Generation of Rapid and High-Quality Serum by Recombinant Prothrombin Activator Ecarin (RAPClot™).

We recently reported the potential application of recombinant prothrombin activator ecarin (RAPClot™) in blood diagnostics. In a new study, we describe RAPClot™ as an additive to develop a novel blood collection prototype tube that produces the highest quality serum for accurate biochemical analyte determination. The drying process of the RAPClot™ tube generated minimal effect on the enzymatic activity of the prothrombin activator. According to the bioassays of thrombin activity and plasma clotting, γ-radiation (>25 kGy) resulted in a 30-40% loss of the enzymatic activity of the RAPClot™ tubes. However, a visual blood clotting assay revealed that the γ-radiation-sterilized RAPClot™ tubes showed a high capacity for clotting high-dose heparinized blood (8 U/mL) within 5 min. This was confirmed using Thrombelastography (TEG), indicating full clotting efficiency under anticoagulant conditions. The storage of the RAPClot™ tubes at room temperature (RT) for greater than 12 months resulted in the retention of efficient and effective clotting activity for heparinized blood in 342 s. Furthermore, the enzymatic activity of the RAPClot™ tubes sterilized with an electron-beam (EB) was significantly greater than that with γ-radiation. The EB-sterilized RAPClot™ tubes stored at RT for 251 days retained over 70% enzyme activity and clotted the heparinized blood in 340 s after 682 days. Preliminary clinical studies revealed in the two trials that 5 common analytes (K, Glu, lactate dehydrogenase (LD), Fe, and Phos) or 33 analytes determined in the second study in the γ-sterilized RAPClot™ tubes were similar to those in commercial tubes. In conclusion, the findings indicate that the novel RAPClot™ blood collection prototype tube has a significant advantage over current serum or lithium heparin plasma tubes for routine use in measuring biochemical analytes, confirming a promising application of RAPClot™ in clinical medicine.

Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics.

We describe here the purification and cloning of a codon-optimized form of the snake prothrombin activator ecarin from the saw scaled viper (Echis carinatus) expressed in mammalian cells. Expression of recombinant ecarin (rEcarin) was carried out in human embryonic kidney cells (HEK) cells under conditions for the development and performance of a novel and scalable recombinant snake ecarin to industry standards. Clotting performance of the rEcarin was established in recalcified citrated whole blood, plasma, and fresh whole blood and found to be comparable to native ecarin (N-Ecarin). Furthermore, hemolysis was observed with N-Ecarin at relatively high doses in both recalcified citrated and fresh whole blood, while clotting was not observed with rEcarin, providing an important advantage for the recombinant form. In addition, rEcarin effectively clotted both recalcified citrated whole blood and fresh whole blood containing different anticoagulants including heparin, warfarin, dabigatran, Fondaparinux, rivaroxaban and apixaban, forming firm clots in the blood collection tubes. These results demonstrate that rEcarin efficiently clots normal blood as well as blood spiked with high concentrations of anticoagulants and has great potential as an additive to blood collection tubes to produce high quality serum for analyte analysis in diagnostic medicine.

PAR2 Activation on Human Kidney Tubular Epithelial Cells Induces Tissue Factor Synthesis, That Enhances Blood Clotting.

Coagulation abnormalities and increased risk of atherothrombosis are common in patients with chronic kidney diseases (CKD). Mechanisms that alter renal hemostasis and lead to thrombotic events are not fully understood. Here we show that activation of protease activated receptor-2 (PAR2) on human kidney tubular epithelial cells (HTECs), induces tissue factor (TF) synthesis and secretion that enhances blood clotting. PAR-activating coagulation-associated protease (thrombin), as well as specific PAR2 activators (matriptase, trypsin, or synthetic agonist 2f-LIGRLO-NH2 (2F), induced TF synthesis and secretion that were potently inhibited by PAR2 antagonist, I-191. Thrombin-induced TF was also inhibited by a PAR1 antagonist, Vorapaxar. Peptide activators of PAR1, PAR3, and PAR4 failed to induce TF synthesis. Differential centrifugation of the 2F-conditoned medium sedimented the secreted TF, together with the exosome marker ALG-2 interacting protein X (ALIX), indicating that secreted TF was associated with extracellular vesicles. 2F-treated HTEC conditioned medium significantly enhanced blood clotting, which was prevented by pre-incubating this medium with an antibody for TF. In summary, activation of PAR2 on HTEC stimulates synthesis and secretion of TF that induces blood clotting, and this is attenuated by PAR2 antagonism. Thrombin-induced TF synthesis is at least partly mediated by PAR1 transactivation of PAR2. These findings reveal how underlying hemostatic imbalances might increase thrombosis risk and subsequent chronic fibrin deposition in the kidneys of patients with CKD and suggest PAR2 antagonism as a potential therapeutic strategy for intervening in CKD progression.

Progress curve analysis of microtitre plate plasma clotting assays. Assessment of tissue factor levels.

MTP plasma clotting assays monitor the time course of fibrin formation in re-calcified plasma by absorbance measurements and are increasingly used as alternatives to traditional one-point clot time assays employed in clinical laboratories to detect thrombotic disorders. The parameters derived from these analyses are analogous to thromboelastography viz. time, rate and maximum extent of clot formation. The derived parameters, based on the whole course of the clotting reaction are more robust, informative and quantitative than single-point clot time assays. However, the parameters themselves are usually obtained arbitrarily by crude graphical analysis of subjectively selected points of progress curves. The current work aimed to investigate the sensitivity and reproducibility of an MTP clotting assay and examine its suitability for measuring tissue factor (TF) levels in cell culture medium and patient urine. The results demonstrate that progress curves can be analysed by fitting a logistic equation, derived from a simplified autocatalytic clot formation model. The parameters, maximum amplitude (Fm), rate constant (k), time to half-maximum amplitude (tm) and maximum rate of clot formation (vm), fit a power curve showing limiting effects with increasing TF concentration. Log/log plots of tm and k against TF concentration provide standard curves for assessment of unknowns.

Oral administration of dermatan sulphate reduces venous thrombus formation in vivo: potential use as a formulation for venous thromboembolism.

Dermatan sulphate (DS) is a sulphated polysaccharide that displays complexity in constituent sulphated disaccharides and interacts with proteins and signalling molecules to modulate numerous biological processes, including inhibition of the coagulation cascade and regulation of blood clotting and fibrinolysis. This study shows the antithrombotic and anticoagulant effects of DS prepared from bovine collagen waste liquor following oral and intravenous administrations in a deep vein thrombosis (DVT) rabbit model. In vitro, the prothrombin time, activated partial thromboplastin time, and thrombin citrated plasma clotting assays revealed that bovine DS had strong antithrombotic and anticoagulant effects comparable to low-molecular-weight heparin [Clexane® (enoxaparin sodium)]. In a DVT rabbit model, animals received intravenous and oral administrations of bovine DS and Clexane® providing further evidence that both agents had strong antithrombotic and anticoagulant effects by significantly reducing or preventing clot formation. Thromboelastography (TEG) assays revealed further that both bovine DS and Clexane® substantially prolonged the clotting time of recalcified citrated whole blood, but only bovine DS could retain clot strength suggesting that bovine DS had less effect on platelet-fibrin interactions. In conclusion, this is the first report that oral administration of DS from bovine collagen waste liquor reduces experimental venous thrombus formation warranting further research into bovine DS as an oral antithrombotic therapeutic.

Venom of the Red-Bellied Black Snake Pseudechis porphyriacus Shows Immunosuppressive Potential.

Venoms act with remarkable specificity upon a broad diversity of physiological targets. Venoms are composed of proteins, peptides, and small molecules, providing the foundation for the development of novel therapeutics. This study assessed the effect of venom from the red-bellied black snake (Pseudechis porphyriacus) on human primary leukocytes using bead-based flow cytometry, mixed lymphocyte reaction, and cell viability assays. We show that venom treatment had a significant immunosuppressive effect, inhibiting the secretion of interleukin (IL)-2 and tumor necrosis factor (TNF) from purified human T cells by 90% or greater following stimulation with mitogen (phorbol 12-myristate 13-acetate and ionomycin) or via cluster of differentiation (CD) receptors, CD3/CD28. In contrast, venom treatment did not inhibit TNF or IL-6 release from antigen-presenting cells stimulated with lipopolysaccharide. The reduced cytokine release from T cells was not associated with inhibition of T cell proliferation or reduction of cell viability, consistent with an anti-inflammatory mechanism unrelated to the cell cycle. Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity. These data suggest that compounds from P. porphyriacus venom may be potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease.

Progress Curve Analysis of the one stage chromogenic assay for ecarin.

Snake venom prothrombin activators such as Ecarin are readily assayed by continuous spectrophotometric monitoring of p-nitroaniline production in a one step assay containing prothrombin and a p-nitroanilide peptide substrate for thrombin. The coupled reactions result in accelerating p-nitroaniline (pNA) production over the course of the assay giving non-linear progress curves, from which initial velocities are not readily obtained. Most studies therefore resort to approximate estimates of activity, based on the absorbance reached at an arbitrary time. A simple kinetic analysis of the coupled reactions shows that the early points of such curves should be fitted by second order polynomials, representing the accelerating reaction rate in µmol pNA/min/min. The first derivative of the polynomial then gives the increasing velocity of pNA production in µmol pNA/min over the time course of the assay. We demonstrate here that, with the substrate S2238, these rates can be converted to absolute thrombin concentrations using the Michaelis-Menten equation, substituted with values for kcat and Km. These thrombin concentrations increase linearly over the time course of the assay allowing the activity to be expressed in units, defined as µmol product/min, most commonly used to report enzyme activity.

Rapid serum tube technology overcomes problems associated with use of anticoagulants.

INTRODUCTION: Failure to obtain complete blood clotting in serum is a common laboratory problem. Our aim was to determine whether snake proth-rombin activators are effective in clotting blood and producing quality serum for analyte measurement in anticoagulated patients. MATERIALS AND METHODS: Whole blood clotting was studied in a total of 64 blood samples (41 controls, 20 Warfarin patients, 3 anticoagulated patients using snake venom prothrombin activator (OsPA)) with plain tubes. Coagulation was analysed using a visual assay, Hyland-Clotek and thromboelastography. Healthy control blood was spiked with a range of anticoagulants to determine the effectiveness of OsPa-induced clotting. A paired analysis of a Dabigatran patient and a control investigated the effectiveness of the OsPA clotting tubes. Biochemical analytes (N = 31) were determined for 7 samples on chemistry and immunoassay analysers and compared with commercial tubes. RESULTS: Snake venom prothrombin activators efficiently coagulated blood and plasma spiked with heparin and commonly used anticoagulants. Clotting was observed in the presence of anticoagulants whereas no clotting was observed in BDRST tubes containing 3 U/mL of heparin. Snake venom prothrombin activator enhanced heparinised blood clotting by shortening substantially the clotting time and improving significantly the strength of the clot. Comparison of 31 analytes from the blood of five healthy and two anticoagulated participants gave very good agreement between the analyte concentrations determined. CONCLUSIONS: Our results showed that the snake venom prothrombin activators OsPA and PtPA efficiently coagulated recalcified and fresh bloods with or without added anticoagulants. These procoagulants produced high quality serum for accurate analyte measurement.

Next-generation rapid serum tube technology using prothrombin activator coagulant: fast, high-quality serum from normal samples.

Background Incomplete blood clotting or latent clotting in serum is a common laboratory problem, especially for patients on anticoagulant therapy or when serum tubes are centrifuged before clotting is completed. We describe a novel approach to producing high-quality serum using snake venom prothrombin activator complex (OsPA) as an additive in blood collection tubes for non-anticoagulated (normal) individuals. Methods Plasma clotting assays were performed using a Hyland-Clotek instrument. Blood clotting was visually observed, and thromboelastography was also performed to determine the important parameters of coagulation. Thrombin generation was assayed using the chromogenic substrate S-2238, and biochemical analytes in the serum were determined on chemistry and immunoassay analysers. Fibrinogen was determined by either ELISA or Clauss fibrinogen assay. Results We initially showed that OsPA had strong coagulation activity in clotting not only recalcified citrated plasma and recalcified citrated whole blood, but also fresh whole blood in a clinical setting. The use of TEG clearly showed improved speed of clotting and generation of a firmer clot. We also showed that the use of OsPA to produce serum did not interfere with the determination of commonly measured biochemical analytes. The underlying clotting mechanism involves a burst of thrombin production at the initial stages of the clotting process upon contact with prothrombin in blood. Conclusions These results demonstrate rapid generation of high-quality serum, contributing to faster turnaround times with standardised quality samples, for accurate analyte determinations in normal individuals.

Does size matter? Venom proteomic and functional comparison between night adder species (Viperidae: Causus) with short and long venom glands.

Night adders (Causus species within the Viperidae family) are amphibian specialists and a common source of snakebite in Africa. Some species are unique in that they have the longest venom glands of any viper, extending approximately 10% of the body length. Despite their potential medical importance and evolutionary novelty, their venom has received almost no research attention. In this study, venoms from a short-glanded species (C. lichtensteinii) and from a long-glanded species (C. rhombeatus) were compared using a series of proteomic and bioactivity testing techniques to investigate and compare the toxin composition and functioning of the venoms of these two species. Both C. rhombeatus and C. lichtensteinii were similar in overall venom composition and inhibition of blood coagulation through non-clotting proteolytic cleavage of fibrinogen. While the 1D gel profiles were very similar to each other in the toxin types present, 2D gel analyses revealed isoformic differences within each toxin classes. This variation was congruent with differential efficacy of South African Institute for Medical Research snake polyvalent antivenom, with C. lichtensteinii unaffected at the dose tested while C. rhombeatus was moderately but significantly neutralized. Despite the variation within toxin classes, the similarity in overall venom biochemistry suggests that the selection pressure for the evolution of long glands served to increase venom yield in order to subjugate proportionally large anurans as a unique form of niche partitioning, and is not linked to significant changes in venom function. These results not only contribute to the body of venom evolution knowledge but also highlight the limited clinical management outcomes for Causus envenomations.

Anti-Coagulant and Anti-Thrombotic Properties of Blacklip Abalone (Haliotis rubra): In Vitro and Animal Studies.

Sulphated polysaccharides with anti-thrombotic and anti-coagulant activities have been found in various marine biota. In this study, a previously characterised anti-thrombotic and anti-coagulant extract from blacklip abalone was fractionated by anion exchange chromatography (AEC), pooled (on a sulphated polysaccharide basis) and administered to Wistar rats via oral gavage (N = 8) for assessment as an oral therapeutic. To ensure that the preparation had anti-coagulant activity prior to oral administration, it was assessed in rat blood by thromboelastography (TEG) significantly increasing reaction (R) time (or time until clot formation). Following in vitro confirmation of anti-coagulant activity, 40 mg of the preparation was orally administered to rats with blood samples collected at 2, 4, and 6 h post-gavage. Assessment of all blood samples by TEG showed some prolongation of R time from 355 to 380 s after 4 h. Dosing of the post-gavage blood samples with the abalone preparation to confirm anti-thrombotic activity in vitro revealed residual anti-coagulant activity, further suggesting that oral administration did increase anti-coagulant potential in the collected blood but that bioavailability was low. Assessment of tissues and haematological parameters showed no obvious harmful effects of the abalone preparation in animals. In summary, even though oral administration of fractionated and pooled blacklip abalone extract to rats delayed clotting after 4 h, bioavailability of the preparation appeared to be low and may be more appropriate for intravenous administration as an anti-thrombotic or anti-coagulant therapeutic.

Differential procoagulant effects of saw-scaled viper (Serpentes: Viperidae: Echis) snake venoms on human plasma and the narrow taxonomic ranges of antivenom efficacies.

Saw-scaled vipers (genus Echis) are one of the leading causes of snakebite morbidity and mortality in parts of Sub-Saharan Africa, the Middle East, and vast regions of Asia, constituting a public health burden exceeding that of almost any other snake genus globally. Venom-induced consumption coagulopathy, owing to the action of potent procoagulant toxins, is one of the most relevant clinical manifestations of envenomings by Echis spp. Clinical experience and prior studies examining a limited range of venoms and restricted antivenoms have demonstrated for some antivenoms an extreme lack of antivenom cross-reactivity between different species of this genus, sometimes resulting in catastrophic treatment failure. This study undertook the most comprehensive testing of Echis venom effects upon the coagulation of human plasma, and also the broadest examination of antivenom potency and cross-reactivity, to-date. 10 Echis species/populations and four antivenoms (two African, two Asian) were studied. The results indicate that the venoms are, in general, potently procoagulant but that the relative dependence on calcium or phospholipid cofactors is highly variable. Additionally, three out of the four antivenoms tested demonstrated only a very narrow taxonomic range of effectiveness in preventing coagulopathy, with only the SAIMR antivenom displaying significant levels of cross-reactivity. These results were in conflict with previous studies using prolonged preincubation of antivenom with venom to suggest effective cross-reactivity levels for the ICP Echi-Tab antivenom. These findings both inform upon potential clinical effects of envenomation in humans and highlight the extreme limitations of available treatment. It is hoped that this will spur efforts into the development of antivenoms with more comprehensive coverage for bites not only from wild snakes but also from specimens widely kept in zoological collections.

In vitro anti-thrombotic and anti-coagulant properties of blacklip abalone (Haliotis rubra) viscera hydrolysate.

Abalone viscera contain sulphated polysaccharides with anti-thrombotic and anti-coagulant activities. In this study, a hydrolysate was prepared from blacklip abalone (Haliotis rubra) viscera using papain and bromelain and fractionated using ion exchange and size exclusion chromatography. Hydrolysates and fractions were investigated for in vitro thrombin inhibition mediated through heparin cofactor II (HCII) as well as anti-coagulant activity in plasma and whole blood. On the basis of sulphated polysaccharide concentration, the hydrolysate inhibited thrombin through HCII with an inhibitor concentration at 50% (IC50) of 16.5 µg/mL compared with 2.1 µg/mL for standard heparin. Fractionation concentrated HCII-mediated thrombin inhibition down to an IC50 of 1.8 µg/mL and improved anti-coagulant activities by significantly delaying clotting time. This study confirmed the presence of anti-thrombotic and anti-coagulant molecules in blacklip abalone viscera and demonstrated that these activities can be enriched with a simple chromatography regime. Blacklip abalone viscera warrant further investigation as a source of nutraceutical or functional food ingredients. Graphical abstract Schematic showing preparation of bioactive extracts and fractions from blacklip abalone.

Evaluation of the Greiner Bio-One serum separator BCA Fast Clot tube.

BACKGROUND: Current commercial tubes have difficulties in producing "true" serum from all blood samples even within the recommended clotting times. Hence, Becton Dickinson (BD) and now Greiner have produced tubes containing thrombin as the procoagulant to reduce the clotting time and increase the possibility of producing serum from anticoagulated blood samples. METHODS: The Greiner BCA Fast Clot (GBBCAFC) tube was evaluated in a hospital environment using 40 participants, (30 healthy and 10 undergoing renal dialysis) for 32 analytes against the Greiner lithium heparin tube and the BD Rapid Serum Tubes (BD RST) tube measured on Beckman DxC 800 and DxI 800 analyzers. Clotting strength was also examined using thromboelastography (TEG). RESULTS: The analytes results showed there was a very close agreement between the BD RST tube and GBBCAFC tube in comparison with lithium heparin plasma. The result comparison data showed equivalent performance with lower levels of hemolysis. The prolonged storage study also showed very similar agreement between the BD RST and the GBBCAFC tubes. Likewise, the TEG data showed there was very little difference in clotting ability between the tubes, and neither was capable of producing true serum from blood spiked with 2 U heparin/mL of blood. CONCLUSIONS: The study showed the GBBCAFC tube with the combination of the two procoagulants blood clotting activator and thrombin produced comparable performance with the lithium heparin plasma and the BD RST serum samples.

A randomised, placebo-controlled trial assessing the efficacy of an oral B group vitamin in preventing the development of chemotherapy-induced peripheral neuropathy (CIPN).

INTRODUCTION: Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect resulting from neurotoxic chemotherapeutic agents. This study aimed to assess the efficacy and safety of an oral B group vitamin compared to placebo, in preventing the incidence of CIPN in cancer patients undergoing neurotoxic chemotherapy. METHODS: A pilot, randomised, placebo-controlled trial was conducted. Newly diagnosed cancer patients prescribed with taxanes, oxaliplatin or vincristine were invited to participate. A total of 71 participants (female 68 %, male 32 %) were enrolled into the study and randomised to the B group vitamin (n = 38) arm or placebo (n = 33). The data from 47 participants were eligible for analysis (B group vitamins n = 27, placebo n = 22). The primary outcome measure was the total neuropathy score assessed by an independent neurologist. Secondary outcome measures included serum vitamin B levels, quality of life, pain inventory and the patient neurotoxicity questionnaires. Outcome measures were conducted at baseline, 12, 24 and 36 weeks. RESULTS: The total neuropathy score (TNS) demonstrated that a B group vitamin did not significantly reduce the incidence of CIPN compared to placebo (p = 0.73). Statistical significance was achieved for patient perceived sensory peripheral neuropathy (12 weeks p = 0.03; 24 weeks p = 0.005; 36 weeks p = 0.021). The risk estimate for the Patient Neurotoxicity Questionnaire (PNQ) was also statistically significant (OR = 5.78, 95 % CI = 1.63-20.5). The European Organisation of Research and Treatment of Cancer (EORTC) quality of life, total pain score and pain interference showed no significance (p = 0.46, p = 0.9, p = 0.37 respectively). A trend was observed indicating that vitamin B12 may reduce the onset and severity of CIPN. CONCLUSION: An oral B group vitamin as an adjunct to neurotoxic chemotherapy regimens was not superior to placebo (p > 0.05) for the prevention of CIPN. Patients taking the B group vitamin perceived a reduction in sensory peripheral neuropathy in the PNQ. Moreover, a robust clinical study is warranted given that vitamin B12 may show potential in reducing the onset and severity of CIPN. Trial number: ACTRN12611000078954 Protocol number: UH2010000749.

Rapid Radiations and the Race to Redundancy: An Investigation of the Evolution of Australian Elapid Snake Venoms.

Australia is the stronghold of the front-fanged venomous snake family Elapidae. The Australasian elapid snake radiation, which includes approximately 100 terrestrial species in Australia, as well as Melanesian species and all the world's sea snakes, is less than 12 million years old. The incredible phenotypic and ecological diversity of the clade is matched by considerable diversity in venom composition. The clade's evolutionary youth and dynamic evolution should make it of particular interest to toxinologists, however, the majority of species, which are small, typically inoffensive, and seldom encountered by non-herpetologists, have been almost completely neglected by researchers. The present study investigates the venom composition of 28 species proteomically, revealing several interesting trends in venom composition, and reports, for the first time in elapid snakes, the existence of an ontogenetic shift in the venom composition and activity of brown snakes (Pseudonaja sp.). Trends in venom composition are compared to the snakes' feeding ecology and the paper concludes with an extended discussion of the selection pressures shaping the evolution of snake venom.

Alteration of blood clot structures by interleukin-1 beta in association with bone defects healing.

The quality of hematomas are crucial for successful early bone defect healing, as the structure of fibrin clots can significantly influence the infiltration of cells, necessary for bone regeneration, from adjacent tissues into the fibrin network. This study investigated if there were structural differences between hematomas from normal and delayed healing bone defects and whether such differences were linked to changes in the expression of IL-1ß. Using a bone defect model in rats, we found that the hematomas in the delayed healing model had thinner fibers and denser clot structures. Moreover, IL-1ß protein levels were significantly higher in the delayed healing hematomas. The effects of IL-1ß on the structural properties of human whole blood clots were evaluated by thrombelastograph (TEG), scanning electronic microscopy (SEM), compressive study, and thrombolytic assays. S-nitrosoglutathione (GSNO) was applied to modulate de novo hematoma structure and the impact on bone healing was evaluated in the delayed healing model. We found that GSNO produced more porous hematomas with thicker fibers and resulted in significantly enhanced bone healing. This study demonstrated that IL-1ß and GSNO had opposing effects on clot architecture, the structure of which plays a pivotal role in early bone healing.

Influence of Interleukin-1 Beta on Platelet-Poor Plasma Clot Formation: A Potential Impact on Early Bone Healing.

OBJECTIVES: Hematoma quality (especially the fibrin matrix) plays an important role in the bone healing process. Here, we investigated the effect of interleukin-1 beta (IL-1ß) on fibrin clot formation from platelet-poor plasma (PPP). METHODS: Five-milliliter of rat whole-blood samples were collected from the hepatic portal vein. All blood samples were firstly standardized via a thrombelastograph (TEG), blood cell count, and the measurement of fibrinogen concentration. PPP was prepared by collecting the top two-fifths of the plasma after centrifugation under 400 × g for 10 min at 20°C. The effects of IL-1ß cytokines on artificial fibrin clot formation from PPP solutions were determined by scanning electronic microscopy (SEM), confocal microscopy (CM), turbidity, and clot lysis assays. RESULTS: The lag time for protofibril formation was markedly shortened in the IL-1ß treatment groups (243.8 ± 76.85 in the 50 pg/mL of IL-1ß and 97.5 ± 19.36 in the 500 pg/mL of IL-1ß) compared to the control group without IL-1ß (543.8 ± 205.8). Maximal turbidity was observed in the control group. IL-1ß (500 pg/mL) treatment significantly decreased fiber diameters resulting in smaller pore sizes and increased density of the fibrin clot structure formed from PPP (P < 0.05). The clot lysis assay revealed that 500 pg/mL IL-1ß induced a lower susceptibility to dissolution due to the formation of thinner and denser fibers. CONCLUSION: IL-1ß can significantly influence PPP fibrin clot structure, which may affect the early bone healing process.

Characterization and structural analysis of a potent anticoagulant phospholipase A2 from Pseudechis australis snake venom.

Pseudechis australis is one of the most venomous and lethal snakes in Australia. Numerous phospholipase A2 (PLA2) isoforms constitute a major portion of its venom, some of which have previously been shown to exhibit not only enzymatic, but also haemolytic, neurotoxic and anticoagulant activities. Here, we have purified a potent anticoagulant PLA2 (identified as PA11) from P. australis venom to investigate its phospholipase, anticoagulant, haemolytic and cytotoxic activities and shown that addition of 11 nM PA11 resulted in a doubling of the clotting time of recalcified whole blood. We have also demonstrated that PA11 has high PLA2 enzymatic activity (10.9 × 10(4) Units/mg), but low haemolytic activity (0.6% of red blood cells hydrolysed in the presence of 1 nM PA11). PA11 at a concentration lower than 600 nM is not cytotoxic towards human cultured cells. Chemical modification experiments using p-bromophenacyl bromide have provided evidence that the catalytic histidine of PA11 is critical for the anticoagulant activity of this PLA2. PA11 that was subjected to trypsin digestion without previous reduction and alkylation of the disulfide bonds maintained enzymatic and anticoagulant activity, suggesting that proteolysis alone cannot abolish these properties. Consistent with these results, administration of PA11 by gavage in a rabbit stasis thrombosis model increased the clotting time of recalcified citrated whole blood by a factor of four. These data suggest that PA11 has potential to be developed as an anticoagulant in a clinical setting.

In vitro Anti-Thrombotic Activity of Extracts from Blacklip Abalone (Haliotis rubra) Processing Waste.

Waste generated from the processing of marine organisms for food represents an underutilized resource that has the potential to provide bioactive molecules with pharmaceutical applications. Some of these molecules have known anti-thrombotic and anti-coagulant activities and are being investigated as alternatives to common anti-thrombotic drugs, like heparin and warfarin that have serious side effects. In the current study, extracts prepared from blacklip abalone (Haliotis rubra) processing waste, using food grade enzymes papain and bromelain, were found to contain sulphated polysaccharide with anti-thrombotic activity. Extracts were found to be enriched with sulphated polysaccharides and assessed for anti-thrombotic activity in vitro through heparin cofactor-II (HCII)-mediated inhibition of thrombin. More than 60% thrombin inhibition was observed in response to 100 µg/mL sulphated polysaccharides. Anti-thrombotic potential was further assessed as anti-coagulant activity in plasma and blood, using prothrombin time (PT), activated partial thromboplastin time (aPTT), and thromboelastography (TEG). All abalone extracts had significant activity compared with saline control. Anion exchange chromatography was used to separate extracts into fractions with enhanced anti-thrombotic activity, improving HCII-mediated thrombin inhibition, PT and aPTT almost 2-fold. Overall this study identifies an alternative source of anti-thrombotic molecules that can be easily processed offering alternatives to current anti-thrombotic agents like heparin.