Viper Venom Research Articles

Detection of enoxaparin and argatroban by use of the novel viscoelastic coagulometer ClotPro

Owing to the simultaneous increase in the risk of thrombosis and bleeding in critically ill patients, point-of-care-available diagnostic tests to guide parenteral anticoagulation are warranted. We evaluated the detection of enoxaparin and argatroban, two commonly used parenteral anticoagulants, using the novel ClotPro viscoelastic coagulometer. For this experimental in vitro study at a tertiary care academic center, blood samples were drawn from twelve (six female, six male) healthy volunteers without intake of antithrombotic medication and no history of hemostatic disorders. Blood samples were spiked with enoxaparin (IU.ml− 1) and argatroban (µg.ml− 1) at increasing concentrations ranging from 0 to 1. The ClotPro Russell’s viper venom (RVV)-test and the ClotPro ecarin (ECA)-test clotting time were performed in parallel with conventional coagulation tests (anti-Xa activity, activated partial thromboplastin time, and diluted thrombin time). We observed a strong correlation between anti-Xa activity and the RVV-test clotting time (r = 0.88 (95% confidence interval (CI) 0.8–0.92; p < 0.001)). Although clotting time cutoff values of 71 and 145 s provided high sensitivity and specificity for detecting anti-Xa activity of ≤ 0.1 and ≥0.6 IU.ml− 1, we found a poor performance at both high and low concentrations. The ECA-test clotting time revealed a very strong correlation with activated partial thromboplastin time (r = 0.96 (95% CI 0.93–0.97; p < 0.001)) and diluted thrombin time (r = 0.97 (95% CI 0.96–0.98; p < 0.001)). The clotting time cutoff values of 86 and 298–431 s provided high sensitivity and specificity for detecting diluted thrombin time values ≤ 0.1 and 0.5-1 µg.ml− 1. Our results suggest that the RVV test is an unreliable method for monitoring enoxaparin treatment, whereas the ECA-test might be an accurate point-of-care alternative for detecting argatroban concentration with potential advantages over standard coagulation tests in terms of point-of-care applicability and turnaround time.

Exploring the Current Potential of Snake Venom Disintegrins as Chemotherapeutic Agents: A Narrative Update on their Clinical Translational Potential

A potential source of multiple enzymatic and nonenzymatic molecules that protect the host is snake venom. In these venoms, several kinds of peptides that have significant beneficial effects were discovered and characterized. Disintegrins act by blocking integrins on transmembrane cell surfaces, inhibiting tumor cells from adhering, migrating, forming new blood vessels, and spreading. This has an important effect on delaying the development, neovascularization, and growth of tumors. These cells are ideal candidates for novel therapies for the management of malignancies due to their tumor selectivity and decreased size. As research findings in various &lt;i&gt;in vivo&lt;/i&gt; &amp; &lt;i&gt;in vitro&lt;/i&gt;, disintegrin proteins are low-molecular-weight polypeptides that are found in the venom of vipers and rattle snakes. They act by blocking the &amp;#946;1 and &amp;#946;3 integrin receptors. Angiogenesis and metastatic processes in cancer are mediated through &amp;#946;1 and &amp;#946;3 integrins. Hence, blocking &amp;#946;1 and &amp;#946;3 integrin receptors plays a prominent role in blocking the progression of cancer, and disintegrins seem to be promising candidates for antineoplastic therapies. The disintegrins with anticancer properties include Crotatroxin 2, Alternagin-C, Rubistatin, Leucurogin, Mojastin- 1, Contortrostatin, Acostatin, Vicrostatin, Tzabcan, Eristostatin, Purpureomaculatus, Saxatilin, Lebein, Salmosin, and Rhodostomin. The above mentioned disintegrins were considered in this study. This review is based on the origins of these disintegrins, their modes of targeting, their categorization, and their inherent anticancer potential.

Sphingomyelin Inhibits Hydrolytic Activity of Heterodimeric PLA2 in Model Myelin Membranes: Pharmacological Relevance.

In this work, the heterodimeric phospholipase A2, HDP-2, from viper venom was investigated for its hydrolytic activity in model myelin membranes as well as for its effects on intermembrane exchange of phospholipids (studied by phosphorescence quenching) and on phospholipid polymorphism (studied by 1H-NMR spectroscopy) to understand the role of sphingomyelin (SM) in the demyelination of nerve fibers. By using well-validated in vitro approaches, we show that the presence of SM in model myelin membranes leads to a significant inhibition of the hydrolytic activity of HDP-2, decreased intermembrane phospholipid exchange, and reduced phospholipid polymorphism. Using AutoDock software, we show that the NHδ+ group of the sphingosine backbone of SM binds to Tyr22(C=Opbδ-) of HDP-2 via a hydrogen bond which keeps only the polar head of SM inside the HDP-2's active center and positions the sn-2 acyl ester bond away from the active center, thus making it unlikely to hydrolyze the alkyl chains at the sn-2 position. This observation strongly suggests that SM inhibits the catalytic activity of HDP-2 by blocking access to other phospholipids to the active center of the enzyme. Should this observation be verified in further studies, it would offer a tantalizing opportunity for developing effective pharmaceuticals to stop the demyelination of nerve fibers by aberrant PLA2s with overt activity - as observed in brain degenerative diseases - by inhibiting SM hydrolysis and/or facilitating SM synthesis in the myelin sheath membrane.

Intraspecific venom variation in the Iberian asp viper (Vipera aspis zinnikeri) across natural and intensive agricultural habitats

Snake venom composition varies at different levels. To date, comparative venom studies have seldom taken into account the role of habitat type in the occurrence of snake venom variation. Here we investigated the presence of venom variation across different populations of the Iberian asp viper (Vipera aspis zinnikeri) inhabiting two contrasting habitats: natural vs. intensive agricultural. We used shotgun proteomics to describe the protein composition of the venoms of six adults from two distinct localities. Furthermore, to test whether local conditions and habitat can alter venom composition in this taxon, we compared the SDS-PAGE profiles of 40 adult venoms from six populations, three in natural habitats and three in intensive agricultural environments. The venoms were composed of 21 toxin families, of which five (CTL, PLA2, VEGF, svSP, and svMP) comprised 69–82 % of each proteome. The relative abundances of toxin families varied considerably at inter- and intra-population levels. Linear regression performed on non-metric multidimensional scaling values showed a significant effect of locality of origin and habitat type on the differences detected between individual SDS-PAGE venom profiles. Our results suggest the presence of regional variation in V. a. zinnikeri venom, potentially reinforcing the role of local pressures in shaping snake venom composition. SignificanceThis work provides the first proteomic characterization of the venom of the Iberian asp viper, Vipera aspis zinnikeri, obtained by means of shotgun proteomics. The statistical analysis of 40 individual SDS-PAGE venom profiles highlights that venom variation in this taxon can be associated with geographical origin and habitat type of the area where each viper was collected. Our results suggest the presence of regional variation in V. a. zinnikeri venom, reinforcing the role that local pressures may play as drivers of snake venom variation.

Venomous Peptides: Molecular Origin of the Toxicity of Snake Venom PLA2-like Peptides.

Snakebite envenoming claims 81-138 thousand lives annually, with vipers responsible for many of those. Phospholipase A2 (PLA2) enzymes and PLA2-like proteins are among the most important viper venom toxins. The latter are particularly intriguing, as three decades after their discovery, their molecular mechanism of toxicity is still poorly understood at best. PLA2-like proteins destabilize eukaryotic cell membranes through an unknown mechanism, causing an uncontrolled influx of Ca2+ ions and ultimately triggering cell death. It is now clear that the C-terminal segment is fundamental to the toxicity, as 13-mer peptides with the same sequence exhibit most or all of the activities of the complete PLA2-like proteins. To finally clarify the mechanism of toxicity of these venom peptides, we have simulated their interaction with model cell membranes. Molecular dynamics simulations showed that peptides initially dispersed across the cell membrane quickly and spontaneously migrated, aggregated, induced membrane thinning, and formed clear and transient membrane pores. We calculated the potentials of the mean force for Ca2+ transfer across the cell membranes through the transient pores. The pores significantly lower the free energy barrier for Ca2+ translocation, an effect that grows with the size of the peptide aggregates and, thus, with the pore radius. Ca2+ flowed across the membrane through the largest pores with almost no barrier. The permeability of Ca2+ through the largest pores exceeded the permeability of pharmaceutical drugs by 4 orders of magnitude, revealing the easiness by which Ca2+ overflows the intracellular medium. These results elucidate the illusive molecular origin of the toxicity of this famous class of snake venom-derived peptides.

Utilizing Artificial Intelligence to Reconcile Indeterminate Lupus Anticoagulant Panel Results

Abstract Lupus anticoagulant (LA) is one laboratory criterion for diagnosing antiphospholipid antibody syndrome (APS), a condition associated with hypercoagulability, pregnancy complications, and death. LA diagnosis requires a complex panel, often performed at a specialized laboratory. Ruling out false positive or transient LAs requires ≥ two positive tests ≥ 12 weeks apart. Most LA tests are negative, with small subsets resulting positive or indeterminate. Indeterminates leave clinicians with limited understanding of appropriate clinical follow-up. Thus, the International Society on Thrombosis and Hemostasis discourages weak resulting nomenclature such as “indeterminate.” Our study used artificial intelligence to predict LA indeterminate results as positive or negative, identifying patients requiring further testing. This IRB approved study reviewed nearly 4000 LA tests performed from 2019-2023. Parameters included CBC, thromboelastography, renal/hepatic function assays, coagulation tests, antiphospholipid antibodies and LA tests. LA test components included screening tests (APTT, Dilute Russel Viper Venom Test (DRVVT) screen), mixing studies (1:1 APTT mix, DRVVT mix), phospholipid-dependent confirmation (DRVVT, hexagonal phase, platelet neutralization), and factor VIII. A positive LA test fulfilled 4 criteria: at least one positive screening test, mixing study and phospholipid-dependent assay and factor VIII inhibitor exclusion. An LA panel meeting 2-3 criteria resulted indeterminate. Studies meeting &amp;lt;2 criteria were negative. This analysis was performed using Machine Intelligence Learning Optimizer (MILO) platform which displays the performance of multiple models. MILO was trained with two classifications: positive and negative cases. Data was split into training (100 training positives; 100 negatives) and testing (primary and secondary validation) sets (100 positives; 1388 negatives). LA panels affected by drug interactions (9.4%) or containing incomplete datasets (21%) were excluded. Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) determined model efficacy. 2903 negative (84%), 318 positive (9.1%), and 246 indeterminate (7.1%) LA results were included from 3467 patients. Only 84 of the 246 indeterminate patients (34%) were retested. Of these, 28 remained indeterminate, 43 became negative and 13 resulted positive. The best performing MILO models were the K-nearest neighbor (KNN) and logistic regression. The ability of these models to determine which indeterminate cases would become positive or negative was evaluated. KNN displayed the highest specificity (0.88) and PPV (0.57). All models had a high NPV and specificity due to substantive exposure to negative cases for the training and testing. Exclusion of some parameters did not improve the performance of the models. However, even the best model (various KNN models) had low sensitivity and PPV due to a small pool of positive training/testing cases and a much smaller group of indeterminate LA with subsequent positive results. Creating models accurately predicting indeterminate cases that may become positive enables appropriate clinical follow-up. Thus, improving model accuracy will require a larger sample size of positive indeterminates with follow-up.

Monitoring the efficiency of reversal on anti-Xa direct oral anticoagulants using point-of-care viscoelastic testing

Bleeding events in patients receiving direct oral anticoagulation (DOAC) can be life-threatening even at therapeutic DOAC plasma concentrations, as anticoagulation impairs hemostasis and should therefore be identified immediately after hospital admission. The anticoagulatory effects of DOAC are typically not measurable in standard coagulation tests, such as PT or aPTT. Specific calibrated anti-FXa-tests allow specific drug monitoring, but they are too time-consuming for critical bleeding events and are commonly not available for 24 h/7 days in routine care. However, recent advances in point-of-care (POC) viscoelastic testing (VET) have shown a promising approach for rapid and quantitative detection of DOAC plasma concentrations using the Russell viper venom factor V activator (RVV for FXa-inhibitors) test or the ecarin clotting time (thrombin inhibitors). In acute bleeding situations, direct FXa inhibitors can be reversed by specific antidote andexanet alfa or hemostasis can be improved by prothrombin complex factor concentrates (PCCs). After reversal, confirmation of reversal efficacy is often requested, but no routine assays are currently available. Thus, the emergency management of bleeding DOAC patients is usually “blinded” with regard to reversal efficacy. POC VET laboratory assays might therefore also be helpful for measuring DOAC effects after reversal. We present a case series demonstrating the usefulness of RVV-clotting time post-DOAC reversal with andexanet alfa.

Individual variation in Macrovipera lebetinus obtusa (blunt-nosed viper) venom from a limited area in Southeastern Anatolia (Türkiye)

Intraspecific variation in snake venom composition has been the subject of many studies, revealing the differences in venom proteomes according to age, diet, sex, and geographic region. Previous research on M. lebetinus venom mainly focused on protein purification, proteomic characterization, and biological activities. Although venom proteomes of its subspecies from different geographical areas were compared, it is a widespread species and there is limited knowledge regarding the individual venom variation of M. lebetinus in the same area. In this study, individual variation in M. lebetinus obtusa venom was investigated in terms of protein profiles, protease, phospholipase A2, ʟ-amino acid oxidase, hyaluronidase and phosphodiesterase, and hemolytic activities based on nine individuals collected in the province of Şanlıurfa (southeastern Türkiye). HPLC analysis indicated more striking differences compared to SDS-PAGE. Phosphodiesterase activity varied most between the individuals, followed by hyaluronidase and phospholipase A2. Hemolytic activity did not show significant alteration based on the blood agar method. The results of this initial study revealed that M. lebetinus venom shows variation between the individuals inhabiting the same habitat, which should be taken into account in the development of antivenoms and the clinical assessment of the bite cases.

The utility of the dilute prothrombin time in the interpretation of antiphospholipid syndrome testing.

To evaluate the utility of the dilute prothrombin time (DPT) in diagnosing antiphospholipid syndrome (APS), alone and when paired with the dilute Russell viper venom time (DRVVT). Dilute prothrombin time and DRVVT testing was performed on plasma samples spiked with apixaban or rivaroxaban, or depleted of vitamin K-dependent clotting factors. A retrospective analysis of all functional APS testing results over a 44-month period at the University of Chicago Medical Center was performed. In spiking studies, the screening clotting time in the DPT (DPTS) is more sensitive to deficiency of vitamin K-dependent factors than is the screening clotting time in the DRVVT (DRVVTS). The converse is true for factor Xa direct oral anticoagulant (DOAC)-spiked plasma. In a 44-month retrospective analysis, only 2.6% of clinical APS panels showed isolated positivity in the DPT-based system. Comparing the DPT-based system with the DRVVT-based system showed utility in identifying false-positive DRVVT results due to anticoagulation. A DRVVTS/DPTS ratio of 0.785 or lower predicted an international normalized ratio of 1.5 or higher (sensitivity, 86.3%; specificity, 60.4%; likelihood ratio, 2.18). Conversely, a DRVVTS/DPTS ratio of 1.165 or higher was the optimal cutoff for predicting the identification of factor Xa DOAC (sensitivity, 61.8%; specificity, 77.8%; likelihood ratio, 2.78). Within the data set that had full DRVVT and DPT results, parameters were identified that could further improve identification of samples with anticoagulation interference. Dilute prothrombin time lupus anticoagulant assay is rarely the sole laboratory functional evidence for APS, but when combined with the DRVVT, the DPT can serve as an effective screen for common anticoagulant interference.

A-161 Diagnosis of Lupus Anticoagulant Cases using Deep Neural Networks

Abstract Background Laboratory identification of lupus anticoagulant (LAC) is critical for diagnosing antiphospholipid antibody syndrome, a common cause of unprovoked thromboembolic events and pregnancy morbidity. Commonly used clot-based assays for LAC include the dilute Russell’s viper venom time (DRVVT) and activated partial thromboplastin time (APTT), but these are susceptible to anticoagulant interferences. Despite standardization efforts, LAC interpretation remains challenging due to pre- and post-analytical complexity and limited expertise. This study explored the use of deep neural networks (DNN) to facilitate LAC diagnosis. Methods Retrospective data for LAC profiling with 13 features performed at Mayo Clinic between 2021 and 2022 was collected. The profiles of 7,202 patients were randomly divided into training (n=4608), validation (n=1153), and test cohorts (n=1441). Diagnostic outputs including LAC positivity (determined by DRVVT or APTT) and the presence of warfarin (WAR) and heparin (HEP), were confirmed by expert assessment. Three DNN architectures were developed and evaluated (Figure 1): (1) four single output DNN models with a domain knowledge driven selection of input features, (2) four single output DNN models using the same 13 inputs across all models, and (3) a multi-output DNN model. Model performance was evaluated using the F1 score and the index of balanced accuracy (IBA) for each output label. Results Architecture 1 achieved high diagnostic accuracy with IBA values of 0.989-1.000 for all four outputs and F1 scores of 0.945-0.991. Architecture 2 exhibited F1 scores of 0.902-0.973 and IBA values of 0.935-0.992. Architecture 3 achieved F1 scores of 0.933-0.967 and IBA values of 0.941-0.983. Conclusions All three architectures accurately classify LAC and common anticoagulation effects. The comparable performance of architectures 1 and 2 suggests that manual feature extraction is unnecessary. Given its relative simplicity and versatility, architecture 3 is the preferred choice for implementation in a clinical laboratory to standardize LAC diagnosis.

Modulation of αv integrins by lebecetin, a viper venom-derived molecule, in experimental neuroinflammation and demyelination models

Several neurodegenerative diseases, such as multiple sclerosis and Parkinson’s disease, are linked to alterations in myelin content or structure. Transmembrane receptors such as integrins could be involved in these alterations. In the present study, we investigated the role of αv-integrins in experimental models of neuroinflammation and demyelination with the use of lebecetin (LCT), a C-lectin protein purified from Macrovipera lebetina viper venom, as an αv-integrin modulator. In a model of neuroinflammation, LCT inhibited the upregulation of αv, β3, β5, α5, and β1 integrins, as well as the associated release of pro-inflammatory factor IL-6 and chemokine CXCL-10, and decreased the expression of phosphorylated NfκB. The subsequent “indirect culture” between reactive astrocytes and oligodendrocytes showed a down-regulation of αv and β3 integrins versus upregulation of β1 one, accompanied by a reduced expression of myelin basic protein (MBP). Treatment of oligodendrocytes with LCT rectified the changes in integrin and MBP expression. Through Western blot quantification, LCT was shown to upregulate the expression levels of PI3K and p-mTOR while downregulating expression levels of p-AKT in oligodendrocytes, suggesting the neuroprotective and pro-myelinating effects of LCT may be related to the PI3K/mTor/AKT pathway. Concomitantly, we found that LCT promoted remyelination by tracking the increased expression of MBP in the brains of cuprizone-intoxicated mice. These results point to an involvement of integrins in not only neuroinflammation but demyelination as well. Thus, targeting αv integrins could offer potential therapeutic avenues for the treatment of demyelinating diseases.

Isolation and Pharmacological Characterisation of Pre-Synaptic Neurotoxins from Thai and Javanese Russell's Viper (Daboia siamensis) Venoms.

The widespread geographical distribution of Russell's vipers (Daboia spp.) is associated with marked variations in the clinical outcomes of envenoming by species from different countries. This is likely to be due to differences in the quantity and potency of key toxins and, potentially, the presence or absence of some toxins in venoms across the geographical spectrum. In this study, we aimed to isolate and pharmacologically characterise the major neurotoxic components of D. siamensis venoms from Thailand and Java (Indonesia) and explore the efficacy of antivenom and a PLA2 inhibitor, Varespladib, against the neuromuscular activity. These data will provide insights into the link between venom components and likely clinical outcomes, as well as potential treatment strategies. Venoms were fractionated using RP-HPLC and the in vitro activity of isolated toxins assessed using the chick biventer cervicis nerve-muscle preparation. Two major PLA2 fractions (i.e., fractions 8 and 10) were isolated from each venom. Fraction 8 from both venoms produced pre-synaptic neurotoxicity and myotoxicity, whereas fraction 10 from both venoms was weakly neurotoxic. The removal of the two fractions from each venom abolished the in vitro neurotoxicity, and partially abolished myotoxicity, of the whole venom. A combination of the two fractions from each venom produced neurotoxic activity that was equivalent to the respective whole venom (10 µg/mL), but the myotoxic effects were not additive. The in vitro neurotoxicity of fraction 8 (100 nM) from each venom was prevented by the pre-administration of Thai Russell's viper monovalent antivenom (2× recommended concentration) or preincubation with Varespladib (100 nM). Additionally, the neurotoxicity produced by a combination of the two fractions was partially reversed by the addition of Varespladib (100-300 nM) 60 min after the fractions. The present study demonstrates that the in vitro skeletal muscle effects of Thai and Javanese D. siamensis venoms are primarily due to key PLA2 toxins in each venom.

Reversible thrombocytopenia of functional platelets after the nose-horned viper venom envenomation is induced by a snaclec

Reversible thrombocytopenia of functional platelets after the nose-horned viper venom envenomation is induced by a snaclec

Species-specific or para-specific antivenoms? On the immunological reactivity and neutralization efficacy for Saw-scaled viper venoms

Species-specific or para-specific antivenoms? On the immunological reactivity and neutralization efficacy for Saw-scaled viper venoms

Complexity of the biological effects of viper venoms: Origin and diversification of snake venom metalloproteinases (SVMPs) in Bothrops snakes

Complexity of the biological effects of viper venoms: Origin and diversification of snake venom metalloproteinases (SVMPs) in Bothrops snakes

Preliminary data on the inhibitory effects of small molecule protease inhibitors against Anatolian viper venoms

Preliminary data on the inhibitory effects of small molecule protease inhibitors against Anatolian viper venoms

Serine protease associated disintegrin from Indian Russell’s viper venom induces apoptosis in MCF7 breast cancer cells

Serine protease associated disintegrin from Indian Russell’s viper venom induces apoptosis in MCF7 breast cancer cells

Importance of the Cysteine-Rich Domain of Snake Venom Prothrombin Activators: Insights Gained from Synthetic Neutralizing Antibodies.

Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus Echis) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms.

A new protein glosaxin consisting of non-catalytic domains of type piii metalloproteinase from the venom of pit viper &lt;i&gt;Gloydius saxatilis&lt;/i&gt; inhibits nicotinic acetylcholine receptor

Previously, we found that the venom of the pit viper Gloydius saxatilis inhibited the muscle-type nicotinic acetylcholine receptor (nAChR). Using liquid chromatography, a protein glosaxin was isolated from the venom that inhibited the binding of the α-bungarotoxin to the nAChR of muscle type from Torpedo californica. The amino acid sequence of the isolated protein was analyzed by high resolution mass spectrometry. Subsequent bioinformatic analysis showed that it is homologous to the amino acid sequences of disintegrin-like proteins, consisting of non-catalytic domains of type PIII metalloproteinases from the venom of pit vipers of genus Gloydius. A study of the biological activity of the isolated protein showed that it inhibits the binding of α-bungarotoxin to Torpedo californica nAChR with IC50 = 51 μM. The protein also inhibited acetylcholine-induced functional responses of the human neuronal nAChR of α3β2 subtype. This is the first evidence of the ability of proteins consisting of non-catalytic domains of snake venom type PIII metalloproteinase to inhibit the nAChR.

Evaluation of an integrated activated partial thromboplastin time (Cephen LS/Cephen) for the detection of lupus anticoagulant.

It is recommended to use two chronometric assays of different principles for the diagnosis of lupus anticoagulant (LA), consisting in diluted Russell Viper Venom Time (dRVVT) and activated Partial Thromboplastin Time (aPTT). Yet, there are only a few integrated aPTT assays; this study aims to evaluate one of them: Cephen LS/Cephen (Hyphen Biomed). 249 samples of patients were included in this study. Normal reference ranges were determined with platelet-poor plasma (PPP) from healthy blood donors. Performances were then evaluated by comparing Cephen LS/Cephen test results to the results of the laboratory's reference assay for the diagnosis of LA and to clinical data, both on non-anticoagulated and anticoagulated patients' samples (Unfractioned heparin (UFH), Low Molecular Weight Heparin (LMWH), Vitamin K Antagonists (VKA) and apixaban). Interference of UFH, LMWH and VKA were also evaluated thanks to spiking experiment of increasing heparin concentrations or factor deficiency. Cephen LS/Cephen test had 48.6% sensitivity towards LA. Although UFH and VKA seemed to interfere with this assay and were likely to cause false negative, LMWH and apixaban did not. Finally, combination of Cephen LS/ Cephen with dRVVT had 89.0% sensitivity. Cephen LS/Cephen seems relevant for LA diagnosis, in combination with dRVVT, and might be used in patients undergoing LMWH or apixaban therapy.