Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A(2) from Bothrops pirajai venom

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Platelet aggregation and antibacterial effects of an L-amino acid oxidase purified from Bothrops alternatus snake venom

The isolation and biochemical/enzymatic characterization of an L-amino acid oxidase, Balt-LAAO-I, from Bothrops alternates snake venom, is described. Balt-LAAO-I is an acidic glycoprotein, pI similar to 5.37, homodimeric, M-r similar to 123, 000, whose Nterminal sequence is ADVRNPLE EFRETDYEVL. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. Bait-LAAO-I induces platelet aggregation and shows bactericidal activity against Escherichia coli and Staphylococcus aureus. In addition, this enzyme is slightly hemorrhagic and induces edema in the mouse paw. Bait-LAAO-I is a multifunctional enzyme with promising relevant biotechnological and medical applications. (C) 2004 Elsevier Ltd. All rights reserved.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI similar to 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an similar to13.7 kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 'SLWQFGKMINYVMJGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0 Angstrom resolution. These crystals are monoclinic and have unit cell dimensions of a = 33.9, b = 63.8, c = 49.1 Angstrom, and beta = 104.0degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 tithes more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation, Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. (C) 2002 Elsevier B.V. All rights reserved.

Crystal structure of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) in the monomeric and dimeric states: insights into its oligomeric state

Phospholipases A(2) belong to the superfamily of proteins which hydrolyzes the sn-2 acyl groups of membrane phospholipids to release arachidonic acid and lysophospholipids. An acidic phospholipase A(2) isolated from Bothrops juraracussu snake venom presents a high catalytic, platelet aggregation inhibition and hypotensive activities. This protein was crystallized in two oligomeric states: monomeric and dimeric. The crystal structures were solved at 1.79 and 1.90 Angstrom resolution, respectively, for the two states. It was identified a Na+ ion at the center of Ca2+-binding site of the monomeric form. A novel dimeric conformation with the active sites exposed to the solvent was observed. Conformational states of the molecule may be due to the physicochemical conditions used in the crystallization experiments. We suggest dimeric state is one found in vivo. (C) 2004 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of BmooPLA(2)-I, a platelet-aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops moojeni venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary diffraction data of a platelet-aggregation inhibitor from the venom of Agkistrodon piscivorus piscivorus (North American water moccasin)

Applaggin (Agkistrodon piscivorus piscivorus platelet-aggregation inhibitor) is a potent inhibitor of blood platelet aggregation derived from the venom of the North American water moccasin, the protein consists of 71 amino acids, is rich in cysteines, contains the sequence-recognition site of adhesion proteins at positions 50-52 (Arg-Gly-Asp) and shares high sequence homology with other snake-venom disintegrins such as echistatin, kistrin and trigramin, Single crystals of applaggin have been grown and X-ray diffraction data have been collected to a resolution of 3.2 Angstrom. The crystals belong to space group P4(1)2(1)2 (or its enantiomorph), with unit-cell dimensions a = b = 63.35, c = 74.18 Angstrom and two molecules per asymmetric unit. Molecular replacement using models constructed from the NMR structures of echistatin and kistrin has not been successful in producing a trial structure for applaggin.

Perioperative administration of vedaprofen, tramadol or their combination does not interfere with platelet aggregation, bleeding time and biochemical variables in cats

A randomized double blind and placebo controlled design was used to investigate the hemostatic, biochemical, gastrointestinal and behavioral effects of pre- and postoperative administration of vedaprofen 0.5 mg/kg PO (V), tramadol 2 mg/kg SC (T), their association (VT) or placebo (P) in 40 adult female cats (3.0 +/- 0.32 kg; 1.8 +/- 0.7 years) distributed in groups of 10. Platelet aggregation and bleeding time were measured before and 52 11 after ovariohysterectomy. Serum urea, creatinine, alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transferase concentrations were measured before and 7 days postoperatively. The occurrence of vomiting, frequency and consistency of feces, and behavior were observed for 7 days postoperatively. Morphine (0.5 mg/kg, IM) was used as rescue analgesic. Laboratory variables did not change. Vomiting was observed only after morphine administration. Mild euphoria was observed in T and VT. The perioperative use of vedaprofen and/or tramadol did not modify the hemostatic, biochemical and gastrointestinal function in cats. (C) 2009 ESFM and AAFP. published by Elsevier Ltd. All rights reserved.

Inhibition of human platelet aggregation by eosinophils

AIMS: The relationship between the activity of eosinophils and platelets has been observed in recent decades by many scientists. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. MAIN METHODS: Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. KEY FINDINGS: Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with l-Nitro-Arginine-Methyl-Ester (l-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP, and the citotoxicity MTT assay proved the viability of tested platelets, showing that the observed inhibition by the ECP protein does not occur simply by cell death. SIGNIFICANCE: Our results indicate that eosinophils play a fundamental role in platelet aggregation inhibition

A randomised determination of the effect of fluvastatin and atorvastatin on top of dual antiplatelet treatment on platelet aggregation after implantation of coronary drug-eluting stents. The EFA-Trial

Drug-drug interaction between statins metabolised by cytochrome P450 3A4 and clopidogrel have been claimed to attenuate the inhibitory effect of clopidogrel. However, published data regarding this drug-drug interaction are controversial. We aimed to determine the effect of fluvastatin and atorvastatin on the inhibitory effect of dual antiplatelet therapy with acetylsalicylic acid (ASA) and clopidogrel. One hundred one patients with symptomatic stable coronary artery disease undergoing percutaneous coronary intervention and drug-eluting stent implantation were enrolled in this prospective randomised study. After an interval of two weeks under dual antiplatelet therapy with ASA and clopidogrel, without any lipid-lowering drug, 87 patients were randomised to receive a treatment with either fluvastatin 80 mg daily or atorvastatin 40 mg daily in addition to the dual antiplatelet therapy for one month. Platelet aggregation was assessed using light transmission aggregometry and whole blood impedance platelet aggregometry prior to randomisation and after one month of receiving assigned statin and dual antiplatelet treatment. Platelet function assessment after one month of statin and dual antiplatelet therapy did not show a significant change in platelet aggregation from 1st to 2nd assessment for either statin group. There was also no difference between atorvastatin and fluvastatin treatment arms. In conclusion, neither atorvastatin 40 mg daily nor fluvastatin 80 mg daily administered in combination with standard dual antiplatelet therapy following coronary drug-eluting stent implantation significantly interfere with the antiaggregatory effect of ASA and clopidogrel.

Effect of the synthetic coumarin, ethyl 2-oxo-2H-chromene-3-carboxylate, on activity of Crotalus durissus ruruima sPLA2 as well as on edema and platelet aggregation induced by this factor

We show that ethyl 2-oxo-2H-chromene-3-carboxylate (EOCC), a synthetic coumarin, irreversibly inhibits phospholipase A(2) (sPLA2) from Crotalus durissus ruruima venom (sPLA2r) with an IC(50) of 3.1 +/- 0.06 nmol. EOCC strongly decreased the V(max) and K(m), and it virtually abolished the enzyme activity of sPLA2r as well as sPLA2s from other sources. The edema induced by 5PLA2r + EOCC was less than that induced by 5PLA2r treated with p-bromophenacyl bromide, which was more efficient at neutralizing the platelet aggregation activity of native 5PLA2r. Native 5PLA2r induced platelet aggregation of 91.54 +/- 9.3%, and sPLA2r +/- EOCC induced a platelet aggregation of 18.56 +/- 6.5%. EOCC treatment also decreased the myotoxic effect of sPLA2r. Mass spectrometry showed that EOCC formed a stable complex with sPLA2r, which increased the mass of native 5PLA2r from 14,299.34 da to 14,736.22 Da. Moreover, the formation of this complex appeared to be involved in the loss of 5PLA2r activity. Our results strongly suggest that EOCC can be used as a pharmacological agent against the 5PLA2 in Crotalus durissus sp. venom as well as other sPLA2s. (C) 2010 Elsevier Ltd. All rights reserved.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A(2) from Bothrops pirajai venom

Background: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A(2) are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A(2) drugs.Methods: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated.Results: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 +/- 0.28 mu g/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA(2) inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid.Conclusion: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.