Viper Venom Research Articles

Complex target SELEX-based identification of DNA aptamers against Bungarus caeruleus venom for the detection of envenomation using a paper-based device

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is β-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

Laboratory Approaches to Test the Function of Antiphospholipid Antibodies

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder caused by the presence of aPLs (antiphospholipid antibodies, i.e., anti-β2-glycoprotein I and anti-cardiolipin). Everyday practice in terms of laboratory diagnostics of APS includes determination of aPLs and well-known functional assays assessing for lupus anticoagulant (LA), in turn using various tests. According to recent guidelines, the recommended method for LA identification or exclusion is based on the Russell Viper Venom test and a sensitive activated partial thromboplastin time assay. Despite the fact that LA can be quantified in laboratory practice in this way, LA is still used as a binary parameter that is just one of the risk factors of thrombosis in APS. As of today, there are no other functional assays to routinely assess the risk of thrombosis in APS. It is well-known that APS patients display a wide range of clinical outcomes although they may express very similar laboratory findings. One way to solve this dilemma, could be if antibodies could be further delineated using more advanced functional tests. Therefore, we review the diagnostic approaches to test the function of aPLs. We further discuss how thrombin generation assays, and rotational thromboelastometry tests can be influenced by LA, and how experimental methods, such as flow cytometric platelet activation, surface plasmon resonance, or nano differential scanning fluorimetry can bring us closer to the puzzling interaction of aPLs with platelets as well as with their soluble protein ligand. These novel approaches may eventually enable better characterization of aPL, and also provide a better linkage to APS pathophysiology.

Snake venom proteomics and antivenomics of two Sundaic lance-headed pit vipers: Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia)

Envenomation by two medically important Sundaic pit vipers, Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia), causes hemotoxic syndrome with a potentially fatal outcome. Research on the compositions and antigenicity of these pit viper venoms is however lacking, limiting our understanding of the pathophysiology and treatment of envenomation. This study investigated the venom proteomes of both species through a protein decomplexation strategy, applying C18 reverse-phase high-performance liquid chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein identification through nano-electrospray ionization liquid chromatography-tandem mass spectrometry (nano-ESI-LCMS/MS) of trypsin-digested peptides. The venom antigenicity was profiled against the Thai Green Pit Viper Antivenom (GPVAV, a hetero-specific antivenom), using indirect enzyme-linked immunosorbent assay (ELISA). The venom proteomes of T. wiroti and T. puniceus consisted of 10 and 12 toxin families, respectively. The major proteins were of diverse snake venom serine proteases (19–30% of total venom proteins), snake venom metalloproteinases (17–26%), disintegrins(9–16%), phospholipases A2 (8–28%) and C-type lectins (~8%). These were putative snake toxins implicated in hemorrhage and coagulopathy, consistent with clinical hemotoxicity. GPVAV showed strong immunorecognition toward high and medium molecular weight proteins (e.g., SVMP and PLA2) in both venoms, while a lower binding activity was observed toward small proteins such as disintegrins. Conserved antigenicity in the major hemotoxins supported toxicity cross-neutralization by GPVAV and indicated that the immunorecognition of low molecular weight toxins may be optimized for improved binding efficacy. Taken together, the study provides insights into the pathophysiology and antivenom treatment of envenomation caused by T. wiroti and T. puniceus in the region.

Potential of herbal cocktail of medicinal plant extracts against ‘big four’ snake venoms from India

BackgroundVenomous snake bites cause acute medical emergencies and are fatal. India accounts for large proportion of snake-bite deaths globally. Medically important ‘BIG FOUR’ snakes of India are Bungarus caeruleus (krait), Naja naja (cobra), Echis carinatus (saw-scaled viper) and Daboia russelii (Russell's viper). Polyherbal formulations have been proved to be effective in treatment of diseases than a single formulation. Objective(s)To evaluate aqueous ethanolic extract cocktail of Azadirachata indica, Butea monosperma, Citrus limon, Clerodendrum serratum and Areca catechu for antidote potential against BIG FOUR venoms in ex vivo and in vivo model. Materials and methodsAnti-hemorrhagic and venom neutralization studies were performed in seven-day old chick embryo model for ex vivo studies. In vivo studies were performed using male Swiss albino mice for antivenom potential of herbal cocktail by performing anti-edematic, anti-hemorrhagic, anti-myotoxic activity, and venom neutralization. ResultsHerbal cocktail exhibited differential venom inhibition potential against four venoms tested. Hemorrhagic activity was completely neutralized by the herbal cocktail; myotoxic activities of krait and Russell's viper venom were neutralized; while anti-edematic activity was observed for krait and cobra venom. Herbal cocktail completely neutralized venom lethality (3∗LD50) of krait and saw-scaled viper venom. ConclusionInhibitions of various venom components of all four venoms suggests presence of phytochemicals in herbal cocktail with therapeutic properties. Further studies would help in the development of a formulation as a first-aid towards treatment of snake bite victims.

Using Electronic Best Practice Alerts to Improve Thrombophilia Testing Based on ASH Choosing Wisely Guidelines

IntroductionThrombophilia testing is costly, and results can be misleading in the setting of acute venous thromboembolism (VTE) or anticoagulation use. It is frequently ordered despite limited impact on clinical decision making. American Society of Hematology (ASH) Choosing Wisely guidelines recommend against thrombophilia testing in patients with VTE in the setting of major transient risk factors (surgery, trauma, prolonged immobility).MethodsA best practice alert (BPA) was implemented in our electronic medical record system to promote awareness of the ASH Choosing Wisely guidelines. The BPA showed the text of the ASH Choosing Wisely guidelines onscreen whenever thrombophilia testing was ordered for a patient with a diagnosis of pulmonary embolism (PE) or deep venous thrombosis (DVT). It was up to the clinician to decide whether the Choosing Wisely guidelines applied in a particular clinical scenario and whether to follow the BPA recommendation and cancel the thrombophilia test or to override the BPA and order testing.Thrombophilia testing orders which would trigger the BPA were protein C and S activity levels, lupus anticoagulant, dilute Russell viper venom test, homocysteine levels, Factor V Leiden, prothrombin gene mutation and activated protein C resistance.The BPA began displaying to providers in the outpatient setting on 7/1/2016 and in the inpatient setting on 9/1/2016. The pre-BPA period was defined as the 12 months prior to activation of the outpatient BPA (7/1/2015 to 6/30/2016). The post-BPA period was defined as the 7 months from the activation of the inpatient BPA (9/1/2016 to 3/31/2017). We reviewed the number of BPAs triggered as well as the actions taken by providers (follow or override). We also compared the total number of thrombophilia tests in the outpatient and inpatient settings in the pre- and post-BPA periods.ResultsThe BPA appeared onscreen 140 times in the outpatient setting and 35 times in the inpatient setting during the study period. The BPA was followed 17.1% of the time in the outpatient setting and 100% of the time in the inpatient setting. Thrombophilia testing volumes stayed unchanged in the outpatient setting (471.5 tests per month pre-BPA vs. 471.6 tests per month post-BPA, p=1.00). In contrast, there was a significant decrease in the average number of thrombophilia tests per month (101.1 vs. 73.3, p=0.038) in the inpatient setting after the implementation of the BPAs. During the study period, inpatient censuses were not statistically different in the pre-BPA and post-BPA periods.DiscussionWe found that the thrombophilia BPA was followed at a very high rate in the inpatient setting compared to the outpatient setting. The BPA is meant to remind providers of the ASH Choosing Wisely guidelines, but providers must still use clinical judgment to determine whether the guideline applies in a given clinical scenario. The high rate of compliance in the inpatient setting suggests that in many cases, thrombophilia testing is being ordered on inpatients with transient risk factors, and that a brief reminder message can effectively change provider behavior. On the other hand, BPA compliance was relatively low in the outpatient setting, though this could be appropriate based on the clinical scenarios which outpatient providers encounter. Future analyses will include manual chart review to determine the appropriateness of BPA overrides in the outpatient setting, as well as a thematic analysis of BPA override comments.There was a significant reduction in the average number of inpatient thrombophilia tests ordered per month in the post-BPA period compared to the pre-BPA period. Although the inpatient thrombophilia BPA had a high rate of compliance, it was only shown 35 times during the study period. Thus the BPA messages alone were unlikely to account for the significant change in total volume of thrombophilia testing. Prior research on blood transfusion BPAs suggests that BPAs can have an educational impact, causing providers to become more aware of guidelines and thus actually reducing the frequency of triggered BPAs over time. Thus, each BPA message may have a multiplicative effect, preventing future unnecessary tests in addition to the original order. DisclosuresNo relevant conflicts of interest to declare.

Centralized Laboratory Testing for Inhibitors in the U.S. Hemophilia Treatment Centers Network: Testing Protocol and Results on 10,178 Specimens

Background: The incidence and prevalence of inhibitors among hemophilia patients in the U.S. has not been determined. The Centers for Disease Control and Prevention (CDC) has established a national inhibitor surveillance program called the Community Counts Registry for Bleeding Disorders Surveillance, through a collaboration with the American Thrombosis and Hemostasis Network and the U.S. Hemophilia Treatment Center (HTC) Network, to collect data and specimens to monitor inhibitors.Methods: A centralized laboratory was established at the CDC to perform inhibitor tests using a protocol based on accepted methods and published findings from a pilot project, the Hemophilia Inhibitor Research Study. Specimens are collected annually and when an inhibitor is detected or suspected locally from patients with hemophilia A (HA), hemophilia B (HB), and Type 3 von Willebrand disease (VWD3) who have received treatment and are enrolled at one of 134 HTCs. In the testing protocol, a modified Nijmegen-Bethesda assay with preanalytical heat inactivation (CDC-NBA) for factor VIII (FVIII) or factor IX (FIX) is first performed. On specimens with low positive or “gray zone” results of 0.5-1.9 Nijmegen-Bethesda units (NBU) for FVIII and 0.3-0.9 NBU for FIX, chromogenic Bethesda assay (CBA) for FVIII, fluorescence immunoassay (FLI) for anti-FVIII or anti-FIX antibodies, and dilute Russell's viper venom test (DRVVT) for lupus anticoagulants are performed. Repeat specimens are requested for positive or ambiguous results in patients with negative history.Results: Initial results on 10,178 specimens from 5,931 HA patients, 1,541 HB patients, and 134 VWD3 patients are shown in the Table. Additional testing on 141 low positive or “gray zone” FVIII specimens showed 74% to be CBA positive and 26% to be CBA negative. Of those CBA positive, 99% were also positive for IgG1 or IgG4 by FLI and were classified as true positives (104/141, 74%). Of CBA-negative specimens, 10 were also IgG1 and IgG4 negative and were classified as false positives (10/141, 7%). FLI results were positive on 26 CBA-negative specimens, which could not be classified (26/141, 18%). Final results on classified specimens are shown in the Table. Fourteen specimens were DRVVT positive, 13 of which were also CBA and FLI positive.Follow up was conducted on two groups of specimens: Arm 1 included specimens with positive or ambiguous results at CDC from patients who were reported to have no history of inhibitor, and Arm 2 included specimens from patients with new inhibitors locally detected at the HTC within the study period. In Arm 1, of 366 positive or ambiguous FVIII specimens, 20 were from patients with negative history and 17 of 18 repeated were confirmed; and of 44 positive FIX specimens, 3 were from patients with negative history and 2 of 3 repeated were confirmed. In Arm 2, 38 locally detected positive FVIII specimens and 6 locally detected positive FIX specimens were confirmed.Conclusions: From December 2013 through December 2016, newly positive test results on 55 FVIII and 8 FIX patients were identified and confirmed in two specimens, at least one of which was tested at the central laboratory. A cohort of 6,456 patients with negative history who were confirmed negative by central testing will be followed prospectively for inhibitor development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Is Lupus Anticoagulant a Significant Feature of COVID-19? A Critical Appraisal of the Literature.

The term "lupus anticoagulant (LA)" identifies a form of antiphospholipid antibodies (aPLs) causing prolongation of clotting tests in a phospholipid concentration-dependent manner. LA is one of the laboratory criteria identified in patients with antiphospholipid (antibody) syndrome (APS). The presence of LA in patients with APS represents a significant risk factor for both thrombosis and pregnancy morbidity. There have been several reports of similarities between some of the pathophysiological features of COVID-19 and APS, in particular the most severe form, catastrophic APS. There have also been many reports identifying various aPLs, including LA, in COVID-19 patients. Accordingly, a very pertinent question arises: "Is LA a feature of COVID-19 pathology?" In this review, we critically appraise the literature to help answer this question. We conclude that LA positivity is a feature of COVID-19, at least in some patients, and potentially those who are the sickest or have the most severe infection. However, many publications have failed to appropriately consider the many confounders to LA identification, being assessed using clot-based assays such as the dilute Russell viper venom time, the activated partial thromboplastin time (aPTT), and the silica clotting time. First, most patients hospitalized with COVID-19 are placed on anticoagulant therapy, and those with prior histories of thrombosis would possibly present to hospital already on anticoagulant therapy. All anticoagulants, including vitamin K antagonists, heparin (both unfractionated heparin and low-molecular-weight heparin), and direct oral anticoagulants affect these clot-based assays. Second, C-reactive protein (CRP) is highly elevated in COVID-19 patients, and also associated with severity. CRP can also lead to false-positive LA, particularly with the aPTT assay. Third, persistence of aPL positivity (including LA) is required to identify APS. Fourth, those at greatest risk of thrombosis due to aPL are those with highest titers or multiple positivity. Most publications either did not identify anticoagulation and/or CRP in their COVID-19 cohorts or did not seem to account for these as possible confounders for LA detection. Most publications did not assess for aPL persistence, and where persistence was checked, LA appeared to represent transient aPL. Finally, high titer aPL or multiple aPL positivity were in the minority of COVID-19 presentations. Thus, at least some of the reported LAs associated with COVID-19 are likely to be false positives, and the relationship between the detected aPL/LA and COVID-19-associated coagulopathy remains to be resolved using larger and better studies.

Impact of a commercially available DOAC absorbent on two integrated procedures for lupus anticoagulant detection

BackgroundLupus anticoagulant (LA)-detection in anticoagulated patients is an unmet need, which becomes even more cogent with the introduction of direct oral anticoagulants (DOAC) that may lead to false-positive results. AimsWe aimed to investigate the effect of a commercially available DOAC absorbent on residual drug concentrations, and on integrated procedures for LA-detection. MethodsBlood from patients treated for atrial fibrillation with either dabigatran (n = 39), rivaroxaban (n = 55), apixaban (n = 47) or edoxaban (n = 47) were collected at peak and trough, and centralized for testing with two LA integrated procedures [i.e., the silica clotting time (SCT) and dilute Russel viper venom (dRVVT)] before and after DOAC absorbent exposure. ResultsThe commercially available DOAC absorbent investigated in this study proved effective in reducing the concentrations of all the investigated DOAC, although small residual drug was detected after absorption, especially in patients on edoxaban.Results mimicking LA were observed in patients on DOAC before absorbent exposure, especially for rivaroxaban when testing was performed with dRVVT (88% rate at peak and 20% at trough) and much less with SCT (12% at peak and 8% at trough). The correspondent rate of results mimicking LA in patients on rivaroxaban after exposure was reduced [dRVVT (peak 8%, trough 4%); SCT (peak and trough 8%)], but not abolished. ConclusionsOverall, in vitro DOAC absorbance by active charcoal compounds is a useful laboratory tool for LA-detection in patients on DOAC. Caution should however be exerted when used in daily practice.

Transcriptomic and functional proteomics analyses to unveil the common and unique pathway(s) of neuritogenesis induced by Russell’s viper venom nerve growth factor in rat pheochromocytoma neuronal cells

ABSTRACT Background : The snake venom nerve growth factor (NGF)-induced signal transduction mechanism has never been explored. Research design and methods : Homology modeling and molecular dynamic studies of the interaction between Russell’s viper venom NGF (RVV-NGFa) and mammalian tropomyosin-receptor kinase A (TrkA) was done by computational analysis. Transcriptomic and quantitative tandem mass spectrometry analyses determined the expression of intracellular genes and proteins, respectively, in RVV-NGFa-treated PC-12 neuronal cells. Small synthetic inhibitors of the signal transduction pathways were used to validate the major signaling cascades of neuritogenesis by RVV-NGFa. Results : A comparative computational analysis predicted the binding of RVV-NGFa, mouse 2.5S-NGF (conventional neurotrophin), and Nn-α-elapitoxin-1 (non-conventional neurotrophin) to different domains of the TrkA receptor in PC-12 cells. The transcriptomic and quantitative proteomic analyses in unison showed differential expressions of common and unique genes and intracellular proteins, respectively, in RVV-NGFa-treated cells compared to control (untreated) mouse 2.5S-NGF and Nn-α-elapitoxin-1-treated PC-12 cells. The RVV-NGFa primarily triggered the mitogen-activated protein kinase-1 (MAPK1) signaling pathway for inducing neuritogenesis; however, 36 pathways of neuritogenesis were uniquely expressed in RVV-NGFa-treated PC-12 cells compared to mouse 2.5S NGF or Nn-α-elapitoxin-1 treated cells. Conclusion : The common and unique intracellular signaling pathways of neuritogenesis by classical and non-classical neurotrophins were identified.

Practical Considerations and Testing Nuances for the Detection of Lupus Anticoagulant: Do Low Phospholipid Screen Results, Assay Type, and Test Ratio Matter?

Lupus anticoagulant (LA) detection requires (1) prolongation of a phospholipid (PL)-dependent clot-based screening assay, (2) noncorrection upon adding normal pooled plasma, and (3) a confirmatory PL dependency test. Paired LA assays run screening and confirmatory tests simultaneously, with their test ratio (TR) or differences used to evaluate test results. We evaluated patients whose paired testing demonstrated PL dependence suggestive of LA, yet the low PL screen was not prolonged. Clinical and laboratory parameters are compared across (1) true positive (screen prolonged, TR positive) vs borderline (screen not prolonged, TR positive); (2) low-, moderate-, and high-TR subgroups; and (3) dilute Russell viper venom time (dRVVT) vs silica clotting time (SCT). Borderline samples are not statistically different from true positives in their rate of repeat LA positivity or association with other anti-PL antibodies. Compared with true positives, borderline dRVVT is more frequent in pregnancy, women, and younger age. Elevated activated partial thromboplastin time is more frequent in true-positive dRVVT and SCT vs borderline and with an increasing dRVVT TR. LA persistence is more frequent with an increasing SCT TR. In addition, dRVVT true positivity is more frequent with thromboembolic events, while SCT is more frequent with autoimmunity and pregnancy complications. Negative low-PL screens may not necessarily lack LA. A reevaluation of the laboratory criteria for LA detection may be needed.

A Diagnostic Solution for Lupus Anticoagulant Testing in Patients Taking Direct Oral FXa Inhibitors Using DOAC Filter.

Background: Direct oral factor Xa (FXa) inhibitors interfere with lupus anticoagulant (LA) assays challenging antiphospholipid syndrome diagnosis in treated patients. We evaluated a new device, called DOAC Filter, and its usefulness in this setting. It is a single-use filtration cartridge in which FXa inhibitor compounds are trapped by non-covalent binding while plasma is filtered through a solid phase. Patient samples were analyzed before and after filtration: 38 rivaroxaban, 41 apixaban, and 68 none. Anticoagulant plasma concentrations were measured using specific anti-Xa assays and HPLC-MS/MS. LA testing was performed using dilute Russell Viper Venom Time (dRVVT) and Silica Clotting Time (SCT). Baseline median [min–max] concentrations were 64.8 [17.6; 311.4] for rivaroxaban and 92.1 ng/mL [37.1; 390.7] for apixaban (HPLC-MS/MS). They were significantly correlated with anti-Xa assay results (r = 0.98 and r = 0.94, respectively). dRVVT was positive in 92% rivaroxaban and 72% apixaban and SCT in 28 and 41% of samples, respectively. Post-filtration, median % of neutralization was 100% with rivaroxaban and apixaban concentrations of, respectively, <2 [<2–2.4] and <2 ng/mL [<2–9.6] using HPLC-MS/MS. No significant effect of DOAC Filter was observed on LA testing in controls (n = 31) and LA-positive (n = 37) non-anticoagulated samples. dRVVT and SCT remained positive in, respectively, 16 and 8% of rivaroxaban and 41 and 18% of apixaban samples. DOAC Filter would be an easy-to-use device allowing FXa inhibitor removal from plasma samples, limiting their interference with LA testing in treated patients.

Association of Lupus Anticoagulant with Brain Atrophy in Gulf War Illness (GWI)

Separate lines of research have documented brain atrophy and evidence of autoimmune mechanisms in Gulf War Illness (GWI), including the presence of lupus anticoagulant (LAC), in veterans with GWI. Here we evaluated the possible association of LAC and brain volume in veterans with GWI. The presence of LAC was determined using Silica Clotting Time and dilute Russell’s Viper Venom Time assays. MRI data was acquired using a Philips 3T MR scanner from which total gray matter, total cortical gray matter, total subcortical gray matter, and total cerebral white matter were derived. The results demonstrated a statistically significant reduction of brain volume in all regions tested in GWI veterans with positive LAC, as compared to those without LAC. These findings add to the literature implicating autoimmune mechanisms in GWI and point to the presence of prothrombotic antiphospholipid antibodies as contributing to brain atrophy in GWI.

Investigation of capillary leak syndrome induced by the eastern (Daboia siamensis) and western (Daboia russelii) Russell’s viper venoms.

Investigation of capillary leak syndrome induced by the eastern (Daboia siamensis) and western (Daboia russelii) Russell’s viper venoms.

Applying index of circulating anticoagulant to mixing tests with lupus anticoagulant screen and confirm reagents can distinguish with high specificity between lupus anticoagulants and direct factor Xa inhibitors.

Lupus anticoagulants (LA) are detected by prolongation of clotting times for dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) screening tests. Direct oral anticoagulants (DOACs) can interfere with both screening and confirmatory tests. The present study aimed to investigate the influence of direct factor Xa inhibitors (DiXaIs) on screen, confirm and mixing tests and establish a method for differentiation from other sample types. A total of 257 samples including nonanticoagulated LA positive, LA positive with DiXaIs, factor deficiency, FVIII inhibitors, warfarin and non-APS DiXaIs were tested. APTT reagents Cephen LS/Cephen and dRVVT reagents LA1/LA2 were used, respectively, to screen/confirm the study group. Index of circulating anticoagulant (ICA) was calculated from clotting times based on the following formula as ICA screening and ICA confirmation. ICA= (1:1 Mix sample - Normal pooled plasma) / Screen patient x 100. An ICA matrix was established which suggested the presence of a DiXaI when both ICA screening and confirmation were above the cut-off. When only ICA screening is elevated, LA is suspected. Sensitivity and specificity of the ICA matrix were 52.2% and 92.8% for DiXaIs and 38.1% and 96.7% for LA in APTT, and 61.2% and 92.9% for DiXaIs and 22.2% and 88.4% for LA in dRVVT, respectively. The ICA matrix achieved high specificity with a lower apparent sensitivity for DiXaI samples comparatively to other devices but due only to less interferences: the matrix could contribute to differentiating DiXaIs from LA in samples where anticoagulation status is unknown.

Evaluation of the effect of apixaban using a viscoelastic coagulation assay with Russell\u2019s viper venom reagent

BackgroundConventional coagulation tests, such as prothrombin time and activated partial thromboplastin time, are not sensitive to anticoagulation by apixaban. We evaluated the antithrombotic effect of apixaban using a Russell viper venom (RVV) test for a patient who underwent posterior spine fusion surgery.Case presentationAn 84-year-old man was scheduled for percutaneous posterior spine fusion. He continued apixaban until the night before surgery and resumed it on the first day after surgery. We performed an RVV test as point-of-care coagulation monitoring in combination with chromogenic anti-activated factor X (anti-Xa) activity, prothrombin time, and activated partial thromboplastin time. Clotting time with the RVV test was prolonged according to the anti-Xa activity of apixaban, which was in the therapeutic range during surgery.ConclusionsAn RVV test might be useful as a point-of-care assay for estimation of the anti-Xa level induced by apixaban during the perioperative period.

28-Year-Old Man With Recurrent Vertigo, Syncope, and Progressive Memory Impairment

28-Year-Old Man With Recurrent Vertigo, Syncope, and Progressive Memory Impairment

Phylogenetically diverse diets favor more complex venoms in North American pitvipers

The role of natural selection in the evolution of trait complexity can be characterized by testing hypothesized links between complex forms and their functions across species. Predatory venoms are composed of multiple proteins that collectively function to incapacitate prey. Venom complexity fluctuates over evolutionary timescales, with apparent increases and decreases in complexity, and yet the causes of this variation are unclear. We tested alternative hypotheses linking venom complexity and ecological sources of selection from diet in the largest clade of front-fanged venomous snakes in North America: the rattlesnakes, copperheads, cantils, and cottonmouths. We generated independent transcriptomic and proteomic measures of venom complexity and collated several natural history studies to quantify dietary variation. We then constructed genome-scale phylogenies for these snakes for comparative analyses. Strikingly, prey phylogenetic diversity was more strongly correlated to venom complexity than was overall prey species diversity, specifically implicating prey species' divergence, rather than the number of lineages alone, in the evolution of complexity. Prey phylogenetic diversity further predicted transcriptomic complexity of three of the four largest gene families in viper venom, showing that complexity evolution is a concerted response among many independent gene families. We suggest that the phylogenetic diversity of prey measures functionally relevant divergence in the targets of venom, a claim supported by sequence diversity in the coagulation cascade targets of venom. Our results support the general concept that the diversity of species in an ecological community is more important than their overall number in determining evolutionary patterns in predator trait complexity.

Intravenous Vipera berus Venom-Specific Fab Fragments and Intramuscular Vipera ammodytes Venom-Specific F(ab')2 Fragments in Vipera ammodytes-Envenomed Patients.

Vipera ammodytes (V. ammodytes) is the most venomous European viper. The aim of this study was to compare the clinical efficacy and pharmacokinetic values of intravenous Vipera berus venom-specific (paraspecific) Fab fragments (ViperaTAb) and intramuscular V. ammodytes venom-specific F(ab’)2 fragments (European viper venom antiserum, also called “Zagreb” antivenom) in V. ammodytes-envenomed patients. This was a prospective study of V. ammodytes-envenomed patients that were treated intravenously with ViperaTAb or intramuscularly with European viper venom antiserum that was feasible only due to the unique situation of an antivenom shortage. The highest venom concentration, survival, length of hospital stay and adverse reactions did not differ between the groups. Patients treated with intravenous Fab fragments were sicker, with significantly more rhabdomyolysis and neurotoxicity. The kinetics of Fab fragments after one or more intravenous applications matched better with the venom concentration in the early phase of envenomation compared to F(ab’)2 fragments that were given intramuscularly only on admission. F(ab’)2 fragments given intramuscularly had 25-fold longer apparent total body clearance and 14-fold longer elimination half-time compared to Fab fragments given intravenously (2 weeks vs. 24 h, respectively). In V. ammodytes-envenomed patients, the intramuscular use of specific F(ab’)2 fragments resulted in a slow rise of antivenom serum concentration that demanded their early administration but without the need for additional doses for complete resolution of all clinical signs of envenomation. Intravenous use of paraspecific Fab fragments resulted in the immediate rise of antivenom serum concentration that enabled their use according to the clinical progress, but multiple doses might be needed for efficient therapy of thrombocytopenia due to venom recurrence, while the progression of rhabdomyolysis and neurotoxic effects of the venom could not be prevented.

Inhibitory Effect of Carissa spinarum Linn Methanolic Leaf Extract Against Vipera russelli

Background: Carissa spinarum Linn has been used as a traditional medicine to treat various ailments, including snakebite. It is found in India, Ceylon and Thailand. Objective: The study was performed to determine the inhibiting potential of Carissa spinarum methanolic leaf extract on the pharmacological effects of Viper venom. Methods: The dose-dependent enzymatic studies, pharmacological and in vivo studies were conducted using standard methods. Results: It neutralized toxic enzymes in a dose-dependent manner with concentrations ranging from 53.3 –1190.4 μg/mL, inhibited lysis of fibrinogen at 1:8 (venom: extract, w/w), and increased the procoagulant activity and lecithin lysis at 1:25 (venom: extract, w/w). The extract neutralized the LD50 of venom in mice and embryos, reduced haemorrhage, myotoxicity and edema induced by the venom in mice. Conclusion: The observed results confirm that the leaf extract possesses adequate phytochemicals that could neutralize the toxic properties of the venom.

The venom proteome of three common scyphozoan jellyfishes (Chrysaora caliparea, Cyanea nozakii and Lychnorhiza malayensis) (Cnidaria: Scyphozoa) from the coastal waters of India

The jellyfish venom stored in nematocysts contains highly toxic compounds comprising of polypeptides, enzymes and other proteins, which form their chemical defence armoury against predators. We have characterized the proteome of crude venom extract from three bloom-forming scyphozoan jellyfish along the south-west coast of India, Chrysaora caliparea, Cyanea nozakii and Lychnorhiza malayensis using a Quadrupole-Time of Flight (Q/TOF) mass spectrometry analysis. The most abundant toxin identified from Chrysaora caliparea and Lychnorhiza malayensis is similar to the pore-forming toxins and metalloproteinases. A protective antioxidant enzyme called peroxiredoxin was found abundantly in Cyanea nozakii. Metalloproteinase identified from the C. caliparea shows similarity with the venom of pit viper (Bothrops pauloensis), while that of L. malayensis was similar to the venom of snakes such as the Bothrops insularis and Bothrops asper. Kininogen-1 is a secreted protein, identified for the first time from the jellyfish L. malayensis. The proteome analysis of Cyanea nozakii, Chrysaora caliparea and Lychnorhiza malayensis contained 20, 12, 8 unique proteins, respectively. Our study characterized the proteome map of crude venom extract from L. malayensis and C. caliparea for the first time, and the venom profile is compared with published information elsewhere. Proteomic data from this study has been made available in the public domain.