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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A remarkable variation in monocyte activation among individuals was observed when blood from different people was incubated with lipopolysaccharides. To elucidate this phenomenon, we studied intracellular signals associated with monocyte activation. This was done by measuring induced thromboplastin synthesis. An inhibitor of phospholipase A2 blocked the lipopolysaccharide induced synthesis of thromboplastin. Thus, release of arachidonic acid (20: 4) seemed to be necessary to activate the monocytes. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, had no effect on the monocyte activation in subjects with a low response to lipopolysaccharides (low responders); this contrasted with nearly 80% inhibition in individuals with very sensitive cells (high responders). Taking aspirin raised monocyte activation by an average of 50%, this was caused by the effect of aspirin on the platelets. Platelets enhanced the lipopolysaccharide activation of monocytes 2-3 fold. The high response phenomenon was partially due to platelets. When platelets in the blood of high responders were substituted with platelets from low responders, the monocyte activation fell by up to 70%. Fatty acids seemed to play a central role in the activation of monocytes. Intake of cod liver resulted in significant reduction of induced thromboplastin synthesis. It is suggested that those who are high responders may be more susceptible to developing atherosclerosis.
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PMID:Fatty acids, platelets and monocytes. Something to do with atherogenesis. 292 3

The placental protein PP41,2 was shown to have thromboplastin-inhibitor activity. We used partial amino acid sequence information from PP4 cyanogen bromide fragments to design oligonucleotide probes for the screening of a human placental cDNA library. In addition to the PP4 cDNA we isolated a cDNA coding for a protein with considerable homology which we subsequently termed PP4-X. PP4 and PP4-X belong to the phospholipase A2 inhibitor family, as judged by their homology to lipocortin I and calpactin I3. The full-length PP4-X cDNA encodes a protein of 321 amino acid residues including a fourfold repeat structure. Northern blot analysis using the PP4-X cDNA reveals two hybridizing RNA species of approximately 1400 nucleotides and 2500 nucleotides, respectively. The shorter one could well represent the PP4-X transcript which is in good agreement with the isolated cDNA insert of 1326 nucleotides. Expression of the PP4-X coding sequence in E. coli resulted in the appearance of a protein which crossreacts with antibodies raised against PP4.
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PMID:Isolation and expression of cDNA coding for a new member of the phospholipase A2 inhibitor family. 297 Feb 57

The 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.
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PMID:Correlation between the enzymatic activity, anticoagulant and antiplatelet effects of phospholipase A2 isoenzymes from Naja nigricollis venom. 314 38

Exposure of phospholipids at the outer surface of activated and control platelets was studied by incubation with a mixture of phospholipase A2 from Naja naja and bee venom, solely or in combination with sphingomyelinase from Staphylococcus aureus, using conditions under which cell lysis remained below 10%. Incubation with phospholipase A2 alone revealed a markedly increased susceptibility of the phospholipids in platelets activated by a mixture of collagen plus thrombin, by the SH-oxidizing compound diamide, or by calcium ionophore A23187, as compared to control platelets or platelets activated separately by collagen or thrombin. Collagen plus thrombin, diamide, and ionophore treated platelets revealed an increased exposure of phosphatidylserine at the outer surface accompanied by a decreased exposure of sphingomyelin, as could be concluded from incubations with a combination of phospholipase A2 and sphingomyelinase. These alterations were much less apparent in platelets activated either by thrombin or by collagen alone. The increased exposure of phosphatidylserine in activated platelets is accompanied by an increased ability of the platelets to enhance the conversion of prothrombin to thrombin by coagulation factor Xa, in the presence of factor Va and calcium. It is concluded that the altered orientation of the phospholipids in the plasma membrane of platelets activated by collagen plus thrombin, by diamide, or by calcium ionophore, is the result of a transbilayer movement. Moreover, the increased exposure of phosphatidylserine in platelets stimulated by the combined action of collagen and thrombin might be of considerable importance for the hemostatic process.
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PMID:Changes in membrane phospholipid distribution during platelet activation. 641 5

Human isolated monocytes possess low levels of procoagulant activity, which was stimulated 10-30 fold by brief (2 hr) exposure to 10 micrograms/ml endotoxin. This activity was expressed in normal or factor XII-deficient plasma, but lost in plasma deficient in factors X or VII, indicating that it was due to thromboplastin. The stimulation of monocyte thromboplastin by endotoxin was inhibited in a dose-dependent manner by two phospholipase A2 inhibitors, 4-bromophenacyl bromide and quinacrine, and by two lipoxygenase inhibitors, eicosatetraynoic acid and nordihydroguaiaretic acid. Two cyclooxygenase inhibitors, aspirin and indomethacin, prevented endotoxin-induced increases in thromboxane B2 production but had no effect on thromboplastin production. These results suggest that a component in the sequence of lipid deacylation, arachidonic acid release, and metabolism via lipoxygenase may mediate the stimulation of monocyte thromboplastin activity by endotoxin.
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PMID:Effects of inhibitors of arachidonic acid metabolism on thromboplastin activity in human monocytes. 642 35

An "A1 type" phospholipase activity with serine-phospholipid preference was released by rat activated platelets. It was distinct from the secretory type II phospholipase A2 [Horigome, K., Hayakawa, M., Inoue, K., and Nojima, S. (1987) J. Biochem. 101, 625-631] and co-purified with the secretory lysophosphatidylserine-selective lysophospholipase activity [Higashi, S., Kobayashi, T., Kudo, I., and Inoue, K. (1988) J. Biochem. 103, 442-447]. Several lines of evidence indicated that a single protein was responsible for the phospholipase A1 and lysophospholipase activities. Marked accumulation of lysophospholipids was observed in rat calcium ionophore-activated washed platelets and both phospholipase A1/lysophospholipase and type II phospholipase A2 were shown to contribute to this phospholipid degradation. A selective inhibitor of type II phospholipase A2 reduced the phospholipid degradation and enhanced the clotting time and prothrombinase activity. These results indicate that secretory platelet phospholipases may play a role in regulation of blood clotting.
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PMID:Phospholipid degradation in rat calcium ionophore-activated platelets is catalyzed mainly by two discrete secretory phospholipase As. 749 Feb 72

It has been suggested (Kini, R. R., and Evans, H. J. (1987) J. Biol. Chem. 262, 14402-14407) that the anticoagulant activity of members of the 14-kDa phospholipase A2 (PLA2) family depends on the presence of basic residues within a variable surface region (residues 54-77) distinct from both the conserved catalytic machinery and surface sites mediating the antibacterial action of these enzymes (see Weiss, J., Inada, M., Elsbach, P., and Crowl, R. M. (1994) J. Biol. Chem. 269, 26331-26337). To further define the determinants of the anticoagulant activity of PLA2, we have analyzed the inhibitory effects of purified native and recombinant PLA2 on cell-free prothrombinase. Both native and recombinant wild-type pig pancreas (net charge -1) and human "secretory" PLA2 (net charge +15) produced similar dose-dependent inhibition of prothrombinase activity that was significantly less potent than a toxic PLA2 purified from snake venom. Site-specific mutations that either increased or decreased PLA2 activity toward bactericidal/permeability-increasing protein-treated Escherichia coli by up to 50-fold had no effect on antiprothrombinase activity. In contrast, substitution of Arg for Asp-59/Gly for Ser-60 in the pig PLA2 increased antiprothrombinase activity by 5-10-fold without affecting catalytic activity toward a range of phospholipid substrates or antibacterial activity. Comparison of antiprothrombinase activity of catalytically active and inactive forms of the PLA2 and under a range of phospholipid conditions revealed that the potent antiprothrombinase activity of native toxic venom PLA2 and of the D59R.S60G mutant pancreatic PLA2 reflect combined catalytic and noncatalytic actions, the latter apparently dependent on basic residues at discrete surface sites in the enzyme.
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PMID:Determinants of the inhibitory action of purified 14-kDa phospholipases A2 on cell-free prothrombinase complex. 792 51

A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.
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PMID:Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli. 822 27

Vesicles that do not contain spectrin were released from human erythrocytes by incubation with dimyristoylphosphatidylcholine. The transbilayer orientation of membrane phospholipids was subsequently determined by two independent methods. Incubation with phospholipase A2 revealed that the phospholipid asymmetry observed in red blood cells was essentially preserved in vesicles. By use of the prothrombinase assay a still highly asymmetric distribution of phosphatidylserine could be demonstrated in spite of its slightly increased exposure on the vesicle surface. These results show that membrane phospholipid asymmetry can be maintained in a system that does not contain an intact membrane skeleton or spectrin.
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PMID:Membrane phospholipid asymmetry in DMPC-induced human red cell vesicles. 822 45

Extracellular phospholipase A2 is secreted from the platelets upon activation by a stimulus such as thrombin. The secreted enzyme has been recently cloned and the recombinant protein produced. Snake venom PLA2 effect on platelet and coagulation has been extensively studied (for review see 3,4) and it has been proposed that the anticoagulant phospholipases may inhibit coagulation by competing with clotting proteins for the lipid surface. Structure function relationship for PLA2s with anticoagulant activity indicates that the activity is conferred by positively charged aminoacids between residues 54 and 77. The corresponding segment of human recombinant secreted platelet PLA2 (r-hnps-PLA2) possesses five positively charged aminoacids in this region being at the identical positions to those of PLA2s with known anticoagulant activities. Here using human and rat plasma we have demonstrated for the first time that the recombinant human extracellular secreted platelet PLA2 increases activated partial thromboplastin time but not prothrombin time.
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PMID:Human recombinant non pancreatic secreted platelet phospholipase A2 has anticoagulant activity in vitro on human plasma. 833 63


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