Purification and characterization of a new platelet aggregation inhibitor with dissociative effect on ADP-induced platelet aggregation, from the venom of Protobothrops elegans (Sakishima-habu)

A platelet aggregation inhibitor, named snake venom platelet aggregation dissociator (SV-PAD)-1, with a dissociative reaction of ADP-induced platelet aggregation, was purified from the venom of Protobothrops elegans (Sakishima-habu) by gel-filtration employing Sephadex G-100, and ion-exchange chromatographies using DEAE-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Mono S. By this procedure, about 1.5 mg of purified protein was obtained from 1.0 g of P. elegans venom. The purified protein showed a single protein band and the molecular weight was about 110 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed four-bands of 7.7, 7.8, 7.95, and 8.15. This protein strongly inhibited ADP-induced platelet aggregation in rabbit platelet-rich plasma (PRP), and its IC₅₀ was about 58 nM. It inhibited ristocetin-induced platelet aggregation in rabbit PRP (IC₅₀: 100 nM), but hardly blocked collagen-induced platelet aggregation. This protein promptly dissociated platelet aggregation in rabbit PRP stimulated by high-concentration ADP.     

Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom

Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets.Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5′RACE and 3′RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the β subunit contained 447 bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120 kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αβ)₄. Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC₅₀ of 0.25 nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.     

Expression,purification and characterization of recombinant Jerdonitin,a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains

Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC₅₀ of 248 nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

Colombistatin: a disintegrin isolated from the venom of the South American snake (<Emphasis Type="Italic">Bothrops colombiensis</Emphasis>) that effectively inhibits platelet aggregation and SK-Mel-28 cell adhesion

Snake venoms are complex mixtures of proteins, which affect the vital biologic systems of prey, as well as humans. Envenomation leads to immobilization by paralysis, cardiac, and circulatory failure. These same venom proteins that cause havoc in the physiologic system could be used as therapeutic agents. Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are non-enzymatic proteins that inhibit cell–cell interactions, cell–matrix interactions, and signal transduction. These proteins may have potential in the treatment of strokes, heart attacks, cancers, osteoporosis, and diabetes. The present study describes the isolation and characterization of a disintegrin (colombistatin) found in the venom of the Venezuelan snake mapanare (Bothrops colombiensis). Colombistatin was purified by a two-step high-performance liquid chromatography procedure, which included reverse phase C18 and size exclusion protein Pak 60. Colombistatin inhibited ADP-induced platelet aggregation, human urinary (T24) and skin melanoma (SK-Mel-28) cancer cell adhesion to fibronectin, and cell migration. Colombistatin contained 72 amino acids with a mass of 7.778 kDa as determined by mass spectrometry. Colombistatin could be used as a therapeutic tool in the treatment of melanoma cancers and also thrombotic diseases.     

NPP-BJ, a nucleotide pyrophosphatase/phosphodiesterase from Bothrops jararaca snake venom, inhibits platelet aggregation

Enzymes of the pyrophosphatase/phosphodiesterase family have multiple roles in extracellular nucleotide metabolism and in the regulation of nucleotide-based intercellular signaling. Snake venoms contain enzymes that hydrolyze nucleic acids and nucleotides, but their function is poorly understood. Here we describe for the first time the isolation and functional characterization of a soluble phosphodiesterase from Bothrops jararaca venom, which shows amino acid sequence similarity to mammalian nucleotide pyrophosphatase/phosphodiesterase 3 (NPP3), and inhibits ADP-induced platelet aggregation. The enzyme, named NPP-BJ, showed an apparent molecular mass of 228 kDa by size exclusion chromatography. NPP-BJ exhibited nuclease activity as well as pyrophosphatase and phosphatase activities, preferentially hydrolyzing nucleoside 5′-triphosphates over nucleoside 5′-diphosphates, but was not active upon nucleoside 5′-monophosphates. Depending on the substrate used, dithiothreitol and EDTA differently inhibited the catalytic activity of NPP-BJ. Platelet aggregation induced by ADP was also abrogated by NPP-BJ, whereas thrombin-induced platelet aggregation was only slightly attenuated. However, polyclonal antibodies raised against NPP-BJ could not abolish the lethal activity of B. jararaca venom. Altogether, these results show that NPP-BJ has a minor contribution to the lethal activity of this venom, but interferes with mechanisms of ADP-induced platelet aggregation.     

S-nitroso-glutathione inhibits platelet activation in vitro and in vivo.

1. The effect of S-nitroso-glutathione (GSNO), a stable nitrosothiol, on platelet activation was examined in vitro and in vivo. 2. The adhesion of human platelets to fibrillar collagen and human endothelial cell monolayers was inhibited by GSNO. 3. GSNO caused a concentration-dependent inhibition of collagen-induced platelet aggregation in vitro and decreased ADP-induced aggregation in the conscious rat. 4. Inhibition of platelet aggregation in vitro correlated with the increase in intraplatelet cyclic GMP levels. 5. The release of NO from GSNO was enhanced by platelet lysate, native glutathione and ascorbate. 6. The results show that GSNO is a carrier of NO and therefore has pharmacological activity as an inhibitor of platelet activation.     

A novel natriuretic peptide from the cobra venom

Natriuretic peptides (NPs) play crucial roles in human physiology and pathophysiology through natriuresis, dieresis and vasorelaxation. NPs are also one of the important components of snake venoms. However, the low abundance in snake venom hampered the investigation. Here, a novel natriuretic peptide named Na-NP was purified from the cobra Naja atra venom. Na-NP consists of 45 amino acid residues and its molecular weight is 4618.5 Da. A full-length cDNA encoding Na-NP was obtained from the cDNA library constructed from the venom gland. The open reading frame of cloned Na-NP was composed of 498 bp and coded for a 165-amino acid residue protein precursor. The nucleotide and deduced protein sequences of Na-NP were remarkably conserved with other elapid NPs while significant different from the viperid NPs. Na-NP showed weak activity to relax the aortic rings precontracted with phenylephrine. Meanwhile, Na-NP showed cGMP-promotion activity against primary cultured rabbit endocardial endothelial cells, but had no effect on human platelet aggregation. In conclusion, this is the first report of a natriuretic peptide from the cobra N. atra venom. Na-NP might be served as a useful tool for the study of human NPs and the development of novel therapeutic drugs.     

Leucurogin, a new recombinant disintegrin cloned from Bothrops leucurus (white-tailed-jararaca) with potent activity upon platelet aggregation and tumor growth

Disintegrins and disintegrins-like proteins are able to inhibit platelet aggregation and integrin-mediated cell adhesion. The aim of this study was to produce one disintegrin-like cloned from Bothrops leucurus venom gland and to characterize it regarding biological activity. The recombinant protein was purified by one step procedure involving anion-exchange chromatography (DEAE-cellulose) and presented a molecular mass of 10.4 kDa. The purified protein was able to inhibit platelet aggregation induced by collagen (IC₅₀ = 0.65 μM) and to inhibit growth of Ehrlich tumor implanted in mice by more than 50% after 7 days administration of 10 μg/day. No effects were observed upon adenosine 5′-diphosphate (ADP)-and arachidonic acid (AA)-induced platelet aggregation. The recombinant protein was recognized by an antibody specific for jararhagin one metalloproteinase isolated from Bothrops jararaca venom, and therefore it was named leucurogin. Anti-angiogenesis effect of leucurogin was evaluated by the sponge implant model. After 7 days administration leucurogin inhibited, in a dose dependent way, the vascularization process in the sponge. Leucurogin represents a new biotechnological tool to understand biological processes where disintegrins-like are involved and may help to characterize integrins that can be involved in development and progression of malignant cells.     

Mechanism of action of the platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom

The platelet aggregation inhibitor purified from Agkistrodon halys snake venom inhibited rabbit platelet aggregations induced by thrombin, sodium arachidonate, collagen or ionophore A-23187. The ic₅₀ was about 11 μg/ml in platelet aggregation regardless of which aggregation inducer was used. β-Mercaptoethanol abolished both the phospholipase A enzymatic and platelet aggregation inhibitory activities of this venom inhibitor. p-Bromophenacyl bromide-treated venom inhibitor lost almost completely its phosphilipase A enzymatic activity, but retained its platelet aggregation inhibitory effect. In the presence of EGTA, the venom inhibitor still showed the same inhibitory activity on thrombin-, sodium arachidonate-, collagen- or ionophore A23187-induced platelet aggregations triggered by successive addition of Ca²⁺. The activation of platelet phospholipase A and the serotonin release reaction triggered by Ca²⁺ influx were unaffected by this venom inhibitor. It also inhibited the clot retraction of platelet-rich plasma. It is concluded that the inhibitory effect of the venom inhibitor on platelet aggregation is independent of its phospholipase A enzymatic activity. Its mode of action is different from those of other known platelet inhibitory drugs. This venom inhibitor possibly acts on a common step subsequent to platelet shape change, leading to inhibition of platelet aggregation.     

Recombinant rubistatin (r-Rub), an MVD disintegrin, inhibits cell migration and proliferation, and is a strong apoptotic inducer of the human melanoma cell line SK-Mel-28

Disintegrins are low molecular weight peptides isolated from viper venom. These peptides bind to integrin receptors using a conserved binding motif sequence containing an RGD or similar motif. As a consequence, disintegrins can inhibit platelet aggregation and inhibit cell migration, proliferation, and initiate apoptosis in cancer cell lines. Rubistatin is a MVD disintegrin cloned from a Crotalus ruber ruber venom gland. The biological activity of MVD disintegrins is poorly understood. Recombinant rubistatin (r-Rub) was cloned into a pET32b plasmid and expressed in reductase-deficient Escherichia coli. Expression was induced with IPTG and the resulting fusion peptide was affinity purified, followed by thrombin cleavage, and removal of vector coded sequences. r-Rub peptide inhibited ADP-induced platelet aggregation by 54% ± 6.38 in whole blood. We assessed the ability of r-Rub to initiate apoptosis in three human cancer cell lines. Cultures of SK-Mel-28, HeLA, and T24 cells were grown for 24 h with 2.5 μM r-Rub followed by Hoechst staining. Chromatin fragmentation was observed in treated SK-Mel-28, but not in T24 or HeLA cells. A TUNEL assay revealed that 51.55% ± 5.28 of SK-Mel-28 cells were apoptotic after 18 h of treatment with 3.5 μM of r-Rub. Cell migration and proliferation assays were performed in order to further characterize the biological effects of r-Rub on SK-Mel-28 cells. At 3 μM, r-Rub inhibited cell migration by 44.4% ± 0.5, while at 3.5 μM it was able to inhibit cell proliferation by 83% ± 6.0.     

A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom

Y.-S. Li, K.-F. Liu, Q.-C. Wang, Y.-L. Rpan and G.-C. Tu. A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom. Toxicon23, 895–903, 1985. — By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G — 75, a potent platelet function inhibitor was purified from Vipera russelli siamensis venom. It appeared as a single protein band on polyacrylamide gel electrophoresis in the presence or absence of SDS, and consists of 123 amino acid residues. Its NH₃-terminal residue is serine. It showed the following characteristics: molecular weight, 13,800; isoelectric point, 10.4; ld(In50), 0.5 ± 0.12 mg/kg (i.v.). The platelet inhibitor exhibited phospholipase A₂ activity with a specific activity of 35 μmoles/min/mg. From 2 g of the venom, 70 mg of the purified inhibitor was obtained. Inhibition of human platelet aggregation induced by ADP or adrenaline was dose-dependent, with id(In50) of 1.14μg/ml or 0.37 μg/ml, respectively. The platelet aggregation induced by thrombin or collagen was also inhibited and the inhibitory activity on platelet aggregation was heat stable (at 100°C, 20 min) in an acidic medium (pH 5.8), while its phospholipase A₂ activity was relatively heat labile under the same condition. The release of ³H-serotonin in platelets stimulated by ADP was also inhibited and this was positively correlated with inhibition of platelet aegregation induced by ADP (r = 0.998, P < 0.002).     

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom.

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. From the deduced amino acid sequence, jerdonin is composed of 71 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonin was determined to be 7483Da by matrix-assisted laser desorption ionization time of flight mass spectrometry. Jerdonin inhibited ADP- and collagen-induced human platelet aggregation with IC(50) of 220 and 240 nM, respectively. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice.     

Hemostatic and toxinological diversities in venom of Micrurus tener tener, Micrurus fulvius fulvius and Micrurus isozonus coral snakes

The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A₂s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD₅₀s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD₅₀ of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD₅₀s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bβ chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders.     

Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell–matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5′-RACE, the 2040 bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3 mg/L of culture medium. The molecular weights were 11 kDa in reduced and 22 kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC₅₀ of 976 nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.     

Effects of 2-methylthioadenosine 5′-β,γ-methylenetriphosphonate and 2-ethylthioadenosine 5′-monophosphate on human platelet activation induced by adenosine 5′-diphosphate

Adenosine 5′-diphosphate (ADP) induces human platelet aggregation, increases intracellular levels of free calcium, and inhibits stimulated adenylate cyclase. These effects of ADP are mediated by P_2T-purinoceptors that are inhibited specifically and competitively by adenosine 5′-triphosphate (ATP). Inhibition of ADP-induced aggregation and increases in calcium by 2-alkylthio analogs of ATP and of adenosine 5′-monophosphate (AMP) are also specific, but the inhibition is non-surmountable. To examine further the nature of inhibition of ADP-induced platelet activation by 2-alkylthio analogs, the effects of 2-methylthioadenosine 5′-β,γ-methylenetriphosphonate (2-MeS-AMP-PCP) and 2-ethylthioadenosine 5′-monophosphate (2-EtS-AMP) were tested on ADP-induced platelet aggregation and inhibition of adenylate cyclase. 2-MeS-AMP-PCP inhibited platelet aggregation induced by ADP but not by epinephrine, arachidonic acid, 5-hydroxytryptamine (5-HT), platelet activating factor (PAF), or 11α,9α-epoxymethano-prostaglandin H₂ (U46619). Inhibition of ADP-induced platelet aggregation by 2-MeS-AMP-PCP was non-surmountable, and it achieved only 50% inhibition of ADP (5 μM)-induced aggregation. 2-MeS-AMP-PCP achieved 100% inhibition of ADP (5 μM)-induced inhibition of prostaglandin E₁-stimulated adenylate cyclase, and Schild analysis showed the inhibition to be potent (pA₂ 7.3) and competitive (slope 1.12). 2-MeS-AMP-PCP inhibited platelet aggregation induced by adenosine 5′-O-2-thiodiphosphate (ADP-β-S), which inhibited stimulated adenylate cyclase activity, but did not inhibit aggregation induced by adenosine 5′-O-1-thiodiphosphate (ADP-α-S), which does not inhibit stimulated adenylate cyclase. 2-EtS-AMP behaved similarly to 2-MeS-AMP-PCP. These results suggest that ADP may induce aggregation by interacting with two forms of the calcium-mobilizing P_2T-purinoceptor, only one of which is coupled to inhibition of adenylate cyclase and at which 2-alkylthio analogs of ATP and AMP are specific competitive antagonists. © 1996 Wiley-Liss, Inc.     

Effect of prenylated flavonoids and chalcones isolated from <Emphasis Type="Italic">Artocarpus</Emphasis> species on platelet aggregation in human whole blood

Five prenylflavonoids and two prenylchalcones from Artocarpus lowii King, A. scortechinii King and A. teysmanii Miq., and acetylated derivatives of cycloheterophyllin and artonin E were investigated for their ability to inhibit arachidonic acid (AA), collagen and adenosine diphosphate (ADP)-induced platelet aggregation in human whole blood by using an electrical impedance method. Among the tested compounds, only cycloheterophyllin inhibited AA-induced platelet aggregation with an IC₅₀ value of 100.9 μM. It also showed strong inhibition against ADP-induced aggregation, with an IC₅₀ value of 57.1 μM. Isobavachalcone, 2′,4′-dihydroxy-4-methoxy-3′-prenyldihydrochalcone, cycloartobiloxanthone, artonin E and artonin E triacetate showed selective inhibition against ADP-induced aggregation, with IC₅₀ values ranging from 55.3 to 192.0 μM, but did not show such effect against other inducers.     

Cloning and characterization of Trimeresurus gracilis venom phospholipases A2: Comparison with Ovophis okinavensis venom and the systematic implications

This study focuses on the structural and functional characterizations of novel venom phospholipases A₂ (PLA₂s) from Trimeresurus gracilis, an endemic Taiwanese pitviper. The PLA₂ cDNAs were cloned from venom glands and sequenced. The majority of the clones encoded a Glu6-containing PLA₂ (designated as Tgc-E6) whose deduced amino acid sequence resembled those of other Crotalinae acidic PLA₂s. Tgc-E6 was also purified and constituted about 6% (w/w) of the total venom proteins. For human platelet rich plasma, Tgc-E6 inhibited the ADP- and collagen-induced aggregation with an IC₅₀ of 272 nM and 518 nM, respectively. Like Ovophis okinavensis venom, T. gracilis venom did not contain any Lys49-PLA₂s, although a cDNA encoding Lys49-PLA₂ has been cloned from each of the species. Their predicted protein sequences are 94% identical, and their pI values 8.3 are lower than those of other Lys49-PLA₂s, mainly due to the acidic substitutions within positions 78-111, which are apparently more similar to those in Tgc-E6 than to those in other Lys49-PLA₂s. This unique structural feature of the venom PLA₂s thus render evidence for close phylogenetic relationship between both species. The structural variations in the venom acidic PLA₂s of the two species possibly have resulted from adaptation to different prey ecology.     

Ex-vivo and In-vivo Antithrombotic Effect of Triflavin,an RGD-containing Peptide

Abstract— Triflavin, an Arg-Gly-Asp-containing snake venom peptide, inhibits platelet aggregation through the blockade of fibrinogen binding to the activated platelets. It binds to fibrinogen receptors associated with the glycoprotein IIb/IIIa complex with a K_d value of 7 × 10^?8 m. In this study, we found that ¹²⁵I-triflavin reached the maximal binding to human platelets within 5 min at 25°C. In addition, when triflavin was intravenously administered at 1·0 mg kg^?1 to rabbits, it reversibly impaired the platelet aggregation of platelet-rich plasma caused by ADP (20 μm) ex-vivo over 30 min. The platelet counts of the experimental rabbits remained unchanged. Triflavin was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at a dose of 2 μg g^?1. Therefore, triflavin was proven to be an effective antithrombotic agent in preventing ADP-induced acute pulmonary thromboembolism in mice and impairing reversibly the platelet function of rabbits when given intravenously.     

Cardiotoxin from Naja naja atra snake venom: A potentiator of platelet aggregation

Cardiotoxin, isolated from Naja naja atra snake venom, potentiates platelet aggregation induced by ADP, thrombin, collagen and venom phospholipase A₂. The malondialdehyde formation caused by ADP, thrombin and venom phospholipase A₂ were also increased in the presence of cardiotoxin. Both potentiation of aggregation and increase in malondialdehyde were blocked by indomethacin or Ca²⁺ (5 mM or 0.05 mM). Cardiotoxin did not potentiate thrombin-induced aggregation of p-bromophenacyl bromide-modified platelets. Thromboxane B₂ formation induced by thrombin or collagen was also increased by cardiotoxin, while that by arachidonate was not affected. As a membrane-active polypeptide, cardiotoxin might augment the Ca²⁺-flux during the activation of the platelet membrane by aggregation inducers and then increase the activation of endogenous phospholipase A₂.