Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells.

BACKGROUND: We evaluated the efficacy of the factor Xa inhibitor rivaroxaban on the differentiation ability of vascular endothelial progenitor cells (EPCs), which play roles in vascular injury repair and atherogenesis. Antithrombotic treatment in patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI) is challenging, and current guidelines recommend oral anticoagulant monotherapy 1 year or more after PCI. However, biological evidence of the pharmacological effects of anticoagulants is insufficient. METHODS: EPC colony-forming assays were performed using peripheral blood-derived CD34-positive cells from healthy volunteers. Adhesion and tube formation of cultured EPCs were assessed in human umbilical cord-derived CD34-positive cells. Endothelial cell surface markers were assessed using flow cytometry, and Akt and endothelial nitric oxide synthase (eNOS) phosphorylation were examined using western blot analysis of EPCs. Adhesion, tube formation and endothelial cell surface marker expression was observed in EPCs transfected with small interfering RNA (siRNA) against protease-activated receptor (PAR)-2. Finally, EPC behaviors were assessed in patients with atrial fibrillation undergoing PCI in whom warfarin was changed to rivaroxaban. RESULTS: Rivaroxaban increased the number of large EPC colonies and increased the bioactivities of EPCs, including adhesion and tube formation. Rivaroxaban also increased vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, Tie-2, and E-selectin expression as well as Akt and eNOS phosphorylation. PAR-2 knockdown increased the bioactivities of EPCs and endothelial cell surface marker expression. Patients in whom the number of large colonies increased after switching to rivaroxaban showed better vascular repair. CONCLUSIONS: Rivaroxaban increased the differentiation ability of EPCs, leading to potential advantages in the treatment of coronary artery disease.

Spike Protein Subunits of SARS-CoV-2 Alter Mitochondrial Metabolism in Human Pulmonary Microvascular Endothelial Cells: Involvement of Factor Xa.

Background: Mitochondria have been involved in host defense upon viral infections. Factor Xa (FXa), a coagulating factor, may also have influence on mitochondrial functionalities. The aim was to analyze if in human pulmonary microvascular endothelial cells (HPMEC), the SARS-CoV-2 (COVID-19) spike protein subunits, S1 and S2 (S1+S2), could alter mitochondrial metabolism and what is the role of FXA. Methods: HPMEC were incubated with and without recombinants S1+S2 (10 nmol/L each). Results: In control conditions, S1+S2 failed to modify FXa expression. However, in LPS (1 µg/mL)-incubated HPMEC, S1+S2 significantly increased FXa production. LPS tended to reduce mitochondrial membrane potential with respect to control, but in higher and significant degree, it was reduced when S1+S2 were present. LPS did not significantly modify cytochrome c oxidase activity as compared with control. Addition of S1+S2 spike subunits to LPS-incubated HPMEC significantly increased cytochrome c oxidase activity with respect to control. Lactate dehydrogenase activity was also increased by S1+S2 with respect to control and LPS alone. Protein expression level of uncoupled protein-2 (UCP-2) was markedly expressed when S1+S2 were added together to LPS. Rivaroxaban (50 nmol/L), a specific FXa inhibitor, significantly reduced all the above-mentioned alterations induced by S1+S2 including UCP-2 expression. Conclusions: In HPMEC undergoing to preinflammatory condition, COVID-19 S1+S2 spike subunits promoted alterations in mitochondria metabolism suggesting a shift from aerobic towards anaerobic metabolism that was accompanied of high FXa production. Rivaroxaban prevented all the mitochondrial metabolic changes mediated by the present COVID-19 S1 and S2 spike subunits suggesting the involvement of endogenous FXa.

Evaluation of Xa inhibitors as potential inhibitors of the SARS-CoV-2 Mpro protease.

Based on previous large-scale in silico screening several factor Xa inhibitors were proposed to potentially inhibit SARS-CoV-2 Mpro. In addition to their known anticoagulants activity this potential inhibition could have an additional therapeutic effect on patients with COVID-19 disease. In this study we examined the binding of the Apixaban, Betrixaban and Rivaroxaban to the SARS-CoV-2 Mpro with the use of the MicroScale Thermophoresis technique. Our results indicate that the experimentally measured binding affinity is weak and the therapeutic effect due to the SARS-CoV-2 Mpro inhibition is rather negligible.

Synthesis of 3-O-Sulfated Heparan Sulfate Oligosaccharides Using 3-O-Sulfotransferase Isoform 4.

Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.

Severe Inflammation, Acute Kidney Injury, and Drug-Drug Interaction: Triple Penalty for Prolonged Elimination of Apixaban in Patients With Coronavirus Disease 2019: A Grand Round.

ABSTRACT: In this article, we present a case of apixaban elimination prolonged by 450% in a patient with coronavirus disease 2019 because of multiple conditions, including drug-drug interaction, severe inflammation, and acute kidney injury. Therapeutic drug monitoring was used to explain unusual routine coagulation assays. This grand round highlights the importance of dialog between the clinician and a therapeutic drug monitoring consultant for optimal patient care.

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation.

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Circulating heparin-like anticoagulants: Case report and review of literature.

We report a case of a 56-year-old woman with a history of idiopathic thrombocytopenic purpura (ITP) following splenectomy on mycophenolate mofetil (MMF), who developed moderate bleeding after stopping MMF. Her laboratory testing suggested the presence of an abnormal circulating heparin-like anticoagulant with demonstrable anti-Xa activity. She was initially treated with antifibrinolytic therapy and was subsequently started on MMF alongside intravenous immunoglobulin, which significantly improved her bleeding symptoms. The presence of abnormal circulating heparin-like anticoagulants is a rare cause of coagulopathy. Few cases exist in the literature, with nearly all occurring in the setting of hematologic or solid-organ malignancy. The mechanism by which these endogenous anticoagulants develop is unclear. Clinical manifestations range from mild bleeding and bruising to life-threatening hemorrhage refractory to conventional therapy. Diagnosis of a heparin-like anticoagulant is based on coagulation testing as well as exclusion of other exogenous anticoagulants, acquired inhibitors, and/or factor deficiencies.

Dosage reduction of low weight heparin in patients with renal dysfunction: Effects on anti-Xa levels and clinical outcomes.

BACKGROUND: To prevent bio-accumulation of low molecular weight heparins (LMWHs) in patients with decreased kidney function, dosage reduction and anti-Xa monitoring has been suggested. The aim of this study was to investigate the effect of pre-emptive dosage reduction of LMWH on anti-Xa levels. Furthermore, we investigated the association between anti-Xa levels and bleeding, thrombotic events and mortality. METHODS: In this single center study, we followed 499 patients with decreased renal function in whom anti-Xa levels were measured. We observed how many patients had anti-Xa levels that fell within the reference range, with a standard protocol of a pre-emptive dosage reduction of LMWH (25% reduction in patients with an estimated glomerular filtration rate (eGFR) between 30 and 60 ml/min/1.73m2 and a reduction of 50% in patients with an eGFR below the 30 ml/min/1.73m2). Furthermore, Cox proportional hazard analyses were used to estimate hazard ratios to investigate the association between anti-Xa levels and major bleeding, thrombotic events and mortality within three months of follow-up. RESULTS: In a cohort of 499 patients (445 dalteparin and 54 nadroparin users), a pre-emptive dosage reduction of LMWH led to adequate levels of anti-Xa in only 19% of the patients (12% for the dalteparin users and 50% for nadroparin users). We did not find an association between anti-Xa levels and bleeding, thrombosis or mortality. CONCLUSION: Pre-emptive dosage reduction of LMWH leads to low anti-Xa levels in a large proportion, but this was not associated with bleeding, thrombosis or mortality.

Perils of Antithrombotic Transitions: Effect of Oral Factor Xa Inhibitors on the Heparin Antifactor Xa Assay.

BACKGROUND: Oral factor Xa inhibitors (OFXais) may interfere with the heparin antifactor Xa (antiXa) assay. The best method to measure heparin activity during the transition from an OFXai to intravenous (IV) unfractionated heparin (UFH) remains unknown. This study aimed to assess the safety and effectiveness of transitioning from an OFXai to UFH. METHODS: A retrospective analysis was conducted of patients with supratherapeutic antiXa levels on UFH who received either apixaban or rivaroxaban within 72 hours before UFH initiation at NYU Langone Health. The primary objective was to identify the incidence of interference on the heparin antiXa assay due to OFXai exposure in the previous 72 hours. The secondary outcomes included the indication for transition to UFH and the rate of thromboembolic and bleeding events. RESULTS: A total of 93 patients with supratherapeutic antiXa activity levels with prior OFXai use were reviewed. Moderate renal impairment, defined as creatinine clearance less than 49 mL/min, was present in 67 (72%) patients. The primary indication for transition from OFXai to UFH was in anticipation for a procedure, and it occurred in 37 (40%) patients. There were 3 major bleeding events and 3 clinically relevant nonmajor bleeding events. No thromboembolic events occurred. CONCLUSIONS: This study assessed the prevalence of supratherapeutic antiXa levels and clinical outcomes during the transition from OFXais to UFH. Health care systems should develop guidelines to assist clinicians in monitoring antiXa activity in patients undergoing a transition from an OFXai to UFH. It is also important to assess the patient's underlying thromboembolic and bleeding risks.

[The role of direct oral anticoagulants in the management of cancer-associated thrombosis in 2020]. / Place des anticoagulants oraux directs dans la prise en charge de la maladie thromboembolique veineuse associée au cancer en 2020.

Direct oral anticoagulants, anti-IIa or anti-Xa, are widely used in the treatment and prevention of venous thromboembolic disease as well as in nonvalvular atrial fibrillation. Direct oral anticoagulants are characterized by a rapid onset of activity, a predictable response and a relatively wide therapeutic window. Nevertheless, theoretical drug interactions exist since direct oral anticoagulants are substrates of the transport protein P-glycoprotein and/or of isoforms of cytochromes P450 pathway. Direct oral anticoagulants do not have a marketing authorization for the treatment of cancer-associated thrombosis unlike low-molecular-weight heparins which remain the gold standard treatment today. However, recent studies have compared low-molecular-weight heparins to direct oral anticoagulants in the treatment of cancer-associated thrombosis. Results of these studies showed a non-inferiority of direct oral anticoagulants in the prevention of recurrent thromboembolic events but at the cost of an increased hemorrhagic risk, in particular for patients with gastrointestinal and urogenital cancers. Thus, international guidelines, unlike French guidelines, integrate them in first line of the therapeutic strategy of cancer patients. We are certainly entering an era of personalized therapy for cancer-associated thrombosis, considering cancer type and also the theoretical risk of drug interactions with anti-cancer treatments or supportive care.

Chromogenic anti-FXa assay calibrated with low molecular weight heparin in patients treated with rivaroxaban and apixaban: possibilities and limitations.

INTRODUCTION: Clinical application of rivaroxaban and apixaban does not require therapeutic monitoring. Commercial anti-activated factor X (anti-FXa) inhibition methods for all anti-FXa drugs are based on the same principle, so there are attempts to evaluate potential clinical application of heparin-calibrated anti-FXa assay as an alternative method for direct FXa inhibitors. We aimed to evaluate relationship between anti-FXa methods calibrated with low molecular weight heparin (LMWH) and with drug specific calibrators, and to determine whether commercial LMWH anti-FXa assay can be used to exclude the presence of clinically relevant concentrations of rivaroxaban and apixaban. MATERIALS AND METHODS: Low molecular weight heparin calibrated reagent (Siemens Healthineers, Marburg, Germany) was used for anti-FXa activity measurement. Innovance heparin (Siemens Healthineers, Marburg, Germany) calibrated with rivaroxaban and apixaban calibrators (Hyphen BioMed, Neuville-sur-Oise, France) was used for quantitative determination of FXa inhibitors. RESULTS: Analysis showed good agreement between LMWH calibrated and rivaroxaban calibrated activity (κ = 0.76) and very good agreement with apixaban calibrated anti-Xa activity (κ = 0.82), respectively. Low molecular weight heparin anti-FXa activity cut-off values of 0.05 IU/mL and 0.1 IU/mL are suitable for excluding the presence of clinically relevant concentrations (< 30 ng/mL) of rivaroxaban and apixaban, respectively. Concentrations above 300 ng/mL exceeded upper measurement range for LMWH anti-FXa assay and cannot be determined by this method. CONCLUSION: Low molecular weight heparin anti-FXa assay can be used in emergency clinical conditions for ruling out the presence of clinically relevant concentrations of rivaroxaban and apixaban. However, use of LMWH anti-FXa assay is not appropriate for their quantitative determination as an interchangeable method.

Synthesis and biological evaluation of Isosteviol derivatives as FXa inhibitors.

Firstly, a series of Isosteviol derivatives were synthesized and evaluated for FXa inhibitory activity. Among these compounds, the inhibitory activity of compounds 22, 35 and 38 on FXa was better than that of Isosteviol. Secondly, surface plasmon resonance (SPR) assays were performed for selected compounds. Compounds 22, 35, 38 have similar kinetic signatures, and affinity values were at µM level. Thirdly, compounds 22 and 35 displayed moderate-to-high anticoagulation activity and showed similar sensitivity to PT and aPTT. These findings will provide new insight into the exploration of FXa inhibition.

Anti-Factor Xa-Based Anticoagulation during Extracorporeal Membrane Oxygenation: Potential Problems and Possible Solutions.

Choices for monitoring of unfractionated heparin (UFH) anticoagulation in extracorporeal membrane oxygenation (ECMO) patients include activated clotting time, activated partial thromboplastin time, reaction times of viscoelastic tests, and anti-factor Xa activity (between 0.3 and 0.7 IU/mL). Recent studies propose the anti-factor Xa to be the gold standard for monitoring UFH anticoagulation in ECMO. However, many extraneous factors combined question the utility of anti-factor Xa as the sole method of monitoring of UFH effects in ECMO. Anti-factor Xa is a chromogenic assay, which may be biased by the frequently elevated values of bilirubin and free hemoglobin in ECMO patients. The test may alternatively underestimate UFH effects in cases of low antithrombin values. More importantly, the anti-factor Xa assay is a plasma-based test which does not take into account the role of platelets and fibrinogen in forming a stable clot. Thrombocytopenia and platelet dysfunction are common features in ECMO patients, and underestimating their role may lead to over-anticoagulation, should only anti-factor Xa guiding be used to adjust the UFH dose. Conversely, fibrinogen is an acute phase protein, and some patients may experience high levels of fibrinogen during the ECMO course. In this case, an UFH monitoring based on anti-factor Xa is insensitive to this condition, although it may potentially be associated with thrombotic complications. Finally, the generally suggested range of 0.3 to 0.7 IU/mL is a somewhat arbitrary estimate, based on the desired range for treating and preventing thrombotic events in non-ECMO patients. In conclusion, anti-factor Xa may offer useful information on the real effects of UFH only when combined with a whole blood test capable of assessing the relative contribution of platelets and fibrinogen to clot formation.

The salivary transcriptome of Limnobdella mexicana (Annelida: Clitellata: Praobdellidae) and orthology determination of major leech anticoagulants.

Bloodfeeding requires several adaptations that allow the parasite to feed efficiently. Leeches and other hematophagous animals have developed different mechanisms to inhibit hemostasis, one of the main barriers imposed by their hosts. Limnobdella mexicana is a member of the leech family Praobdellidae, a family of host generalists known for their preference to attach on mucosal membranes of mammals, such as those in nasopharyngeal cavities, bladders and ocular orbits. Previous studies have hypothesized a positive relationship between diversity of anticoagulants and diversity of hosts in bloodfeeding leeches. However, orthology determination of putative anticoagulants and the lack of standardization of sequencing effort and method hinder comparisons between publicly available transcriptomes generated in different laboratories. In the present study, we examine the first transcriptome of a praobdellid leech and identify 15 putative anticoagulants using a phylogeny-based inference approach, amino-acid conservation, Pfam domains and BLAST searches. Our phylogenetic analyses suggest that the ancestral leech was able to inhibit factor Xa and that some hirudins that have been reported in previous studies on leech anticoagulants may not be orthologous with the archetypal hirudin.

Early initiation of a factor Xa inhibitor can attenuate tissue repair and neurorestoration after middle cerebral artery occlusion.

The timing of anti-coagulation therapy initiation after acute cardioembolic stroke remains controversial. We investigated the effects of post-stroke administration of a factor Xa inhibitor in mice, focusing on tissue repair and functional restoration outcomes. We initiated administration of rivaroxaban, a Xa inhibitor, immediately after permanent distal middle cerebral artery occlusion (pMCAO) in CB-17 mice harboring few leptomeningeal anastomoses at baseline. Rivaroxaban initiated immediately after pMCAO hindered the recovery of blood flow in ischemic areas by inhibiting leptomeningeal anastomosis development, and led to impaired restoration of neurologic functions with less extensive peri-infarct astrogliosis. Within infarct areas, angiogenesis and fibrotic responses were attenuated in rivaroxaban-fed mice. Furthermore, inflammatory responses, including the accumulation of neutrophils and monocytes/macrophages, local secretion of pro-inflammatory cytokines, and breakdown of the blood-brain barrier, were enhanced in infarct areas in mice treated immediately with rivaroxaban following pMCAO. The detrimental effects were not found when rivaroxaban was initiated after transient MCAO or on day 7 after pMCAO. Collectively, early post-stroke initiation of a factor Xa inhibitor may suppress leptomeningeal anastomosis development and blood flow recovery in ischemic areas, thereby resulting in attenuated tissue repair and functional restoration unless occluded large arteries are successfully recanalized.

The effect of DOAC-Stop on lupus anticoagulant testing in plasma samples of venous thromboembolism patients receiving direct oral anticoagulants.

Background Direct oral anticoagulants (DOACs) cause false positive lupus anticoagulant (LA) results. We assessed the impact of DOAC-Stop, reversing in vitro effects of DOACs, on LA testing in anticoagulated patients. Methods We assessed 75 venous thromboembolism patients aged 44.5±14.6 years. Blood samples were collected 2-28 h since intake of DOACs, including 50 patients on rivaroxaban, 20 on dabigatran and five on apixaban. LA testing was performed at baseline and after DOAC-Stop treatment. Positive LA was defined as the normalized (patient/standard plasma clotting time) LA screening and screening (LA1)/confirmation (LA2) ratios exceeding 1.2. Results LA diluted Russell's viper venom time (dRVVT) normalized screening test revealed abnormal results in 73 (97.3%) and activated partial thromboplastin time (APTT)-LA in 49 (65.3%) patients. In six (8%) patients, antiphospholipid syndrome (APS) was diagnosed. dRVVT LA1/LA2 was abnormal in 35 (50.7%) patients taking DOACs. The APTT ratio was normal in all studied subjects. DOAC-Stop completely removed dabigatran and reduced by 98% rivaroxaban and by 92.3% apixaban concentrations (all p<0.05). After DOAC-Stop screening dRVVT remained prolonged in 34 (49.3%) patients (p<0.001), while dRVVT LA1/LA2 was abnormal in six (8.7%) subjects, with no association with DOAC concentrations at baseline and after DOAC-Stop. The APTT-LA screening test remained prolonged in five (7.2%) patients, while the APTT LA1/LA2 ratio was normal in those subjects. DOAC-Stop did not influence LA testing in APS patients. Conclusions Application of DOAC-Stop effectively reduced plasma DOAC concentrations leading to appropriate dRVVT results in up to 97% of VTE patients.

Full Study Report of Andexanet Alfa for Bleeding Associated with Factor Xa Inhibitors.

BACKGROUND: Andexanet alfa is a modified recombinant inactive form of human factor Xa developed for reversal of factor Xa inhibitors. METHODS: We evaluated 352 patients who had acute major bleeding within 18 hours after administration of a factor Xa inhibitor. The patients received a bolus of andexanet, followed by a 2-hour infusion. The coprimary outcomes were the percent change in anti-factor Xa activity after andexanet treatment and the percentage of patients with excellent or good hemostatic efficacy at 12 hours after the end of the infusion, with hemostatic efficacy adjudicated on the basis of prespecified criteria. Efficacy was assessed in the subgroup of patients with confirmed major bleeding and baseline anti-factor Xa activity of at least 75 ng per milliliter (or ≥0.25 IU per milliliter for those receiving enoxaparin). RESULTS: Patients had a mean age of 77 years, and most had substantial cardiovascular disease. Bleeding was predominantly intracranial (in 227 patients [64%]) or gastrointestinal (in 90 patients [26%]). In patients who had received apixaban, the median anti-factor Xa activity decreased from 149.7 ng per milliliter at baseline to 11.1 ng per milliliter after the andexanet bolus (92% reduction; 95% confidence interval [CI], 91 to 93); in patients who had received rivaroxaban, the median value decreased from 211.8 ng per milliliter to 14.2 ng per milliliter (92% reduction; 95% CI, 88 to 94). Excellent or good hemostasis occurred in 204 of 249 patients (82%) who could be evaluated. Within 30 days, death occurred in 49 patients (14%) and a thrombotic event in 34 (10%). Reduction in anti-factor Xa activity was not predictive of hemostatic efficacy overall but was modestly predictive in patients with intracranial hemorrhage. CONCLUSIONS: In patients with acute major bleeding associated with the use of a factor Xa inhibitor, treatment with andexanet markedly reduced anti-factor Xa activity, and 82% of patients had excellent or good hemostatic efficacy at 12 hours, as adjudicated according to prespecified criteria. (Funded by Portola Pharmaceuticals; ANNEXA-4 ClinicalTrials.gov number, NCT02329327.).

Discordant Partial Thromboplastin Time (PTT) vs Anti-Xa Heparin Activity.

OBJECTIVES: To determine the relationship between baseline variations in the partial thromboplastin time (PTT) and the discordance between the PTT and anti-Xa heparin activity (anti-Xa) during heparin therapy. METHODS: The baseline PTT on heparin was determined using automated heparin neutralization with protamine (prPTT). The prPTT was used to calculate a baseline-corrected PTT on heparin to reduce discordance with anti-Xa measurements. RESULTS: The prPTT removed up to 1 U/mL of heparin, returning baseline values for normal, factor-deficient, and lupus inhibitor plasmas. A prolonged prPTT was seen in 97 (53%) of 182 samples from heparinized patients. The heparinized PTT was discordant compared with anti-Xa in 64 (35%) of 182 samples and 43 (67%) of 64 discordant samples, and 46% of concordant samples showed a prolonged prPTT. A baseline-corrected PTT reduced discordance with anti-Xa measurements by 64%. CONCLUSIONS: PTT/anti-Xa discordance due to baseline PTT prolongation could be reduced using a baseline-corrected PTT.

Mud in the blood: Novel potent anticoagulant coagulotoxicity in the venoms of the Australian elapid snake genus Denisonia (mud adders) and relative antivenom efficacy.

Due to their potent coagulotoxicity, Australian elapid venoms are unique relative to non-Australian members of the Elapidae snake family. The majority of Australian elapids possess potent procoagulant venom, while only a few species have been identified as possessing anticoagulant venoms. The majority of research to-date has concentrated on large species with range distributions overlapping major city centres, such as brown snakes (Pseudonaja spp.) and taipans (Oxyuranus spp.). We investigated the venom from the poorly studied genus Denisonia and documented anticoagulant activities that were differentially potent on amphibian, avian, and human plasmas. Both species were potently anticoagulant upon amphibian plasma, consistent with these snakes preying upon frogs as their primary food source. While D. devisi was only relatively weakly active on avian and human plasma, D. maculata was potently anticoagulant to amphibian, avian, and human plasma. The mechanism of anticoagulant action was determined to be the inhibition of prothrombin activation by Factor Xa by blocking the formation of the prothrombinase complex. Fractionation of D. maculata venom followed by MS sequencing revealed that the toxins responsible were Group I phospholipase A2. As no antivenom is produced for this species or its near relatives, we examined the ability of Seqirus Australian snake polyvalent antivenom to neutralise the anticoagulant effects, with this antivenom shown to be effective. These results contribute to the body of knowledge regarding adaptive evolution of venom, revealing a unique taxon-specific anticoagulant effect for D. devisi venom. These results also reveal the potential effects and mechanisms behind envenomation by the potently acting D. maculata venom on human plasma, while the discovery of the efficacy of an available antivenom provides information crucial to the design of snakebite management strategies.