Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Crude 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-mercaptoethanol, 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, lysophosphatidylcholine 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.
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PMID:Mechanism of inhibitory action on platelet activation of a phospholipase A2 isolated from Lachesis muta (Bushmaster) snake venom. 940 22

Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 (6 H)-tetrol) inhibited thrombin-,collagen- and ADP-induced aggregation of washed rat platelets. Thrombin- and collagen-induced ATP release were also inhibited by brazilin in a concentration-dependent manner. Brazilin inhibited the formation of platelet thromboxane A2 caused by thrombin, whereas it had no effect on the prostaglandin D2 formation. Brazilin inhibited [3H]-arachidonic acid liberation from membrane phospholipids of thrombin-stimulated platelets. Brazilin inhibited the rise of intracellular free calcium caused by thrombin. These results indicate that the inhibition of phospholipase (PLA2) activity and [Ca2+]i elevation might be at least a part of antiplatelet mechanism of brazilin.
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PMID:Effects of Brazilin on the phospholipase A2 activity and changes of intracellular free calcium concentration in rat platelets. 986 55

The alphaII(b)beta3 integrin and FcgammaRII receptors mediate, respectively, platelet adhesion and spreading on fibrinogen and immunoglobulin (IgG) coated surfaces. Platelet adhesion to fibrinogen resulted in a partial conversion of the faster to the slower migrating (phosphorylated) form of Ca(+2)-sensitive cytosolic phospholipase A2(cPLA2) but failed to trigger arachidonic acid (AA) release. Full mobility shift of cPLA2 and a massive release of AA release were stimulated by platelet adhesion to IgG or addition of thrombin to the fibrinogen adherent platelets. IgG and thrombin induced AA production were blocked by methyl arachidonyl fluorophosphonate (MAFP), an irreversible inhibitor of cPLA2 and the Ca(+2)-independent phospholipase A2 (iPLA2). In contrast, bromoenol lactone (BEL), a specific inhibitor of iPLA2 had no effect on the release of AA. MAFP and BEL prevented pp125FAK phosphorylation and platelet spreading on fibrinogen having no effect on pp125FAK phosphorylation or platelet spreading on immobilized IgG. We conclude that alpha(IIb)beta3-mediated pp125FAK phosphorylation and platelet spreading on fibrinogen are regulated by PLA2 enzymes.
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PMID:Phospholipase A2 enzymes regulate alpha IIb beta3-mediated, but not Fc gammaRII receptor-mediated, pp125FAK phosphorylation in platelets. 1023 50

Several biochemical and biological activities such as phospholipase A2, arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA2) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA2 had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA2 was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.
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PMID:Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A2. 1182 58

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph1P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. 1) Platelet phospholipids were labeled using [32P]orthophosphate, and the production of [32P]Sph1P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph1P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from platelets in vitro. Only 10% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA1 and PLA2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A1 and A2 and then to LPA by plasma lysophospholipase D.
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PMID:Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood. 1192 70

Bites by the Indian cobra (Naja naja naja) are common in India and Sri Lanka because of its close association with humans. Cobra venoms are complex and contain several toxic components, including neurotoxins that cause post-synaptic neuromuscular blockade with respiratory paralysis and even death. Bites may also cause extensive local necrosis by mechanisms not fully elucidated. Although no significant coagulopathy has been reported, N.n. naja venom can form blood clots in vitro by activating prothrombin as demonstrated by thrombin-specific chromogenic substrate. Scanning electron microscopy demonstrates that the clots formed by venom lack the thin fibrin strands of normal blood clots formed by thromboplastin or glass contact. Rheometry shows that clots formed by venom have abnormally low elasticity over an extended period and then, as the platelets contract, a retarded and more feeble increase in elasticity. Purified N.n. naja venom PLA2 inhibits platelet aggregation in PRP and explains the decreased clot retraction and retarded and compromised elasticity build up. The present study shows that the PLA2 and the prothrombin activator from N.n. naja venom have effects on haemostasis and blood clotting, although such effects are not observed systemically in envenomed humans.
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PMID:In vitro procoagulant and anticoagulant properties of Naja naja naja venom. 1455 74

We have previously demonstrated that thrombin stimulation of endothelial cells results in increased membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) activity, accelerated hydrolysis of membrane plasmalogen phospholipids, and production of several biologically active phospholipid metabolites, including prostacyclin and platelet-activating factor (PAF) that is abolished by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. This study was designed to further investigate the role of alternative PLA2 inhibitors, including methyl arachidonyl fluorophosphonate (MAFP, an inhibitor of cytosolic PLA2 isoforms), on phospholipid turnover and PAF production from thrombin-stimulated human coronary artery endothelial cells (HCAECs). Paradoxically, pretreatment of HCAEC with MAFP (5-25 microM) resulted in a significant increase in PAF production in both unstimulated and thrombin-stimulated cells that was found to be a direct result of inhibition of PAF acetylhydrolase (PAF-AH) activity. Pretreatment with MAFP did not significantly inhibit HCAEC PLA2 activity, possibly due to the localization of PLA2 activity in the membrane fraction rather than the cytosol. Bromoenol lactone did not inhibit PAF-AH activity, even at concentrations as high as 20 microM. We conclude that MAFP augments thrombin-stimulated PAF production by inhibition of PAF catabolism without affecting membrane-associated iPLA2 activity.
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PMID:Inhibition of platelet-activating factor (PAF) acetylhydrolase by methyl arachidonyl fluorophosphonate potentiates PAF synthesis in thrombin-stimulated human coronary artery endothelial cells. 1456 38

Thrombin exerts multiple actions on cardiomyocytes leading to increased intracellular Na+ and Ca2+ concentrations, and to activation of a Ca2+-independent PLA2, and has been proposed to favor the genesis of arrhythmias and ischemic injury in acute coronary syndromes. However, the influence of thrombin on cardiomyocyte cell death during ischemia-reperfusion has not been studied. A beneficial influence of low thrombin concentrations has been described in other cell types. HL-1 cardiomyocytes were subjected to simulated ischemia (SI) and reperfusion (SR) and cell death was assessed by means of LDH release to the incubation media. Thrombin dose-dependently increased cell death in normoxic cells, in cells subjected to SI, and in cells subjected to SR (by 20+/-8%, 95+/-32% and 35+/-9%, respectively, at 100 U/ml). The effects of thrombin were associated to increased cytosolic Ca2+ overload, mimicked by 100 microM PAR-1 agonist peptide SFLLRNPNDKYEPF, and reversed by the direct thrombin inhibitor lepirudin (IC50=1.3+/-0.2 microg/ml). The presence of thrombin during simulated ischemia-reperfusion increases cardiomyocyte cell death by a mechanism that involves activation of PAR-1 receptors and can be prevented by the direct thrombin inhibitor lepirudin.
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PMID:Thrombin increases cardiomyocyte acute cell death after ischemia and reperfusion. 1603 7

Snake venoms contain saccharide-binding lectins. In this work, we examined the biological activities of a lectin (BjcuL) purified from Bothrops jararacussu snake venom by chromatography on non-derivatized Sepharose 4B and Sephacryl S-200 HR. The protein, a homodimer with subunits of 14.5 kDa, gave a single immunoprecipitin line in immunoelectrophoresis and cross-reacted in ELISA with antivenoms raised against Bothrops spp. (lanceheads), Micrurus spp. (coral snakes), Crotalus durissus terrificus (South American rattlesnake), and arthropod (Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus) venoms. BjcuL agglutinated human formaldehyde-fixed erythrocytes at > or = 100 ng/ml and was inhibited by lactose and EDTA (> or = 2 mM) and high concentrations (> 100 mM) of glucose and sucrose, but not by N-acetylglucosamine. BjcuL had no direct hemolytic activity and was devoid of esterase, PLA2 and proteolytic activities. The lectin (up to 200 microg/ml) did not aggregate human platelet-rich plasma (PRP) or washed platelets (WP), nor did it alter the aggregation induced by ADP in PRP or by thrombin in WP. When injected into mouse hind paws, BjcuL (10-100 microg/paw) caused edema and increased vascular permeability, with a maximum effect after 1h that persisted for up to 6 h (edema) or gradually decreased after the peak interval (vascular permeability). No hemorrhage was observed in BjcuL-injected paws. In anesthetized rats, B. jararacussu venom (200 microg/kg, i.v.) produced sustained hypotension (maximum decrease of approximately 60%) whereas a similar dose of BjcuL decreased the blood pressure by approximately 15%, with a rapid return to the resting level.
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PMID:Biological activities of a lectin from Bothrops jararacussu snake venom. 1630 23

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 microM/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.
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PMID:Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom. 1672 48


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