Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A2 Isolated from Lachesis muta (Bushmaster) Snake Venom

1997 ◽  
Vol 78 (05) ◽  
pp. 1372-1380 ◽  
Author(s):  
André L Fuly ◽  
Olga L T Machado ◽  
Elias W Alves ◽  
Célia R Carlinis
Keyword(s):  
Platelet Aggregation ◽  
Enzymatic Activity ◽  
Phospholipase A2 ◽  
Inhibitory Activity ◽  
Thermal Inactivation ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Crude Venom ◽  
Lachesis Muta

SummaryCrude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources.LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercapto-ethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lyso-phosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet’s response to several agonists.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

Human Leucocyte Urokinase Inhibitor - Purification, Characterization and Comparative Studies Against Different Plasminogen Activators

1985 ◽  
Vol 54 (04) ◽  
pp. 750-755 ◽  
Author(s):  
M Kopitar ◽  
B Rozman ◽  
J Babnik ◽  
V Turk ◽  
D E Mullins ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Inhibitory Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Plasminogen Activator Inhibitor ◽  
Exchange Chromatography ◽  
Sds Page ◽  
Peripheral Leukocytes ◽  
Ph Range ◽  
Activator Inhibitor

SummaryA plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pi of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37° C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.

Exogenous Inhibitors of Platelet Aggregation from Animal Sources

2001 ◽  
Vol 85 (01) ◽  
pp. 179-181 ◽  
Author(s):  
Geraldine Chow ◽  
R. Manjunatha Kini
Keyword(s):  
Platelet Aggregation ◽  
Enzymatic Activity ◽  
Phospholipase A2 ◽  
Enzymatic Activities ◽  
Inhibit Platelet Aggregation

SummaryThis updated inventory includes all inhibitors of platelet aggregation from animal sources until mid-1999. They are proteins or glycoproteins with Mr from 5000 to tens of thousands, and inhibit platelet aggregation by different mechanisms. Most lack enzymatic activity, but some exhibit enzymatic activities, such as phospholipase A2 (PLA2), proteinase and nucleotidase.

scholarly journals Novel Pharmacological Properties of Dinoponera quadriceps Giant Ant Venom

2015 ◽  
Vol 10 (9) ◽  
pp. 1934578X1501000 ◽  
Author(s):  
Juliana da Costa Madeira ◽  
Yves Patric Quinet ◽  
Dayanne Terra Tenório Nonato ◽  
Paloma Leão Sousa ◽  
Edna Maria Camelo Chaves ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Anticoagulant Activity ◽  
Adenosine Diphosphate ◽  
South American ◽  
Crude Venom ◽  
Leucocyte Migration ◽  
Ant Venom ◽  
Hymenopteran Species ◽  
Anti Inflammatory

The South American giant ant, Dinoponera quadriceps (Hymenoptera, Formicidae, Ponerinae), produces proteinaceous venom that has antinociceptive, neuroprotective and antimicrobial effects, thereby supporting the popular use of these ants to treat asthma, rheumatism, earache and back pain. Anticoagulant activity is another biological property that has been shown for the venom of other hymenopteran species, like wasps. The aim of this study was to assess the anti-inflammatory, anticoagulant and antiplatelet properties of D. quadriceps venom (DqV). DqV anti-inflammatory activity was assessed by intravenous administration in Swiss mice in the models of paw edema and peritonitis. In vitro, DqV was assessed in coagulation (activated partial thromboplastin time) and platelet aggregation tests. DqV inhibited (27–33%) the edema elicited by carrageenan and the leucocyte migration (43%) elicited by zymosan. DqV decreased by 57% and 42%, respectively, the content of malondialdehyde and nitrite in the peritoneal fluid. DqV prolonged (1.8x) the clotting time and decreased (27%) the platelet aggregation induced by adenosine diphosphate. The crude venom of D. quadriceps presents an anti-inflammatory effect in mice and in vitro anticoagulant and antiplatelet effects.

Correlation Between the Enzymatic Activity, Anticoagulant and Antiplatelet Effects of Phospholipase A2 Isoenzymes from Naja nigricollis Venom

1988 ◽  
Vol 60 (02) ◽  
pp. 170-173 ◽  
Author(s):  
R Manjunatha Kini ◽  
Herbert J Evans
Keyword(s):  
Catalytic Activity ◽  
Platelet Aggregation ◽  
Enzymatic Activity ◽  
Phospholipase A2 ◽  
Blood Coagulation ◽  
Anticoagulant Effect ◽  
Clotting Time ◽  
Inhibition Of Platelet Aggregation ◽  
Class B ◽  
Inhibitory Activities

SummaryThe three phospholipase A2 isoenzymes from Naja nigricollis crawshawii snake venom inhibit both blood coagulation and platelet aggregation. To investigate the correlation between phospholipid splitting ability of these enzymes and their inhibitory activities, the effects of various preincubation times and the inclusion of EDTA were examined. Preincubation of plasma and thromboplastin with the phospholipase isoenzymes resulted in an increase in Ca2+-initiated clotting time with time of preincubation. Incubation of the isoenzymes with EDTA before their addition to the plasma-thromboplastin mixture reduced the anticoagulant effect. These results indicate that the catalytic activity contributes at least partially to the anticoagulant effect. However, inhibition of platelet aggregation appears to be independent of enzymatic activity since there is no increase in inhibition with time of preincubation of platelets and phospholipases, and inclusion of EDTA has no significant effect on inhibition of aggregation. All three enzymes appear to belong to class B of the platelet affector PLA2 enzymes as determined by platelet effects, since they do not initiate platelet aggregation.

Characterisation and Some Properties of the Protein C Activator from Agkistrodon Contortrix Contortrix Venom

1988 ◽  
Vol 59 (01) ◽  
pp. 040-044 ◽  
Author(s):  
Thomas Exner ◽  
Riita Vaasjoki
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Apparent Molecular Weight ◽  
Single Chain ◽  
Crude Venom ◽  
Agkistrodon Contortrix ◽  
Normal Plasma ◽  
Agkistrodon Contortrix Contortrix ◽  
Contact Mechanism

SummaryThe protein C activator (PCA) detectable in the venom of Agkistrodon Contortrix Contortrix (ACCV, Southern Copperhead) by specific immunochromometric assay and anticoagulant activity has been isolated and partially characterized. Chromatog raphy of the crude venom on SP-Sephadex followed by Con A Sepharose and finally on hydroxylapatite was necessary to achieve an electrophoretically – pure product. The isolated PCA is a single chain glycoprotein with strong positive charge and an apparent molecular weight of 36,000.It had an immediate-inhibiting effect on the activated partial thromboplastin time (APTT) of normal plasma with no noticeable eilect Oii the piOtliiumbm time, its piolonging eifect oil the APTT was dependent on protein C and il appeared to interfere with the contact mechanism rather than with factors V and VIII. It had enzymatic activity on some tripeptide chromogenic substrates sensitive to thrombin and kallikrein. When mixed with normal plasma it generated activity on substrates sensitive to activated protein C and should be useful for studies of protein C.

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

The Action Mechanism of the Purified Platelet Aggregation Principle of Trimeresurus mucrosquamatus Venom

1979 ◽  
Vol 41 (03) ◽  
pp. 475-490 ◽  
Author(s):  
Chaoho Ouyang ◽  
Che-Ming Teng
Keyword(s):  
Platelet Aggregation ◽  
Adenine Nucleotides ◽  
Muscle Relaxants ◽  
Prostaglandin Synthesis ◽  
Action Mechanism ◽  
Crude Venom ◽  
Platelet Factor ◽  
Induce Platelet Aggregation ◽  
Minimal Concentration ◽  
Aggregation Principle

SummaryThe minimal concentration of the platelet aggregation principle (Platelet Aggregoserpen- tin, PAS) necessary to induce platelet aggregation was 10 ng/ml, about one-hundredth of that of the crude venom. PAS induced the release of platelet factors 3 and 4 from platelets, but the released platelet factor 3 was easily inactivated by the anti-phospholipid effect of PAS. Pretreatment of platelets with neuraminidase potentiated PAS-induced platelet aggregation. PAS-induced platelet aggregation was independent on released ADP; it could occur in the ADP-removing systems, such as apyrase or a combination of phosphoenolpyruvate and pyruvate kinase. However, PAS-induced platelet aggregation could be inhibited by adenine nucleotides and adenosine.PAS-induced platelet aggregation was inhibited by some anti-inflammatory agents, antimalarial drugs, local anesthetics, antihistamine and smooth muscle relaxants. After deaggregation of PAS-treated platelets, thrombin and sodium arachidonate could further induce platelet aggregation, but ADP and second dose of PAS could not. It is concluded that PAS-induced platelet aggregation is due to prostaglandin synthesis. Recent literatures on the mechanism of platelet aggregation were surveyed and the actions of PAS were discussed.

Impairment of Platelet Aggregation by Echis colorata Venom Mediated by L-Amino Acid Oxidase or H2O2

1982 ◽  
Vol 48 (03) ◽  
pp. 277-282 ◽  
Author(s):  
I Nathan ◽  
A Dvilansky ◽  
T Yirmiyahu ◽  
M Aharon ◽  
A Livne
Keyword(s):  
Hydrogen Peroxide ◽  
Amino Acid ◽  
Platelet Aggregation ◽  
Snake Venom ◽  
Oxidase Activity ◽  
Enzyme Preparation ◽  
Acid Oxidase ◽  
Crude Venom ◽  
Amino Acid Oxidase ◽  
Hemostatic Disorders

SummaryEchis colorata bites cause impairment of platelet aggregation and hemostatic disorders. The mechanism by which the snake venom inhibits platelet aggregation was studied. Upon fractionation, aggregation impairment activity and L-amino acid oxidase activity were similarly separated from the crude venom, unlike other venom enzymes. Preparations of L-amino acid oxidase from E.colorata and from Crotalus adamanteus replaced effectively the crude E.colorata venom in impairment of platelet aggregation. Furthermore, different treatments known to inhibit L-amino acid oxidase reduced in parallel the oxidase activity and the impairment potency of both the venom and the enzyme preparation. H2O2 mimicked characteristically the impairment effects of L-amino acid oxidase and the venom. Catalase completely abolished the impairment effects of the enzyme and the venom. It is concluded that hydrogen peroxide formed by the venom L-amino acid oxidase plays a role in affecting platelet aggregation and thus could contribute to the extended bleeding typical to persons bitten by E.colorata.

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