarding values <30 ng/mL, 30-50 ng/mL, or ≥50 ng/mL, the agreement probability between dTT and ECA-II was of 90.6% [88.4-92.5] (Cohen's kappa coefficient 0.84). CONCLUSION: dTT and ECA-II assays rapidly provide accurate dabigatran-level results for clinical practice, both assays being suitable in emergency, taking into account the thrombin inhibitory effect of dabigatran metabolites.

Is there a role for the laboratory monitoring in the management of specific antidotes of direct oral anticoagulants?

Given the growing number of patients receiving direct oral anticoagulant (DOAC), patients requiring rapid neutralization is also increasing in case of major bleedings or urgent surgery/procedures. Idarucizumab is commercialized as a specific antidote to dabigatran while andexanet alfa has gained the Food and Drug Administration and the European Medicines Agency approval as an oral anti-factor Xa inhibitors antidote. Other antidotes or hemostatic agents are still under preclinical or clinical development, the most advanced being ciraparantag. DOAC plasma levels measurement allows to appropriately select patient for antidote administration and may prevent unnecessary prescription of expensive molecules in some acute clinical settings. However, these tests might be inconclusive after some antidote administration, namely andexanet alfa and ciraparantag. The benefit of laboratory monitoring following DOAC reversal remains unclear. Here, we sought to provide an overview of the key studies evaluating the safety and efficacy of DOAC reversal using the most developed/commercialized specific antidotes, to discuss the potential role of the laboratory monitoring in the management of patients receiving DOAC specific antidotes and to highlight the areas that deserve further investigations in order to establish the exact role of laboratory monitoring in the appropriate management of DOAC specific antidotes.

The use of 1E12, a monoclonal anti-platelet factor 4 antibody, to improve the diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral-based vaccine against SARS-CoV-2 due to prothrombotic immunoglobulin (Ig) G antibodies to platelet factor 4 (PF4) and may be difficult to distinguish from heparin-induced thrombocytopenia (HIT) in patients treated with heparin. OBJECTIVES: We assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIAs) in this context. METHODS: The ability of F(ab')2 fragments of 1E12, 1C12, and 2E1, 3 monoclonal anti-PF4 antibodies, to inhibit the binding of human VITT or HIT antibodies to PF4 was evaluated using EIAs. Alanine-scanning mutagenesis was performed to define the amino acids involved in the interactions between the monoclonal antibodies and PF4. RESULTS: A strong inhibition of VITT IgG binding to PF4 was measured with 1E12 (median inhibition, 93%; n = 8), whereas it had no effect on the binding of HIT antibodies (median, 6%; n = 8). In contrast, 1C12 and 2E1 inhibited VITT (median, 74% and 76%, respectively) and HIT antibodies (median, 68% and 53%, respectively) binding to PF4. When a competitive anti-PF4 EIA was performed with 1E12 for 19 additional VITT samples, it strongly inhibited IgG binding to PF4, except for 1 patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key amino acids on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition. CONCLUSION: A simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible.

Minimally modified human blood coagulation factor X to bypass direct factor Xa inhibitors.

BACKGROUND: Direct oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity. OBJECTIVES: The ability of FXa variants to bypass the direct oral FXa inhibitors was assessed. METHODS: Human FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. RESULTS: F174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients. CONCLUSION: These human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.

Practical Nomogram Predicting Apixaban or Rivaroxaban Concentrations from Low-Molecular-Weight Heparin Anti-Xa Values: Special Interest in Acute Ischemic Stroke Patients.

BACKGROUND AND PURPOSE: In patients with acute ischemic stroke (AIS) using a direct oral factor-Xa anticoagulant (DOAC) during the last 48 hours, a fixed plasma heparin-calibrated anti-Xa activity (0.5 IU/mL) was proposed as a threshold below which patients could be eligible for thrombolysis and/or thrombectomy. Besides, specific DOAC-calibrated anti-Xa thresholds up to 50 ng/mL have been proposed. However, specific DOAC assays are not widely available contrarily to low-molecularweight heparin (LMWH) anti-Xa activity. We developed and validated a nomogram for predicting apixaban and rivaroxaban concentrations based on LMWH anti-Xa assay. METHODS: Our prospective study included apixaban (n=325) and rivaroxaban (n=276) patients. On the same sample, we systematically measured specific DOAC concentration and LMWH anti-Xa activity, using STA®-Liquid-Anti-Xa (Stago) and specific DOAC- or LMWH-calibrators, respectively. The nomogram was built using quantifiable values for both assays on the derivation cohorts with a log-linear regression model. Model performances including sensitivity, specificity, and true positive rate for different thresholds were checked on the validation cohorts. RESULTS: The models built from the derivation cohorts predicted that values <30 ng/mL and <50 ng/ mL DOAC thresholds corresponded to LMWH-anti-Xa values <0.10 IU/mL and <0.64 IU/mL for apixaban; <0.10 IU/mL and <0.71 IU/mL for rivaroxaban. The model accurately predicted apixaban/ rivaroxaban concentrations in the validation cohort. CONCLUSIONS: This easy-to-use nomogram, developed with our reagent, allowed accurately predicting DOAC concentrations based on LMWH-anti-Xa results in emergency situations such as AIS when drug-specific assessments are not rapidly available. Using DOAC <50 ng/mL equivalent threshold, instead of the fixed LMWH <0.5 IU/mL one, would allow proposing thrombolysis to more patients.

Bothrops venom-induced hemostasis disorders in the rat: Between Scylla and Charybdis.

Hemostasis impairment represents the most threatening consequence of Viperidae envenoming, notably with Bothrops genus. In the French departments of America, B. atrox envenomation in French Guiana may lead to bleeding while B. lanceolatus envenomation in Martinique to thrombosis. Bleeding related to B. atrox envenomation is attributed to vascular damage mediated by venom metalloproteinases and blood uncoagulable state resulting from thrombocytopenia and consumptive coagulopathy. Thrombosis related to B. lanceolatus envenomation are poorly understood. We aimed to compare the effects of B. atrox and B. lanceolatus venoms in the rat to identify the determinants of the hemorrhagic versus thrombotic complications. Viscoelastometry (ROTEM), platelet count, plasma fibrinogen, thrombin generation assay, fibrinography, endothelial (von Willebrand factor, ADAMTS13 activity, ICAM-1, and soluble E-selectin), and inflammatory biomarkers (IL-1ß, IL-6, TNF-α, MCP-1, and PAI-1) were determined in blood samples obtained at H3, H6, and H24 after the subcutaneous venom versus saline injection. In comparison to the control, initial fibrinogen consumption was observed with the two venoms while thrombocytopenia and reduction in the clot amplitude only with B. atrox venom. Moreover, we showed an increase in thrombin generation at H3 with the two venoms, an increase in fibrin generation accompanied with hyperfibrinogenemia at H24 and an increase in inflammatory biomarkers with B. lanceolatus venom. No endothelial damage was found with the two venoms. To conclude, our data support two-sided hemostasis complications in Bothrops envenoming with an initial risk of hemorrhage related to platelet consumption and hypocoagulability followed by an increased risk of thrombosis promoted by the activated inflammatory response and rapid-onset fibrinogen restoration.

Assessment of DOAC in GEriatrics (Adage Study): Rivaroxaban/Apixaban Concentrations and Thrombin Generation Profiles in NVAF Very Elderly Patients.

BACKGROUND: Although a growing number of very elderly patients with atrial fibrillation (AF), multiple conditions, and polypharmacy receive direct oral anticoagulants (DOACs), few studies specifically investigated both apixaban/rivaroxaban pharmacokinetics and pharmacodynamics in such patients. AIMS: To investigate: (1) DOAC concentration-time profiles; (2) thrombin generation (TG); and (3) clinical outcomes 6 months after inclusion in very elderly AF in-patients receiving rivaroxaban or apixaban. METHODS: Adage-NCT02464488 was an academic prospective exploratory multicenter study, enrolling AF in-patients aged ≥80 years, receiving DOAC for at least 4 days. Each patient had one to five blood samples at different time points over 20 days. DOAC concentrations were determined using chromogenic assays. TG was investigated using ST-Genesia (STG-ThromboScreen, STG-DrugScreen). RESULTS: We included 215 patients (women 71.1%, mean age: 87 ± 4 years), 104 rivaroxaban and 111 apixaban, and 79.5% receiving reduced-dose regimen. We observed important inter-individual variabilities (coefficient of variation) whatever the regimen, at C max [49-46%] and C min [75-61%] in 15 mg rivaroxaban and 2.5 mg apixaban patients, respectively. The dose regimen was associated with C max and C min plasma concentrations in apixaban (p = 0.0058 and p = 0.0222, respectively), but not in rivaroxaban samples (multivariate analysis). Moreover, substantial variability of thrombin peak height (STG-ThromboScreen) was noticed at a given plasma concentration for both xabans, suggesting an impact of the underlying coagulation status on TG in elderly in-patients. After 6-month follow-up, major bleeding/thromboembolic event/death rates were 6.7%/1.0%/17.3% in rivaroxaban and 5.4%/3.6%/18.9% in apixaban patients, respectively. CONCLUSION: Our study provides original data in very elderly patients receiving DOAC in a real-life setting, showing great inter-individual variability in plasma concentrations and TG parameters. Further research is needed to understand the potential clinical impact of these findings.

Bothrops atrox and Bothrops lanceolatus Venoms In Vitro Investigation: Composition, Procoagulant Effects, Co-Factor Dependency, and Correction Using Antivenoms.

Bothrops venoms are rich in enzymes acting on platelets and coagulation. This action is dependent on two major co-factors, i.e., calcium and phospholipids, while antivenoms variably neutralize venom-related coagulopathy effects. Our aims were (i) to describe the composition of B. atrox and B. lanceolatus venoms; (ii) to study their activity on the whole blood using rotational thromboelastometry (ROTEM); (iii) to evaluate the contribution of calcium and phospholipids in their activity; and (iv) to compare the effectiveness of four antivenoms (Bothrofav™, Inoserp™ South America, Antivipmyn™ TRI, and PoliVal-ICP™) on the procoagulant activity of these two venoms. Venom composition was comparable. Both venoms exhibited hypercoagulant effects. B. lanceolatus venom was completely dependent on calcium but less dependent on phospholipids than B. atrox venom to induce in vitro coagulation. The four antivenoms neutralized the procoagulant activity of the two venoms; however, with quantitative differences. Bothrofav™ was more effective against both venoms than the three other antivenoms. The relatively similar venom-induced effects in vitro were unexpected considering the opposite clinical manifestations resulting from envenomation (i.e., systemic bleeding with B. atrox and thrombosis with B. lanceolatus). In vivo studies are warranted to better understand the pathophysiology of systemic bleeding and thrombosis associated with Bothrops bites.