Two Acidic,Anticoagulant PLA2 Isoenzymes Purified from the Venom of Monocled Cobra Naja kaouthia Exhibit Different Potency to Inhibit Thrombin and Factor Xa via Phospholipids Independent,Non-Enzymatic Mechanism

Background

The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A₂ (PLA₂; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA₂ isoenzymes, Nk-PLA₂α (13463.91 Da) and Nk-PLA₂β (13282.38 Da) purified from the venom of N. kaouthia.

Principal Findings

By LC-MS/MS analysis, these PLA₂s showed highest similarity (23.5% sequence coverage) with PLA₂ III isolated from monocled cobra venom. The catalytic activity of Nk-PLA₂β exceeds that of Nk-PLA₂α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA₂ isoenzymes. The anticoagulant potency of Nk-PLA₂α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA₂β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA₂s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA₂α and Nk-PLA₂β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA₂ isoenzymes inhibit their “pharmacological target(s)” by uncompetitive mechanism without the requirement of phospholipids/Ca²⁺. The anticoagulant potency of Nk-PLA₂β which is higher than that of Nk-PLA₂α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA₂ isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA₂β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.

Conclusion/Significance

In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA₂α and Nk-PLA₂β for the treatment and/or prevention of cardiovascular disorders are proposed.     

Purification and partial characterization of an anticoagulant PLA2 from the venom of Indian Daboia russelii that induces inflammation through upregulation of proinflammatory mediators

The present study describes the purification and partial characterization of a basic anticoagulant PLA₂ enzyme named as Rv(i) PLA₂ from the venom of Indian Daboia russelii. The molecular mass of the protein was found to be 13,659.65 Da, and peptide mass fingerprinting revealed that it belongs to group II PLA₂ family. The peptide sequence showed similarity to uncharacterized basic PLA₂ enzyme having an accession no. of P86368 reported from Sri Lankan D. russelii. Rv(i) PLA₂ exhibited strong phospholipase A₂ and anticoagulant activity. It also induced expression of COX‐2 and TNF‐α mRNA in a dose‐dependent manner in phorbol 12‐myristate 13‐acetate differentiated THP‐1 cells, which play a crucial role during inflammation. Chemical modification of His residue in Rv(i) PLA₂ with p‐bromophenacyl bromide abolished the enzymatic, anticoagulant, and inflammatory activities. The result indicates that the catalytic site of Rv(i) PLA₂ might play a vital role in inducing inflammation at the bite site during D. russelii envenomation.     

Studies, with a luminogenic peptide substrate, on blood coagulation Factor X/Xa produced by mouse peritoneal macrophages

The formation and secretion of coagulation Factor X/Xa by mouse peritoneal macrophages was studied with a luminogenic peptide substrate (S-2613; t-butyloxycarbonylisoleucylglutamyl-γ-piperidylglycylarginylisoluminol). Amidolysis was quantified by measuring the light emitted during oxidation of isoluminol, released by Factor Xa. A lower detection limit of about 0.5ng of Factor Xa was established; the assay was linear with enzyme concentration up to at least 100ng/ml. Factor X was determined after treatment with the Factor X-activating component of Russell's-viper (Vipera russelli) venom. Macrophages, cultured in the absence of serum, released Factor X/Xa into the culture medium. The concentration of coagulation enzyme in the medium increased in an essentially linear fashion over a period of at least 3 days, at a rate corresponding to 6–8ng produced/24h per 10⁶ cells. The ratio of Factor Xa/X+Xa varied from about 60 to 100%, showing that activation of Factor X to Xa is not prerequisite to release of the enzyme from the cells. Factor Xa activity was suppressed in the presence of warfarin [3-(α-acetonylbenzyl)-4-hydroxycoumarin; 12.5μg/ml of medium], but could be restored by adding vitamin K (0.1μg/ml) along with the warfarin. Cultures to which Sepharose beads containing covalently bound anti-(Factor X) antibodies had been added showed decreased amounts of free Factor X/Xa in the culture medium. The missing activity could be demonstrated by incubating the recovered conjugate with the substrate peptide S-2613. Factor Xa produced by the macrophages was efficiently inactivated by heparin in the presence of antithrombin, heparin with high affinity for antithrombin being more effective than the corresponding low-affinity species.     

Purification and characterization of the anticoagulant principle of Trimeresurus mucrosqualatus venom

By means of CM-Sephadex column chromatography, Trimeresurus mucrosquamatus venom was separated into 20 fractions. Fraction XX had the marked anticoagulant action. This fraction was refractionated three times on Sephadex G-75, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 1.61 S was obtained by ultracentrifugation. It was a single peptide chain with a molecular weight of 11 700. The isoelectric point was higher than pH 10.The anticoagulant principle possesses phospholipase A activity and was calcium ion dependent. It did not possess proteolytic, tosyl-L-arginine methyl ester esterase, phosphodiesterase and alkaline phosphomonoesterase activities of the crude venom. The phospholipase A activity was heat-labile at pH 7.4, but was heat-stable at pH 5.6. The anticoagulant activity was more resistant to heat treatment as compared with phospholipase A activity.The anticoagulant action of the purified principle was competitively inhibited by platelet phospholipid, tissue thromboplastin and cephalin, and was neutralized by antiserum. The anticoaugulant principle inhibited platelet aggregation induced by ADP. It did not destroy fibrinogen, Factor X, prothrombin and thrombin; nor did it induce fibrinolysis nor interfere with the interaction between thrombin and fibrinogen. It is concluded that the anticoagulant action of this phospholipase A was due to the inhibition of the activations of Factors X and II through the activation of the procoagulant activity of phospholipids mediated partly by phospholipid-binding activity of this venom enzyme and partly by its enzymatic hydrolysis of phospholipids.     

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity,a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host''s immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.     

Effects of the bumblebee (Bombus ignitus) venom serine protease inhibitor on serine protease and phospholipase A2 of B. ignitus venom

Bumblebee venom contains serine proteases and serine protease inhibitors. In this study, we characterized whether the bumblebee (Bombus ignitus) venom serine protease inhibitor (Bi-KTI) inhibits B. ignitus venom serine protease (Bi-VSP) or phospholipase A₂ (Bi-PLA₂). Bi-KTI did not inhibit Bi-VSP activity at pH 5.4 or 7.4, whereas Bi-KTI slightly inhibited Bi-VSP activity at pH 7.4 after a 30 min preincubation. The Bi-VSP activity that converts prothrombin into thrombin and fibrin into fibrin degradation products was not significantly affected by Bi-KTI. Additionally, Bi-KTI or Bi-VSP did not inhibit Bi-PLA₂ activity. These findings indicate that each bee venom component appears to a play a toxic role via a unique function.     

Targeting of venom phospholipases: the strongly anticoagulant phospholipase A(2) from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex.

The strongly anticoagulant basic phospholipase A(2) (CM-IV) from Naja nigricollis venom has previously been shown to inhibit the prothrombinase complex of the coagulation cascade by a novel nonenzymatic mechanism (S. Stefansson, R. M. Kini, and H. J. Evans Biochemistry 29, 7742-7746, 1990). That work indicated that CM-IV is a noncompetitive inhibitor and thus it interacts with either factor Va or factor Xa, or both. We further examined the interaction of CM-IV and the protein components of the prothrombinase complex. Isothermal calorimetry studies indicate that CM-IV does not bind to prothrombin or factor Va, but only to factor Xa. CM-IV has no effect on the cleavage of prothrombin by factor Xa in the absence of factor Va. However, in the presence of factor Va, CM-IV inhibits thrombin formation by factor Xa. With a constant amount of CM-IV, raising the concentration of factor Va relieved the inhibition. The phospholipase A(2) enzyme inhibits by competing with factor Va for binding to factor Xa and thus prevents formation of the normal Xa-Va complex or replaces bound factor Va from the complex. Thus factor Xa is the target protein of this anticoagulant phospholipase A(2), which exerts its anticoagulant effect by protein-protein rather than protein-phospholipid interactions.     

Functional characterization of single-chain factor X from rat liver

¹⁴C-Labeled single-chain factor X prepared by vitamin K-dependent carboxylation in vitro was partially purified by adsorption to BaSO₄ and chromatography on DEAE-Sephacel. Known activators of factor X were analyzed for their effect on the single-chain molecule. ¹⁴C-Labeled factor X antigens were recovered immunochemically from incubation mixtures and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation with trypsin resulted in the generation of factor Xa clotting activity, and the ¹⁴C-labeled product migrated after reduction with an apparent molecular weight of 22,500 ± 1500 (mean ± 1 SD). The light chain produced by factor Xa was similar to that produced by trypsin (M_r 24,500 ± 1500; mean ± 1 SD). Incubation of single-chain factor X with factor VII and thromboplastin, factor IXa, or the factor X activating enzyme from Russell's viper venom gave a reducible product with a light chain of higher apparent molecular weight (M_r 37,000–38,000). Incubation with factor VII and thromboplastin also resulted in the generation of factor Xa clotting activity. Incubation of single-chain factor X with platelets resulted in the binding of about 20% of the ¹⁴C. The bound ¹⁴C-labeled factor X antigen released by freezing and thawing in the presence of EDTA was reduced to give a ¹⁴C-labeled polypeptide with M_r 31,000. Walker 256 tumor cells bound about 30% of the ¹⁴C. The bound material, after reduction, gave a ¹⁴C-labeled polypeptide with M_r 23,000.     

Alpha1-adrenoceptors trigger the snake venom production cycle in secretory cells by activating phosphatidylinositol 4,5-bisphosphate hydrolysis and ERK signaling pathway

Loss of venom from the venom gland after biting or manual extraction leads to morphological changes in venom secreting cells and the start of a cycle of production of new venom. We have previously shown that stimulation of both α- and β-adrenoceptors in the secretory cells of the venom gland is essential for the onset of the venom production cycle in Bothrops jararaca. We investigated the signaling pathway by which the α-adrenoceptor initiates the venom production cycle. Our results show that the α₁-adrenoceptor subtype is present in venom gland of the snake. In quiescent cells, stimulation of α₁-adrenoceptor with phenylephrine increased the total inositol phosphate concentration, and this effect was blocked by the phospholipase C inhibitor U73122. Phenylephrine mobilized Ca²⁺ from thapsigargin-sensitive stores and increased protein kinase C activity. In addition, α₁-adrenoceptor stimulation increased the activity of ERK 1/2, partially via protein kinase C. Using RT-PCR approach we obtained a partial sequence of a snake α₁-adrenoceptor (260 bp) with higher identity with α_1D and α_1B-adrenoceptors from different species. These results suggest that α₁-adrenoceptors in the venom secreting cells are probably coupled to a G_q protein and trigger the venom production cycle by activating the phosphatidylinositol 4,5-bisphosphate and ERK signaling pathway.     

Biophysical characterization of anticoagulant hemextin AB complex from the venom of snake Hemachatus haemachatus

Hemextin AB complex from the venom of Hemachatus haemachatus is the first known natural anticoagulant that specifically inhibits the enzymatic activity of blood coagulation factor VIIa in the absence of factor Xa. It is also the only known heterotetrameric complex of two three-finger toxins. Individually only hemextin A has mild anticoagulant activity, whereas hemextin B is inactive. However, hemextin B synergistically enhances the anticoagulant activity of hemextin A and their complex exhibits potent anticoagulant activity. In this study we characterized the nature of molecular interactions leading to the complex formation. Circular dichroism studies indicate the stabilization of β-sheet in the complex. Hemextin AB complex has an increased apparent molecular diameter in both gas and liquid phase techniques. The complex formation is enthalpically favorable and entropically unfavorable with a negative change in the heat capacity. Thus, the anticoagulant complex shows less structural flexibility than individual subunits. Both electrostatic and hydrophobic interactions are important for the complexation; the former driving the process and the latter helping in the stabilization of the tetramer. The tetramer dissociates into dimers and monomers with the increase in the ionic strength of the solution and also with increase in the glycerol concentration in the buffer. The two dimers formed under each of these conditions display distinct differences in their apparent molecular diameters and anticoagulant properties. Based on these results, we have proposed a model for this unique anticoagulant complex.     

Inhibition of Nicotinic Acetylcholine Receptors,a Novel Facet in the Pleiotropic Activities of Snake Venom Phospholipases A2

Phospholipases A₂ represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A₂, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A₂ from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A₂ content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A₂, which add a new target to the numerous activities of these enzymes.     

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with ∼40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31–41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited ∼5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with ∼4-fold decreased k_cat and ∼2-fold decreased K_m, although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.     

Thermodynamic and Kinetic Characterization of the Protein Z-dependent Protease Inhibitor (ZPI)-Protein Z Interaction Reveals an Unexpected Role for ZPI Lys-239

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), circulates in blood as a tight complex with its cofactor, protein Z (PZ), enabling it to function as a rapid inhibitor of membrane-associated factor Xa. Here, we show that N,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled K239C ZPI is a sensitive, moderately perturbing reporter of the ZPI-PZ interaction and utilize the labeled ZPI to characterize in-depth the thermodynamics and kinetics of wild-type and variant ZPI-PZ interactions. NBD-labeled K239C ZPI bound PZ with ∼3 nm K_D and ∼400% fluorescence enhancement at physiologic pH and ionic strength. The NBD-ZPI-PZ interaction was markedly sensitive to ionic strength and pH but minimally affected by temperature, consistent with the importance of charged interactions. NBD-ZPI-PZ affinity was reduced ∼5-fold by physiologic calcium levels to resemble NBD-ZPI affinity for γ-carboxyglutamic acid/EGF1-domainless PZ. Competitive binding studies with ZPI variants revealed that in addition to previously identified Asp-293 and Tyr-240 hot spot residues, Met-71, Asp-74, and Asp-238 made significant contributions to PZ binding, whereas Lys-239 antagonized binding. Rapid kinetic studies indicated a multistep binding mechanism with diffusion-limited association and slow complex dissociation. ZPI complexation with factor Xa or cleavage decreased ZPI-PZ affinity 2–7-fold by increasing the rate of PZ dissociation. A catalytic role for PZ was supported by the correlation between a decreased rate of PZ dissociation from the K239A ZPI-PZ complex and an impaired ability of PZ to catalyze the K239A ZPI-factor Xa reaction. Together, these results reveal the energetic basis of the ZPI-PZ interaction and suggest an important role for ZPI Lys-239 in PZ catalytic action.     

Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord

An anticoagulant fraction was isolated from the homogenate of human umbilical cord arteries, using Sephadex gel filtration and DEAE-Sephacel chromatography. Analysis with dodecyl sulfate/polyacrylamide gel electrophoresis and inactivation studies using proteolytic enzymes indicate that the anticoagulant activity is associated with a polypeptide with an apparent Mr of 32 000. The anticoagulant inhibits thromboplastin as well as factor Xa induced clotting but does not affect thrombin initiated fibrin formation. The anticoagulant inhibits the activation of prothrombin by the complete prothrombinase complex, by phospholipid bound factor Xa but not by free factor Xa. The inhibition is instantaneous and independent of the incubation time over the whole range of concentrations tested. Therefore, the anticoagulant is unlikely to be a phospholipase or a protease. Its action does not resemble that of the plasma protease inhibitors, but it probably interferes with the phospholipid--clotting factor interactions.     

Cloning and Characterization of a P2X Receptor Expressed in the Central Nervous System of Lymnaea stagnalis

P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC₅₀ 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αβmeATP was a weak agonist. BzATP was a partial agonist with an EC₅₀ of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC₅₀ values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn²⁺ and Cu²⁺ ions exhibited a biphasic effect, potentiating currents up to 100 µM and inhibiting at higher concentrations. Quantitative RT-PCR and in situ hybridization detected expression of LymP2X mRNA in neurones of all CNS ganglia suggesting this ion channel may have widespread roles in Lymnaea CNS function.     

Correlation between the phospholipids domains of the target cell membrane and the extent of Naja kaouthia PLA2-induced membrane damage: Evidence of distinct catalytic and cytotoxic sites in PLA2 molecules

Two phospholipase A₂ (PLA₂) enzymes (NK-PLA₂-A and NK-PLA₂-B) were purified from the venom of the monocled cobra Naja kaouthia. The molecular weights of NK-PLA₂-A and NK-PLA₂-B, as estimated by mass spectrometry, were 13,619 and 13,303 Da respectively. Both phospholipases were highly thermostable, had maximum catalytic activity at basic pH, and showed preferential hydrolysis of phosphatidylcholine. Intravenous injection of either PLA₂ up to a dose of 10 mg/kg body weight was non-toxic to mice and did not show neurotoxic symptoms. The N. kaouthia PLA₂s displayed anticoagulant and cytotoxic activity, but poor hemolytic activity. Both the PLA₂s were more toxic to Sf9 and Tn cells compared to VERO cells. NK-PLA₂ exhibited selective lysis of wild-type baculovirus-infected Sf9 cells compared to normal cells. Amino acid modification studies and heating experiments suggest that separate sites in the NK-PLA₂ molecules are responsible for their catalytic, anticoagulant and cytotoxic activities.     

Procoagulant,Tissue Factor-Bearing Microparticles in Bronchoalveolar Lavage of Interstitial Lung Disease Patients: An Observational Study

Coagulation factor Xa appears involved in the pathogenesis of pulmonary fibrosis. Through its interaction with protease activated receptor-1, this protease signals myofibroblast differentiation in lung fibroblasts. Although fibrogenic stimuli induce factor X synthesis by alveolar cells, the mechanisms of local posttranslational factor X activation are not fully understood. Cell-derived microparticles are submicron vesicles involved in different physiological processes, including blood coagulation; they potentially activate factor X due to the exposure on their outer membrane of both phosphatidylserine and tissue factor. We postulated a role for procoagulant microparticles in the pathogenesis of interstitial lung diseases. Nineteen patients with interstitial lung diseases and 11 controls were studied. All subjects underwent bronchoalveolar lavage; interstitial lung disease patients also underwent pulmonary function tests and high resolution CT scan. Microparticles were enumerated in the bronchoalveolar lavage fluid with a solid-phase assay based on thrombin generation. Microparticles were also tested for tissue factor activity. In vitro shedding of microparticles upon incubation with H₂O₂ was assessed in the human alveolar cell line, A549 and in normal bronchial epithelial cells. Tissue factor synthesis was quantitated by real-time PCR. Total microparticle number and microparticle-associated tissue factor activity were increased in interstitial lung disease patients compared to controls (84±8 vs. 39±3 nM phosphatidylserine; 293±37 vs. 105±21 arbitrary units of tissue factor activity; mean±SEM; p<.05 for both comparisons). Microparticle-bound tissue factor activity was inversely correlated with lung function as assessed by both diffusion capacity and forced vital capacity (r² = .27 and .31, respectively; p<.05 for both correlations). Exposure of lung epithelial cells to H₂O₂ caused an increase in microparticle-bound tissue factor without affecting tissue factor mRNA.Procoagulant microparticles are increased in interstitial lung diseases and correlate with functional impairment. These structures might contribute to the activation of factor X and to the factor Xa-mediated fibrotic response in lung injury.     

II — Subunit structure of a potent platelet-activating glycoprotein isolated from the venom of Crotalus durissus cascavella

The potent platelet-activating factor isolated from the venom of Crotalus durissus cascavella is an acidsoluble multisubunit glycoprotein of Mr 72,000 built up of two types of subunits, α and β, linked by disulphide bonds. The mean apparent Mr of the reduced complex was around 12,000 by gel filtration under denaturating conditions. The Mrs of the α and β subunits, with an apparent ratio of 1/1, were 12,600 and 13,580 by SDS-PAGE respectively. The Mr 72,000 glycoprotein is thought to be an α₃ β₃ complex. The urea dissociated glycoprotein (Mr 72,000) retained its platelet-stimulating activity. It is concluded that the Mr 300,000 form isolated at acidic pH under native conditions, and showing a rosette - like, ring-shaped structure in the electron microscope as well as the Mr 144,000 form isolated at physiological pH under native conditions and active on platelets [1] were the tetrameric and dimeric states of the molecule respectively.     

Pharmacokinetics of Naja sumatrana (Equatorial Spitting Cobra) Venom and Its Major Toxins in Experimentally Envenomed Rabbits

Background

The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom''s composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A₂, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits.

Principal findings

The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A₂ and whole venom (T_max = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (F_i.m. = 81.5%) compared to that of the phospholipase A₂ (F_i.m. = 68.6%) or cardiotoxin (F_i.m. = 45.6%). The incomplete absorption of the phospholipase A₂ and cardiotoxin may infer the toxins'' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions.

Conclusion/Significance

Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.