Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product.

Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.

Phenolic fractions from nine Trifolium species modulate the coagulant properties of blood plasma in vitro without cytotoxicity towards blood cells.

OBJECTIVE: The study covers an evaluation of the influence of extracts (1-50 µg/ml), isolated from aerial parts of nine Trifolium L. species (i.e. T. alexandrinum, T. fragiferum, T. hybridum, T. incarnatum, T. pallidum, T. pratense, T. resupinatum var. majus, T. resupinatum var. resupinatum and T. scabrum) on haemostatic properties of blood plasma. METHODS: The clot formation and fibrinolysis assay (CFF), blood clotting times, the extrinsic and intrinsic coagulation pathway-dependent polymerization of plasma fibrin were measured. The effects of plant extracts on amidolytic activity of thrombin were also evaluated and compared with argatroban, an antithrombotic drug. Cytotoxicity was assessed in a model of blood platelets and as the viability of peripheral blood mononuclear cells. KEY FINDINGS: While no changes in blood clotting times or fibrinolytic properties of blood plasma were found, some fractions impaired the blood plasma coagulation induced by the intrinsic coagulation pathway. Reduction in the maximal velocity of fibrin polymerization was also observed in the clot formation and fibrinolysis assay. No cytotoxicity of Trifolium extracts towards the investigated cells was recorded. CONCLUSIONS: The most efficient anticoagulant activity in plasma was found for T. fragiferum and T. incarnatum extracts, while the T. alexandrinum fraction was the most effective inhibitor of thrombin amidolytic activity.

EC-PIII, a novel non-hemorrhagic procoagulant metalloproteinase: Purification and characterization from Indian Echis carinatus venom.

Procoagulant snake venom toxins find extensive use as reagents in laboratory tests and diagnostic kits. In the present study we report a novel P-III class procoagulant SVMP, EC-PIII from Echis carinatus venom. EC-PIII was purified using a combination of gel-filtration and anion-exchange chromatography. It has a molecular mass of 110kDa and is a dimeric protein as determined by SDS-PAGE. DLS results show that the protein is homogenous and stable in solution. Peptide mass fingerprinting revealed that the peptides obtained show high homology to the other members of SVMP family. The enzymatic studies revealed that EC-PIII shows protease activity and is inhibited by metalloproteinase inhibitors such as EDTA. EC-PIII exhibits procoagulant effect under in-vitro conditions. Local toxicity studies revealed that EC-PIII is devoid of hemorrhagic as well as myotoxic activities. This is the first report of a non-hemorrhagic SVMP to be identified from Indian Echis carinatus venom. EC-PIII can find potential use in diagnostic and other therapeutic uses owing to its biochemical and pharmacological properties.

Experience of a new high-purity factor X concentrate in subjects with hereditary factor X deficiency undergoing surgery.

INTRODUCTION: Maintaining haemostasis in surgery is challenging for hereditary rare bleeding disorders in which multi-coagulation-factor concentrates are the only therapeutic option. Hereditary factor X (FX) deficiency affects 1:500 000 to 1:1 000 000 individuals, and no specific replacement FX concentrate has been available. A high-purity, plasma-derived FX concentrate (pdFX) has been developed for patients with hereditary FX deficiency. AIM: Our objective was to assess the safety and efficacy of pdFX in subjects with FX deficiency undergoing surgery. METHODS: Subjects with hereditary mild-to-severe FX deficiency (basal plasma FX activity [FX:C] <20 IU dL(-1) ) undergoing surgery received pdFX preoperatively to raise FX:C to 70-90 IU dL(-1) and postoperatively to maintain levels >50 IU dL(-1) until the subject was no longer at risk of bleeding due to surgery. Efficacy of pdFX was assessed by blood loss during surgery, requirement for blood transfusion, postoperative bleeding from the surgical or other sites, and changes in haemoglobin levels. Safety was assessed by adverse events (AEs), development of inhibitors, and clinically significant changes in laboratory parameters. RESULTS: Five subjects (aged 14-59 years) underwent seven surgical procedures (four major and three minor). Treatment duration was 1-15 days. For each procedure, pdFX treatment was assessed as "excellent" in preventing bleeding and achieving haemostasis. No blood transfusions were required, no AEs related to pdFX were observed, and no clinically significant trends were found in any laboratory parameters. CONCLUSION: These data demonstrate that pdFX is safe and effective as replacement therapy in five subjects with mild-to-severe FX deficiency undergoing surgery on seven occasions.

Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

Label-Free (XIC) Quantification of Venom Procoagulant and Neurotoxin Expression in Related Australian Elapid Snakes Gives Insight into Venom Toxicity Evolution.

This study demonstrates a direct role of venom protein expression alteration in the evolution of snake venom toxicity. Avian skeletal muscle contractile response to exogenously administered acetylcholine is completely inhibited upon exposure to South Australian and largely preserved following exposure to Queensland eastern brown snake Pseudonaja textilis venom, indicating potent postsynaptic neurotoxicity of the former and lack thereof of the latter venom. Label-free quantitative proteomics reveals extremely large differences in the expression of postsynaptic three-finger α-neurotoxins in these venoms, explaining the difference in the muscle contractile response and suggesting that the type of toxicity induced by venom can be modified by altered expression of venom proteins. Furthermore, the onset of neuromuscular paralysis in the rat phrenic nerve-diaphragm preparation occurs sooner upon exposure to the venom (10 µg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic ß-neurotoxins. The study also finds that the onset of rat plasma coagulation is faster following exposure to the venoms with higher expression of venom prothrombin activator subunits. This is the first quantitative proteomic study that uses extracted ion chromatogram peak areas (MS1 XIC) of distinct homologous tryptic peptides to directly show the differences in the expression of venom proteins.

Extraction and application of starch-based coagulants from sago trunk for semi-aerobic landfill leachate treatment.

Malaysia is one of the highest starch producers. In this study, sago starch was utilized as a natural coagulant aid to reduce the dosage of aluminum-based coagulant in leachate treatment. The potential of native sago trunk starch (NSTS) and commercial sago starch (CSS) was evaluated as sole coagulant and coagulant aid in the presence of polyaluminum chloride (PACl) in the removal of color, suspended solids (SS), NH3-N, turbidity, chemical oxygen demand, organic UV254, Cd, and Ni. Leachate was sampled from Pulau Burung Landfill Site, one of the semi-aerobic landfills in Malaysia. The optimum dosage for PACl in the presence of NSTS or CSS as coagulant aid was reduced from 3100 to 2000 mg/L. In the presence of 2000 mg/L PACl with 6000 mg/L NSTS and 2000 mg/L PACl with 5000 mg/L CSS, the removal performance for color, SS, and turbidity are 94.7, 99.2, and 98.9%, respectively. Similar results were obtained with the use of 3100 mg/L PACl alone. Therefore, CSS and NSTS can be used as coagulant aid.

Comparative analysis of procoagulant and fibrinogenolytic activity of crude protease fractions of turmeric species.

ETHNAOPHARMACOLOGIAL RELEVANCE: Turmeric rhizome is a traditional herbal medicine, which has been widely used as a remedy to stop bleeding on fresh cuts and for wound healing by the rural and tribal population of India. AIM OF THE STUDY: To validate scientific and therapeutic application of turmeric rhizomes to stop bleeding on fresh cuts and its role in wound healing process. MATERIALS AND METHODS: The water extracts of thoroughly scrubbed and washed turmeric rhizomes viz., Curcuma aromatica Salisb., Curcuma longa L., Curcuma caesia Roxb., Curcuma amada Roxb. and Curcuma zedoria (Christm.) Roscoe. were subjected to salting out and dialysis. The dialyzed crude enzyme fractions (CEFs) were assessed for proteolytic activity using casein as substrate and were also confirmed by caseinolytic zymography. Its coagulant activity and fibrinogenolytic activity were assessed using human citrated plasma and fibrinogen, respectively. The type of protease(s) in CEFs was confirmed by inhibition studies using specific protease inhibitors. RESULTS: The CEFs of C. aromatica, C. longa and C. caesia showed 1.89, 1.21 and 1.07 folds higher proteolytic activity, respectively, compared to papain. In contrast to these, C. amada and C. zedoria exhibited moderate proteolytic activity. CEFs showed low proteolytic activities compared to trypsin. The proteolytic activities of CEFs were confirmed by caseinolytic zymography. The CEFs of C. aromatica, C. longa and C. caesia showed complete hydrolysis of Aα, Bß and γ subunits of human fibrinogen, while C. amada and C. zedoria showed partial hydrolysis. The CEFs viz., C. aromatica, C. longa, C. caesia, C. amada and C. zedoria exhibited strong procoagulant activity by reducing the human plasma clotting time from 172s (Control) to 66s, 84s 88s, 78s and 90s, respectively. The proteolytic activity of C. aromatica, C. longa, C. caesia and C. amada was inhibited (>82%) by PMSF, suggesting the possible presence of a serine protease(s). However, C. zedoria showed significant inhibition (60%) against IAA and moderate inhibition (30%) against PMSF, indicating the presence of cysteine and serine protease(s). CONCLUSION: The CEFs of turmeric species exhibited strong procoagulant activity associated with fibrinogenolytic activity. This study provides the scientific credence to turmeric in its propensity to stop bleeding and wound healing process practiced by traditional Indian medicine.

Elucidation of procoagulant mechanism and pathophysiological significance of a new prothrombin activating metalloprotease purified from Daboia russelii russelii venom.

The procoagulant proteases present in Russell's Viper venom (RVV) are responsible for promoting consumption coagulopathy in victims. In this study, a procoagulant metalloprotease (Rusviprotease) possessing prothrombin activating and α-fibrinogenase properties has been purified and characterized from RVV. Rusviprotease is a 26.8 kDa glycoprotein which also exists in other multimeric forms. The peptide mass fingerprinting and secondary structure analyses of Rusviprotease revealed its similarity with snake venom prothrombin activators and metalloproteases. Similar to group A prothrombin activators, Rusviprotease cleaved prothrombin independent of any co-factor requirement generating meizothrombin which is further cleaved to form thrombin. The Km and Vmax values of Rusviprotease towards prothrombin were determined to be 1.73 µM, and 153.5 nM thrombin generated/min/µmoles of Rusviprotease, respectively. The Km and Vmax values of Rusviprotease towards fibrinogen were calculated to be 3.14 µM and 78.7 nmol/min, respectively. Spectrofluorometric study provided the evidence of interaction between Rusviprotease and factor Xa with a Kd value of 6.64 nM. This interaction augmented the prothrombin activating property of the factor Xa-prothrombinase-Rusviprotease complex by 2.5 fold. Intravenous injection of Rusviprotease to BALB/c mice (0.1 mg/kg) resulted in in vivo defibrinogenation rendering the blood incoagulable. In conclusion, Rusviprotease is the first example of a prothrombin activator with fibrinogenolytic property purified from Daboia russelii russelii venom.

Flebogamma(®) DIF (intravenous immunoglobulin) purification process effectively eliminates procoagulant activities.

BACKGROUND: Studies have demonstrated that traces of activated factor XI (FXIa) present in specific brands of intravenous immunoglobulin (IVIG) concentrates may pose a thrombogenic risk. AIM: To characterize procoagulant activity during fractionation and the elimination capacity of the Flebogamma(®) DIF (Grifols' IVIG) manufacturing process. METHODS: Flebogamma(®) DIF fractionation steps included cryoprecipitate supernatant (Cryo/S), Fraction (Fr) I supernatant, and Fr II + III suspension. Purification steps included ultrafiltrate I, acid treatment, and pasteurization. Samples were assessed for total protein, IgG, and procoagulant activation markers. RESULTS: Cryo/S showed no procoagulant activity for prekallikrein activator (PKA), kallikrein-like, and non-activated partial thromboplastin time (NaPTT) with normal (-PPP) or FXI-deficient (-FXI) platelet poor plasma. Thrombin generation test (TGT)-PPP and TGT-FXI were <83-148 and <53-197 nM thrombin, respectively. Shortened NaPTTs (100-296 s), high PKA (51-119 IU/mL), kallikrein-like activities (0.043-0.075 ΔAU/min), positive TGTs (98-298 nM), and FXIa (9.5-14.0 ng/mL) were detected in Fr II + III. After pasteurization, no residual evidence of any procoagulant activity marker was observed, including the final IVIG concentrate at 5% or 10% protein. Results were similar in Fr II + III from different IVIG manufacturing facilities. CONCLUSIONS: The Flebogamma(®) DIF production process is capable of eliminating procoagulant activity because of its purification steps.

New insights into the complex mixture of latex cysteine peptidases in Calotropis procera.

The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and ß(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.

Procoagulant and prothrombotic effects of the herbal medicine, Dipsacus asper and its active ingredient, dipsacus saponin C, on human platelets.

BACKGROUND: In spite of the growing popularity of herbal medicines and natural food supplements, their effects on cardiovascular homeostasis remain largely unknown, especially regarding pro-thrombotic risks. OBJECTIVE: In the present study, 21 herbal tea extracts were screened for the procoagulant activities on platelets, an important promoter of thrombosis to examine if herbal medicines or natural products may have prothrombotic risks. We discovered that Dipsacus asper (DA), known to have analgesic and anti-inflammatory effects, potently induced procoagulant activities in platelets. We tried to identify the active ingredient and elucidate the underlying mechanism. RESULTS: Among 10 major ingredients of DA, dipsacus saponin C (DSC) was identified as a key active ingredient in DA-induced procoagulant activities. DSC-induced procoagulant activities were achieved by the exposure of phosphatidylserine (PS) and PS-bearing microparticle generation that were caused by the alteration in the activities of phospholipid translocases: scramblase and flippase. These events were initiated by increased intracellular calcium and ATP depletion. Notably, DSC induced a series of apoptotic events including the disruption of mitochondrial membrane potential, translocation of Bax and Bak, cytochrome c release and caspase-3 activation. The key roles of apoptotic pathway and caspase activation were demonstrated by the reversal of DSC-induced PS exposure and procoagulant activities with the pretreatment of caspase inhibitors. Interestingly, EGTA reversed DSC-induced procoagulant activities and apoptotic events suggesting that an intracellular calcium increase may play a central role. These results were also confirmed in vivo where platelets of the rats exposed to DSC or DA exhibited PS exposure. Most importantly, DSC or DA administration led to increased thrombus formation. CONCLUSION: These results demonstrate that herbal medicines or natural products such as DA or DSC might have prothrombotic risks through procoagulant activation of platelets.

Moringa coagulant as a stabilizer for amorphous solids: Part I.

Stabilization of amorphous state is a focal area for formulators to reap benefits related with solubility and consequently bioavailability of poorly soluble drugs. In the present work, an attempt has been made to explore the potential of moringa coagulant as an amorphous state stabilizer by investigating its role in stabilization of spray-dried (amorphous) ibuprofen, meloxicam and felodipine. Thermal studies like glass forming ability, glass transition temperature, hot stage microscopy and DSC were carried out for understanding thermodynamic stabilization of drugs. PXRD and dissolution studies were performed to support contribution of moringa coagulant. Studies showed that hydrogen bonding and electrostatic interactions between drug and moringa coagulant are responsible for amorphous state stabilization as explored by ATR-FTIR and molecular docking. Especially, H-bonding was found to be predominant mechanism for drug stabilization. Therein, arginine (basic amino acid in coagulant) exhibited various interactions and played important role in stabilization of aforesaid amorphous drugs.

Characterization of superparamagnetic iron oxide nanoparticles and its application in protein purification.

The application of surface modified magnetic adsorbent particles in combination with magnetic separation techniques has received considerable awareness in recent years. There is a particular need in protein purification and analysis for specific, functional and generic methods of protein binding on solid supports. Nanoscale superparamagnetic iron oxide particles have been used to purify a natural coagulant protein extracted from Moringa oleifera seeds. Spectrophotometric analysis of the coagulant protein was performed using synthetic clay solution as substrate. Protein binding with carboxyl and silica surface modified superparamagnetic iron oxide nanoparticles (SPION) were compared with the known carboxyl methyl cellulose (CMC) beads of approximately 1 microm. SPION modified with carboxyl surface showed higher binding capacity towards the coagulant protein compared to the CMC beads. The high surface area to volume ratio of the carboxyl-coated SPION resulted in high binding capacity and rapid adsorption kinetics of the crude protein extract. The purification and molecular weight of coagulant protein is analyzed by SDS-PAGE. This approach utilizes the most efficient, feasible and economical method of coagulant protein purification and it can also be applicable to other proteins that possess similar properties.

Comparative study of anticoagulant and procoagulant properties of 28 snake venoms from families Elapidae, Viperidae, and purified Russell's viper venom-factor X activator (RVV-X).

Snake venoms consist of numerous molecules with diverse biological functions used for capturing prey. Each component of venom has a specific target, and alters the biological function of its target. Once these molecules are identified, characterized, and cloned; they could have medical applications. The activated clotting time (ACT) and clot rate were used for screening procoagulant and anticoagulant properties of 28 snake venoms. Crude venoms from Daboia russellii siamensis, Bothrops asper, Bothrops moojeni, and one Crotalus oreganus helleri from Wrightwood, CA, had procoagulant activity. These venoms induced a significant shortening of the ACT and showed a significant increase in the clot rate when compared to the negative control. Factor X activator activity was also measured in 28 venoms, and D. r. siamensis venom was 5-6 times higher than those of B. asper, B. moojeni, and C. o. helleri from Wrightwood County. Russell's viper venom-factor X activator (RVV-X) was purified from D. r. siamensis venom, and then procoagulant activity was evaluated by the ACT and clot rate. Other venoms, Crotalus atrox and two Naja pallida, had anticoagulant activity. A significant increase in the ACT and a significant decrease in the clot rate were observed after the addition of these venoms; therefore, the venoms were considered to have anticoagulant activity. Venoms from the same species did not always have the same ACT and clot rate profiles, but the profiles were an excellent way to identify procoagulant and anticoagulant activities in snake venoms.

Improvement of virus safety of an antihemophilc factor IX by virus filtration process.

Viral safety is an important prerequisite for clinical preparations of plasma-derived pharmaceuticals. One potential way to increase the safety of therapeutic biological products is the use of a virus-retentive filter. In order to increase the viral safety of human antihemophilic factor IX, particularly in regard to non-enveloped viruses, virus removal process using a polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. The most critical factor affecting the filtration efficiency was operating pH and the optimum pH was 6 or 7. Flow rate increased with increasing operating pressure and temperature. Recovery yield in the optimized production-scale process was 96%. No substantial changes were observed in the physical and biochemical characteristics of the filtered factor IX in comparison with those before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production-scale cartridges and to test if it could remove several experimental model viruses for human pathogenic viruses, including human hepatitis A virus (HAV), porcine parvovirus (PPV), murine encephalomyocarditis virus (EMCV), human immunodeficiency virus type 1 (HIV), bovine viral diarrhea virus (BVDV), and bovine herpes virus (BHV). Nonenveloped viruses (HAV, PPV, and EMCV) as well as enveloped viruses (HIV, BVDV, and BHV) were completely removed during filtration. The log reduction factors achieved were >or=6.12 for HAV, >or=4.28 for PPV, >or=5.33 for EMCV, >or=5.51 for HIV, >or=5.17 for BVDV, and >or=5.75 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of factor IX.

Characterization of a novel pro-coagulant metalloprotease (RVBCMP) possessing alpha-fibrinogenase and tissue haemorrhagic activity from venom of Daboia russelli russelli (Russell's viper): evidence of distinct coagulant and haemorrhagic sites in RVBCMP.

A novel, basic pro-coagulation metalloprotease (Russell's viper basic coagulant metalloprotease, RVBCMP) with an approximate molecular weight of 15kDa was purified from the venom of Daboia russelli russelli (Russell's viper) from eastern India. RVBCMP exerted dose-dependent coagulation of platelet-poor human plasma; however, RVBCMP possessed less coagulant activity as compared with the coagulant activity of crude Russell's viper venom (RVV). RVBCMP did not show oedema induction, direct haemolysis of washed erythrocytes, hydrolysis of human plasma albumin or globulin, and thrombin-like activity, but exhibited caseinolytic, alpha-fibrinogenolytic, and liver tissue haemorrhagic activities. Inhibition of coagulant and protease activities of RVBCMP by EDTA suggested a metalloprotease nature of this protein. RVBCMP showed antigenicity as was evident from the immunoblotting experiment. None of the tested plant extracts, except Leucus lavandulaefolia, inhibited the coagulant or haemorrhagic activity of RVBCMP. Interestingly, aqueous extracts of the tested plants as well as the commercial polyvalent antivenom raised against crude RVV differentially inhibited the coagulant and tissue haemorrhagic activity of RVBCMP. The current investigation provides a fairly good indication that RVBCMP possesses a distinct, perhaps overlapping, site for coagulant and tissue haemorrhagic activity.

Infrared and circular dichroism spectroscopic characterisation of secondary structure components of a water treatment coagulant protein extracted from Moringa oleifera seeds.

The secondary structure of a water treatment coagulant protein extracted from Moringa oleifera (MO) seeds has been investigated by Fourier transform infrared spectroscopy (FTIR) in the dried state, and by circular dichroism (CD) spectroscopy. The FTIR and CD spectra indicate that the secondary structure of the protein is dominated by alpha-helix. The FTIR spectrum recorded two distinct and strong absorption bands at 1656 cm(-1) and 1542 cm(-1), in the usual range of absorption of helices of proteins. The CD spectrum showed the shape of mainly alpha-helical secondary structure (estimated to be 58+/-4%) characteristic of negative ellipticity bands near 222 nm and 208 nm and a positive band at 192 nm. The beta-sheet structure composition was estimated to be 10+/-3% whereas unordered structures were around 33%. Changes in solution pH affected the protein secondary structure significantly only at pH values above 10, as indicated by CD spectra, whereas ionic strength had minimal effect. CD data also showed that sodium dodecyl sulphate (SDS) interacts with the coagulant protein and modifies the protein conformation. The surfactant-induced conformational change of the coagulant protein was confirmed by quenching of tryptophan fluorescence of the protein.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper.

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.