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Background: Testing for lupus anticoagulant (LA) is not recommended in case of inflammation as C-reactive protein (CRP) can interfere in vitro with the phospholipids present in the activated partial thromboplastin time test used to detect an LA. However, the potential interference of an acute phase protein (ie, CRP) in LA testing using the dilute Russell's viper venom (DRVV) test is poorly studied. Objectives: To study the effect of inflammation, as evidenced by increased CRP levels, on DRVV tests. Methods: First, a retrospective analysis (2013-2023) was performed: data on all LA workups were retrieved, and the association between CRP levels and DRVV screen, mix, and confirm clotting times was studied. Second, data on DRVV panels and CRP levels were extracted from 2 prospective studies involving intensive care unit patients to study the association between both variables. Third, CRP was added to normal pooled plasma at 6 relevant concentrations (up to 416 mg/L) to study the association between CRP itself and DRVV coagulation times. Results: In the retrospective analysis, DRVV screen and confirm clotting times significantly increased as CRP increased (increase of 0.11 seconds and 0.03 seconds per 1 mg/L increase of CRP level, respectively). In the prospective analysis, only DRVV screen was prolonged with high CRP levels (increase of 0.06 seconds for a 1 mg/L increase in CRP level); DRVV screen/confirm ratio was also increased with high CRP levels. In vitro, the addition of CRP did not significantly increase any DRVV clotting times. Conclusion: LA testing should be performed with much caution in the presence of inflammation as it may be associated with prolongation of both activated partial thromboplastin time and DRVV clotting times.

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Envenoming resulting from snakebites is recognized as a priority neglected tropical disease by The World Health Organization. The Bothrops genus, consisting of different pitviper species, is considered the most medically significant taxa in Central and South America. Further research into Bothrops venom composition is important to aid in the development of safer and more effective snakebite treatments. In addition, the discovery of Bothrops toxins that could potentially be used for medical or diagnostic purposes is of interest to the pharmaceutical industry. This study aimed to employ high-throughput (HT) venomics to qualitatively analyze venom composition while utilizing coagulation bioassays for identifying coagulopathic toxins and characterizing coagulopathic activity in various Bothrops venoms. Using the recently demonstrated HT venomics workflow in combination with post-column coagulopathic bioassaying, focus was placed at anticoagulant toxins. Well-known procoagulant toxins were also investigated, taking into account that using the HT venomics workflow, procoagulant toxins are especially prone to denaturation during the reversed-phase chromatographic separations performed in the workflow. The findings revealed that the venoms of B. atrox and B. jararaca harbored procoagulant toxins, whereas those of B. alternatus and B. neuwiedi contained both procoagulant and anticoagulant toxins. In general, anticoagulation was associated with phospholipases A2s, while procoagulation was associated with snake venom metalloproteinases and snake venom serine proteases. These results showed the identification of coagulopathic venom toxins in the Bothrops venoms analyzed using multiple analytical methods that complement each other. Additionally, each venom underwent qualitative characterization of its composition.

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INTRODUCTION: The activity of direct oral anticoagulants (DOAC) is important in acute clinical situations. Recent studies have suggested a strong influence of DOAC on the diluted Russel's Viper Venom Time (dRVVT). Therefore, it may be a suitable screening parameter for antithrombotic plasma activity of different DOAC. This prospective study aims to evaluate the sensitivity and specificity of dRVVT to detect residual DOAC activity at recommended plasma level thresholds. METHODS: A total of 80 patients were recruited, with 20 each treated with one of the four approved DOAC (apixaban, edoxaban, rivaroxaban or dabigatran), respectively. Blood plasma was collected before (baseline), at plasma peak time, and 6 and 12 h after DOAC. DRVVT was measured using the screen (LA1) and confirm (LA2) assay for lupus anticoagulant and compared with DOAC plasma levels. A reference range was calculated based on the dRVVT values of 61 healthy blood donors. RESULTS: All DOAC significantly prolonged the dRVVT especially at higher DOAC plasma levels. The LA1 time ≥41 s had a sensitivity ≥98% to detect edoxaban, dabigatran and rivaroxaban plasma levels ≥30 ng/mL but it was only 87% for apixaban. Sensitivity was ≥98% for all DOAC with the LA2 assay ≥36 s. The negative predictive value of a DOAC plasma level <30 ng/mL and dRVVT LA2 <36 s was 99%. CONCLUSIONS: The dRVVT confirm assay (LA2) reliably detects residual DOAC plasma levels ≥30 ng/mL and could be useful to rapidly rule out relevant DOAC activity in emergency situations and to guide treatment decisions.

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This work aimed to purify the proteins that cause blood coagulation in the venom of the Iranian Echis carinatus snake species in a comprehensive manner. Gel filtration chromatography (GFC), Ion exchange chromatography (IEC), and Size Exclusion High-Performance Liquid Chromatography (SEC-HPLC) were utilized in the purification of the coagulation factors. The prothrombin clotting time (PRCT) and SDS-PAGE electrophoresis were performed to confirm the coagulative fractions. The fraction with the shortest coagulation time was selected. The components of this designated fraction were identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) following thorough purification. Circular dichroism (CD) was employed to determine the second structure of the coagulation factor. The crude venom (CV) was analyzed and had a total protein concentration of 97%. Furthermore, the PRCT of the crude venom solution at a concentration of 1 mg/ml was determined to be 24.19 ± 1.05 s. The dosage administered was found to be a factor in the venom's capacity to induce hemolysis. According to CD analysis, the protein under investigation had a helical structure of 16.7%, a beta structure of 41%, and a turn structure of 9.8%. CHNS proved that the purified coagulant protein had a Carbon content of 77.82%, 5.66% Hydrogen, 3.19% Nitrogen, and 0.49% Sulphur. In the present investigation, a particular type of snake venom metalloproteinase (SVMP) has undergone the process of purification and characterization and has been designated as EC-124. This purified fraction shows significant efficacy as a procoagulant. Our findings have shown that this compound has a function similar to factor X and most likely it can cause blood coagulation by activating factor II (FII).

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The search for mechanism-based anti-inflammatory therapies is of fundamental importance to avoid undesired off-target effects. Phospholipase A2 (PLA2) activity is a potential molecular target for anti-inflammatory drugs because it fuels arachidonic acid needed to synthesize inflammation mediators, such as prostaglandins. Herein, we aim to investigate the molecular mechanism by which ß-keto amyrin isolated from a methanolic extract of Cryptostegia grandiflora R. Br. Leaves can inhibit inflammation caused by Daboia russellii viper (DR) venom that mainly contains PLA2. We found that ß-keto amyrin neutralizes DR venom-induced paw-edema in a mouse model. Molecular docking of PLA2 with ß-keto amyrin complex resulted in a higher binding energy score of -8.86 kcal/mol and an inhibition constant of 611.7 nM. Diclofenac had a binding energy of -7.04 kcal/mol and an IC50 value of 620 nM, which predicts a poorer binding interaction than ß-keto amyrin. The higher conformational stability of ß-keto amyrin interaction compared to diclofenac is confirmed by molecular dynamics simulation. ß-keto amyrin isolated from C. grandiflora inhibits the PLA2 activity contained in Daboia russellii viper venom. The anti-inflammatory property of ß-keto amyrin is due to its direct binding into the active site of PLA2, thus inhibiting its enzyme activity.

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Lupus anticoagulant (LA) is a well-established risk factor for the clinical manifestations of antiphospholipid syndrome (APS). Accurate LA detection is an essential prerequisite for optimal diagnosis and management of patients with APS or aPL carriers. Variability remains a challenge in LA testing, with reliable detection influenced by multiple factors, including pre-analytical conditions, anticoagulation treatment, choice of tests and procedures performed, as well as interpretation of results, that can lead to false-positives or negatives. A standardised approach to LA testing, following current guidance, based on published data and international consensus, and with attention to detail, is required to underpin accurate detection of LA. Future work should focus on better characterisation of the nature of LA, which may ultimately lead to improved diagnosis and management of patients with APS and aPL carriers. This article reviews current practice and challenges, providing an overview on detection of LA.

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Background: The laboratory tests for lupus anticoagulant (LA) detection comprise complex and multistep coagulation testing procedures. There is no established gold standard assay or direct comparison of algorithms as recommended by different guidelines. Objectives: This study aimed to evaluate and compare the LA detection performance of different laboratory algorithms suggested by the existing guidelines. Methods: The routine LA test data of 1801 plasma samples, including 188 LA-positive and 1613 LA-negative samples, were re-evaluated by applying the algorithms recommended by existing guidelines and were interpreted using various methods. Diagnostic performance indices for each LA detection algorithm were compared with those of the other algorithms. The efficacies of the different interpretation methods were analyzed to determine a suitable interpretation methodology for each assay. Results: The diagnostic performance for detecting LA varied by the algorithm and method of interpretation used. All laboratory algorithms displayed exceptional diagnostic performance with all diagnostic parameters of >90.0%. Nearly perfect agreement was observed in all algorithms when compared to the Clinical and Laboratory Standards Institute 2014 guideline interpreted by normalized screen-to-confirm ratio (NSCR) and mixing test-specific cutoff (MTC), as a reference assay (Cohen's kappa coefficient, >0.90 [range, 0.94-1.00]). A combination of the index of circulating anticoagulant and NSCR was optimal for interpreting the activated partial thromboplastin time-based test, whereas a combination of the MTC and NSCR was suitable for the diluted Russell's viper venom time-based test. Conclusion: All laboratory algorithms showed equivalent diagnostic performance. Establishing the best method of interpretation for each assay is recommended to improve LA detection performance.

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Daboia russelii is a category-I medically important snake throughout the Indian sub-continent contributing to majority of snakebite incidences in this part of the world. As such, extensive studies on its venom composition and search of efficient and appropriate interventions for its treatment become crucial. In this study, the proteome of Daboia russelii venom from Tanore, Rajshahi, Bangladesh was profiled using a combination of chromatographic and mass spectrometric techniques. A total of 37 different proteins belonging to 11 different snake venom protein families were detected. Proteomics analysis revealed the presence of major phospholipase A2 toxins. Daboiatoxin (both A and B subunits), the main lethal PLA2 toxin in the venom of Daboia siamensis (Myanmar viper) which is neurotoxic, myotoxic and cytotoxic was detected. Presence of Daboxin P, which is a major protein in the venom of Indian Daboia russelii with strong anticoagulant activity, was also observed. Inconsistent distribution of such lethal toxins in the venom of same species calls for more investigations of snake venoms from lesser explored regions and formulation of better alternatives to the current antivenom therapy for efficient treatment.

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OBJECTIVES: To determine the impact of residual platelets on dilute Russell's viper venom time (DRVVT) assay in frozen-thawed plasma submitted for lupus anticoagulant (LAC) testing. METHODS: We measured platelet counts in frozen-thawed samples submitted for LAC testing and evaluated the association between platelet count and the DRVVT screening time and ratios. We also spiked platelets into a LAC-positive sample to observe the effect on the DRVVT. RESULTS: Progressive increase in platelet count resulted in a statistically significant shortening of the DRVVT assay results on plasma after 1 freeze-thaw cycle. A similar effect was noted on the LAC-positive sample. CONCLUSIONS: Residual platelets in plasma samples result in shortening of DRVVT assay after 1 freeze-thaw cycle. This may result in a false-negative LAC test result.

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Recurrent pregnancy loss (RPL) can be a devastating experience for individuals trying to have children. Various potential causes contribute to the multifactorial pathogenesis of RPL, including chromosomal anomalies, endocrine conditions, autoimmunity, thrombophilias, and infectious agents. Antinuclear antibodies (ANAs) offer an unspecific clue to the underlying autoimmune pathogenic etiology of RPL. This case details a 40-year-old female with a history of RPL, fibromyalgia, and ANA positivity, who spontaneously developed lupus anticoagulant antibodies during her second trimester of pregnancy. Although the recommended American Society of Reproductive Medicine (ASRM) diagnostic criteria for initiating a thrombophilia evaluation was not formally met, the patient's maintenance of low-molecular-weight heparin (LMWH) throughout her pregnancy may have contributed to the success of the pregnancy. When treating a patient with RPL, consideration of the comprehensive clinical picture should precede the need to strictly adhere to published criteria for using non-proven clinical interventions. A risk-benefit analysis ought to be considered when offering patients additional medications that may come with some risks but could significantly improve the chances of a successful clinical outcome, such as live birth. We aim to provide evidence to promote greater flexibility in guidelines so that a patient's unique autoimmune etiologies of RPL are not overlooked.

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BACKGROUND: Triple positivity for all 3 criteria antiphospholipid antibodies confers high risk of symptom development in carriers, and recurrence in antiphospholipid syndrome (APS). Most triple-positivity studies report lupus anticoagulant (LA) testing as positive without distinguishing between positivity with dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) and single-assay positivity or only perform dRVVT. Single LA assay repertoires remain in use in some centers, which risks missing some triple positives. Positivity with both assays may identify higher risk. OBJECTIVES: The aim of this study is to investigate the frequency of single LA assay positivity in triple-positive patients. METHODS: Three hundred forty-two triple-positive profiles from nonanticoagulated patients (237 APS, 45 systemic lupus erythematosus without APS symptoms, and 60 nonclinical criteria) were identified from laboratory databases and assessed for LA positivity by dRVVT and/or APTT. RESULTS: Seventy-three of 237 (30.8%) APS samples were LA-positive with 1 assay, 40/237 (16.9%) by dRVVT only, and 33/237 (13.9%) with APTT only. Nineteen of 45 (42.2%) were LA-positive with 1 assay in the systemic lupus erythematosus cohort; 12/45 (26.7%) with dRVVT only and 7/45 (15.5%) with APTT only. Thirty-three of 60 (55.0%) were LA-positive with 1 assay in the nonclinical criteria cohort; 24/60 (40.0%) with dRVVT only and 9/60 (15.0%) with APTT only. The most common solid-phase assay profile was elevated immunoglobulin G aCL and aß2GPI. CONCLUSION: Up to 55.0% of triple-positive samples were positive in 1 LA assay, representing significant potential for misdiagnosis and inappropriate management via single LA assay repertoires.

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Daboxin P, reported earlier from the venom of Daboia russellii, disturbs the blood coagulation cascade by targeting factor X and factor Xa. The present study exhibits that Daboxin P also inhibits platelet aggregation induced by various agonists. The thrombin-induced platelet aggregation was inhibited maximum whereas inhibition of collagen-induced platelet aggregation was found to be 50% and no inhibition of adenosine diphosphate (ADP) and arachidonic acid-induced aggregation was observed. Daboxin P dose-dependently inhibited the thrombin-induced platelet aggregation with Anti-Aggregation 50 (AD50 ) dose of 55.166 nM and also reduced the thrombin-mediated calcium influx. In-silico interaction studies suggested that Daboxin P binds to thrombin and blocks its interaction with its receptor on the platelet surface. Quenching of thrombin's emission spectrum by Daboxin P and electrophoretic profiles of pull-down assay further reveals the binding between Daboxin P and thrombin. Thus, the present study demonstrates that Daboxin P inhibits thrombin-induced platelet aggregation by binding to thrombin.

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Vipegrin is a 6.8 kDa Kunitz-type serine proteinase inhibitor purified from Russell's viper (Vipera russelii russelii) venom. Kunitz-type serine proteinase inhibitors are non-enzymatic proteins and are ubiquitous constituents of viper venoms. Vipegrin could significantly inhibit the catalytic activity of trypsin. It also posseses disintegrin-like properties and could inhibit collagen and ADP-induced platelet aggregation in a dose-dependent manner. Vipegrin is cytotoxic to MCF7 human breast cancer cells and restricts its invasive property. Confocal microscopic analysis revealed that Vipegrin could induce apoptosis in MCF7 cells. Vipegrin disrupts cell to cell adhesion of MCF7 cells through its disintegrin-like activity. It also causes disruption of attachment of MCF7 cells to synthetic (poly L-lysine) and natural (fibronectin, laminin) matrices. Vipegrin did not cause cytotoxicity on non-cancerous HaCaT, human keratinocytes. The observed properties indicate that Vipegrin may help the development of a potent anti-cancer drug in future.

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Activated protein C resistance (APCR) reflects a hemostatic state defined by a reduced ability of activated protein C (APC) to affect an anticoagulant response. This state of hemostatic imbalance is characterized by a heightened risk of venous thromboembolism. Protein C is an endogenous anticoagulant that is produced by the hepatocytes and undergoes proteolysis-mediated activation to APC. APC in turn degrades activated Factors V and VIII. APCR describes a state of resistance by activated Factors V and VIII to APC-mediated cleavage of these factors, thereby promoting amplified thrombin production and a potentially procoagulant state. This resistance of APC may be inherited or acquired. Mutations in Factor V are responsible for the most frequent form hereditary APCR. The predominant mutation, a G1691A missense mutation at Arginine 506, the so-called Factor V Leiden [FVL], causes a deletion of an APC-targeted cleavage site in Factor Va, thereby rendering it resistant to inactivation by APC. There are a variety of laboratory assays for APCR, but this chapter focuses on a procedure using a commercially available clotting assay that utilizes a snake venom and ACL TOP analyzers.

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Serine Proteinase Associated Disintegrin-1 (SPAD-1) is a low molecular mass (26 kDa) positively charged protein purified from Russell's viper venom (RVV) possessing cytotoxic activity on MCF7, human breast cancer cells. Primary sequence analysis of the protein confirms that it is a novel Snake Venom Serine Proteinase (SVSP) and a member of the trypsin family. SPAD-1 contains a conserved triad of Histidine (H), Aspartic acid(D) and Serine(S) residues at its active site for proteinase activity and also an adjacent histidine-glycine-aspartic acid (HGD) disintegrin-like motif. The serine proteinase and disintegrin parts are functionally active and independent. SPAD-1 showed proteolytic digestion of fibrinogen and fibronectin, but laminin digestion was below the detectable limit. Proteolytically inactivated SPAD-1 inhibited collagen and ADP-induced platelet aggregation. This study proposes considering Serine Proteinase Associated Disintegrin (SPAD) as a new group of snake venom proteins. Members of this group contain a serine proteinase catalytic triad and a disintegrin-like motif. SPAD-1 caused visible morphological changes in MCF7 cells, including a reduction of the cell-to-cell attachments, rounding of cell shape and death, in vitro. SPAD-1 also showed a dose-dependent significant decrease in the invasive potency of breast cancer cells. Confocal microscopic analysis revealed the breakage of nuclei of the SPAD-1-treated cells. SPAD-1 also increased cell detachment from the poly L-lysine-coated, laminin-coated and fibronectin-coated culture plate matrices, confirming the disintegrin activity. This study concludes that SPAD-1 may be a good candidate for anti-tumour drug design in the future.

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Disintegrins are small peptides possessing a tripeptide motif capable of binding to integrins. These were first isolated from viper venoms and are now also found in many other hematophagous organisms. Many integrins have been studied for their role in the onset of disease and the interaction of disintegrins with these receptors makes them potential therapeutic molecules. Disintegrins are also used as molecular scaffolds to design effective drugs for cardiovascular diseases and cancer. Even the gene and protein sequencing data of disintegrins have provided insights into understanding the molecular complexity of disintegrins. In this review, we try to summarize the structural and functional importance of disintegrins in identifying the biological targets and triggering various signaling pathways involved in platelet aggregation and cancer. Also, we have tried to elucidate a possible molecular mechanism behind the action of disintegrins on platelet aggregation and cancer. This understanding will help us to design and to explore more of these integrin-binding molecules.

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Lupus anticoagulant (LA) is composed of heterogeneous autoantibodies, which have a close association with thrombotic events. Due to its heterogeneity, two methods for increasing sensitivity are recommended for LA. An investigation of the thrombotic risk and anticardiolipin (aCL) and anti-ß2-glycoprotein I (aB2GPI) antibody profiles was conducted based on the results of using two parallel methods (dilute Russell viper venom time (dRVVT), silica clotting time (SCT)) in a real world clinical laboratory. Of 5120 patients, 684 patients (13%) were LA positive, and 422 patients (8%) experienced thrombotic events including pregnancy complication. Development of thrombotic events was more likely to occur in patients who were positive for both dRVVT and SCT compared with those who were positive for dRVVT or SCT only. In addition, significantly higher positive rates of aCL and aB2GPI and the persistently positive rate of LA at intervals of 12 weeks or longer were observed in patients who were positive for both dRVVT and SCT compared with those who were positive for dRVVT or SCT only. Considering three laboratory tests (LA, aCL, and aB2GPI), high thrombotic risk was observed for patients with both dRVVT and SCT positive LA results. A report on LA results that divides LA positive into two types (LA-single positive and LA-both positive) may be beneficial to clinicians in detection of high-risk thrombotic patients.

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Anticoagulant rodenticides (ARs) continue to be used across the United States as a method for controlling pest rodent species. As a consequence, wild birds of prey are exposed to these toxicants by eating poisoned prey items. ARs prevent the hepatic recycling of vitamin K and thereby impede the post-translational processing of coagulation factors II, VII, IX, and X that are required for procoagulant complex assembly. Through this mechanism of action, ARs cause hemorrhage and death in their target species. Various studies have documented the persistence of these contaminants in birds of prey but few have attempted to use affordable and accessible diagnostic tests to diagnose coagulopathy in free-ranging birds of prey. In our study free-ranging red-tailed hawks were found to be exposed to difethialone and brodifacoum. Eleven of sixteen (68%) livers tested for AR exposure had detectable residues. Difethialone was found in 1/16 (6%), and brodifacoum was detected in 10/16 (62%) liver samples that were tested for rodenticide residues. Difethialone was found at a concentration of 0.18 ug/g wet weight and brodifacoum concentrations ranged from 0.003-0.234 ug/g wet weight. Two out of 34 (6%) RTHA assessed for blood rodenticide had brodifacoum in serum with measured concentrations of 0.003 and 0.006 ug/g. The range of clotting times in the prothrombin time (PT) and Russell's viper venom time assays for control RTHA were 16.7 to 39.7 s and 11.5 to 91.8 s, respectively. One study bird was diagnosed with clinical AR intoxication with a brodifacoum levels in blood of 0.006 and 0.234 ug/g wet weight in blood and liver respectively, a packed cell volume (PCV) of 19%, and PT and RVVT times of >180 s. No correlation was found between PT and RVVT in the control or free-range RTHA, and there was no relationship found between the presence of liver anticoagulant residues and clotting times in the PT and RVVT.

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BACKGROUND: Captive reptiles often present with clinical signs suggestive of a clotting disorder or severe illness that can induce or exacerbate a coagulopathy. However, coagulopathies in reptiles are difficult to characterize due to lack of species-appropriate reagents to perform coagulation tests. The objective of this study was to develop screening tests to evaluate the extrinsic and common pathways of coagulation in green iguanas (Iguana iguana). KEY FINDINGS: Reptile and avian thromboplastin, extracted from reptile and avian brains, respectively, were used to initiate coagulation in prothrombin time (PT) assays and commercially available reagents were used to determine Russell's viper venom time, thrombin time, and fibrinogen using the Clauss method. Coagulation assays were performed on citrate-anticoagulated plasma from 18 healthy green iguanas. Results were summarized as median (minimum-maximum): PT (reptile thromboplastin), 34.8 seconds (27.1-42.1 s), PT (avian thromboplastin), 78.5 seconds (51.6-114.23 s), Russell's viper venom time, 56.15 seconds (18.4-79.7 s), thrombin time, 10 seconds (7.0-36.5 s), and fibrinogen, 258 mg/dl (89-563.0) (2.58 [0.89-5.63 g/L]). SIGNIFICANCE: Commercial reagents can be used to evaluate the common pathway and fibrinogen; however, avian- or reptile-sourced thromboplastin is preferred for a reliable coagulation trigger to perform the PT assay and evaluate the extrinsic pathway.

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