Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Platelets release large quantities of plasminogen activator inhibitor 1 (PAI-1) that plays an important role in maintaining the integrity of fibrin-rich thrombi. In addition, tissue-type plasminogen activator (t-PA), a key physiological regulator of fibrinolysis, has been detected in platelet alpha-granules at low abundance. This information raises the possibility of enhancing t-PA expression in megakaryocytes as a means to enhance the fibrinolytic properties of platelet alpha-granules and target PAs directly to fibrin clots. This study was initiated to investigate adenovirus (Ad)-mediated expression and packaging of t-PA into alpha-granules-like structures in the megakaryocytic cell line MEG-01. Ad/t-PA infection of phorbol myristate acetate (PMA)-differentiated MEG-01 cells increased cellular t-PA levels by 120 fold (1580 +/- 130 ng/10(6) cells at 5 MOI) in comparison to non-or Ad/beta-gal-infected cells. Fluorescence-activated cell sorter (FACS) analysis indicates that Ad/t-PA-infected cells yielded a homogenous shift in the t-PA staining profile with a 4-fold shift in mean fluorescence in comparison to non- or Ad/beta-gal-infected cells. For the isolation of alpha-granule-like structures, MEG-01 cell homogenates were fractionated by differential centrifugation and two consecutive Percoll density gradients. Fibrin autography of storage granules revealed a prominent lytic zone at Mr 66 kD comigrating with free t-PA. Quantitative analyses indicate that a 16-fold elevation in t-PA antigen within storage granules in comparison to non- or Ad/beta-gal-infected cells. To document the ability of t-PA to be stored in a rapidly-releasable form in these cells, we isolated platelet-like particles from the supernatant of differentiated cells and determined that particles from Ad/t-PA-infected cells display a 4-8 fold enhanced secretion of t-PA following treatment with the clasical secretagogue calcium ionphore 23187, ADP, or thrombin. Confocal immunofluoresence microscopy analysis indicates that Ad/t-PA mediated productive expression of t-PA in murine megakaryocytes. These data provide support for the concept of increasing the expression of t-PA in megakaryocytes as a means to alter the hemostatic properties of alpha-granules.
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PMID:Adenovirus-mediated expression and packaging of tissue-type plasminogen activator in megakaryocytic cells. 1143 88

To investigate the role of active plasminogen activator inhibitor 1 (PAI-1) in the evolution of a microthrombus generated in the arteriolar microcirculation, the monoclonal antibody, 33H1F7, which transforms active PAI-1 to a tissue type plasminogen activator (t-PA) substrate, was evaluated in an arteriolar thrombosis model in the rat mesentery. Arterioles (200-300 um) were stimulated electrically to create an endothelial lesion; ADP was then perfused for 2 min to induce the formation of a platelet-rich thrombus which lysed spontaneously in 140 +/- 24 s. Two successive ADP superfusions produced comparable thrombi which lysed in comparable times. Different doses of 33H1F7 were infused to rats for 30 min and the dose which inactivates rapidly and totally active rat PAI-1 (300 microg/kg/min) was selected to be tested on the thrombosis model. Infusion of 33H I F7 beginning 10 min before the ADP application significantly reduced the lysis time in comparison to the control (123 +/- 30 s versus 169 +/- 33 s, P < 0.05, paired Student's t-test) and the cumulative thrombus area during the lysis period was decreased by 56 +/- 7%. These results demonstrate that inactivation of PAI-1 is able to accelerate lysis of a platelet-rich clot in a mesenteric arteriole of the rat. Thus active PAI-1 most likely participates to the resistance to thrombolysis in the arteriolar microcirculation and its inactivation may shorten ischemic periods after microvascular obstruction such as e.g. during cerebral stroke.
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PMID:Inactivation of plasminogen activator inhibitor-1 accelerates thrombolysis of a platelet-rich thrombus in rat mesenteric arterioles. 1177 23

The effect of a new domestic polyunsaturated fatty acid (PUFA) concentrate called epaden on the blood coagulation system was studied in comparison with acetylsalicylic acid (ASA). The antithrombotic potential of the blood vessel wall was determined by release of the inhibitors of thrombocyte aggregation, fibrinolysis activators, and anticoagulants in rabbits under immobilization induced stress conditions. In the control group, the immobilization stress resulted in a decrease of the collagen-induced platelet aggregation, a drop in the fibrinogen level, and an increase in the tissue-type plasminogen activator (t-PA) activity. The administration of ASA and epaden reduced a drop in the fibrinogen level caused by the immobilization stress. Animals receiving epaden showed a decrease in the ADP-induced platelet aggregation and an increase in the t-PA activity in comparison with the levels before modeling stressed conditions. No such effect was observed in the group treated with ASA. It is suggested that the additional antithrombotic effect of epaden observed under the immobilization stress conditions is related to a protective action of this substance on the vessel wall.
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PMID:[Effect of chronic administration of epaden and acetylsalicylic acid on the antithrombotic potential of blood vessel walls in rabbits]. 1202 86

A phospholipase A(2) (PLA(2)), called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 micro mol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 micro g/ml enzyme was incubated for 90 min in the presence of PC and Ca(2+). No detectable hemolysis was noticed when PC was not added. Ca(2+) was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca(2+) was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC(50) of 1.0 micro M. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D 49 PLA(2)s. Also, the residues concerned to Ca(2+) binding were conserved. This is the first report of cDNA sequence of T. jerdonii venom PLA(2).
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PMID:Characterization and cloning of a novel phospholipase A(2) from the venom of Trimeresurus jerdonii snake. 1222 Jul 17

Expression of tissue factor (TF) by activated monocytes may initiate thrombotic episodes associated with diseases, such as thrombosis and atherosclerosis. In this study, steps in the regulatory pathways of lipopolysaccharide (LPS)-induced monocyte TF activity and released TNF-alpha in human whole blood were probed for using an array of inhibitors, comprising specific inhibitors of cytosolic phospholipase A(2) (PLA(2)) (AACOCF(3)), secretory PLA(2) (SB-203347), protein kinase (PK) (staurosporine), PKC (GF-109203; BIM), and serine protease (Pefabloc SC), antagonists of thromboxane prostanoid (TP) receptor (R) (SQ-29548), platelet activating factor (PAF) R (BN-52021), leukotriene B(4) R (SC-41930), serotonin R (cyproheptadine), fibronectin/fibrinogen R (RGDS), and finally, creatine phosphate/creatine phosphokinase (CP/CPK) which removes ADP. Whereas when added alone neither of these agents significantly inhibited LPS-induced TF or TNF-alpha, when presented as a reference cocktail comprising all the agents, TF activity and TNF-alpha were reduced by 77% and 49%, respectively. By subsequently testing a series of incomplete inhibitory cocktails equal to the reference except for deleted single agents or combinations of two or three active agents, the inhibitory effect of the reference cocktail could be shown to depend on the presence of the protease inhibitor and the thromboxane A(2) and PAF antagonists.
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PMID:The central role of thromboxane and platelet activating factor receptors in ex vivo regulation of endotoxin-induced monocyte tissue factor activity in human whole blood. 1223 Sep 18

A phospholipase A(2)(PLA(2)) was purified from the venom of the snake Trimeresurus stejnegeri Schmidt by Sephadex G-75 gel filtration, Mono Q ion exchange chromatography and Superose-12 gel filtration with FPLC and proved to be homogeneous as shown in SDS-PAGE and IEF. Its molecular weight is around 18 000 and isoelectric point is 4.7. Amino acid composition of PLA(2) was determined. Apart from hydrolyzing phosphatidylcholine, the enzyme has a potent inhibitory effect on platelet aggregation induced by ADP or collagen with half-inhibitory concentration of 10-15 &mgr;g/ml and 50-80 &mgr;g/ml respectively.
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PMID:Purification and Characterization of Phospholipase A(2) from the Venom of Snake Trimeresurus stejnegeri Schmidt. 1223 3

The effects of progestogens on hemostatic function seems to be minimal, however, in this longterm surveillance study of the effects of Norplant among 100 healthy, nonsmoking, nonalcohol drinking, and nonlactating Singaporean women indicates the possibility of an increased predisposition of thrombosis. Clinical assessment and blood sampling were taken prior to insertion and at 6, 12, 24, 36, and 48 months. Laboratory tests included prothrombin time (PT), activated partial thromboplastic time (APTT), hematocrit and platelet count as hemostatic measures. Also measured were fibrinogen, coagulation factor II, a 1-stage assay for factors V and VII, factor VII, factor X, antithrombin III, plasminogen activator activity on the fibrin plate and FDP, platelet aggregation, factor VIIIR. 20 normal controls not on any medication were used and international standards were applied in most cases. The coefficients of variation of the various tests with reference control plasma or sera are summarized. The paired t test was used to assess statistical differences. Skewed results were analyzed with the Wilcoxon signed rank test. % changes between pre- and postinsertion levels were also utilized. The results were that the hemoglobin concentration increased from 12.1 gm/dl to 13.4 gm/dl in the 1st year, and then decreased to preinsertion levels within 3 years, and increased to 13.3 gm/dl in the 4th year. The 4th year also reflected no menstrual disorder. PT and APTT appeared significantly shortened in year 1. PT continued to shorten in 3 more years, while APTT increased to the preinsertion mean. Vitamin K dependent factors II and VII decreased significantly in year 1, while factors V and X increased significantly. Factor II increased minimally within 2 more years, but decreased again in year 4. Factor VII continued to decrease over the 4 years. The concentration of factors II and VII were significantly lower after 4 years. Factor V increased 14.4% in year 1, but decreased below preinsertion levels in the next 3 years, and then decreased to preinsertion levels in year 4. No significant changes in fibrinolytic activity occurred in 4 years. Major changes occurred in mean platelet count, which rose significantly in year 1 and increased concentration in year 4. In 4 years, platelet aggregation using ADP or equine collagen rose significantly. Serious thought must be given to the risk of thrombosis and potential for hypercoagulation.
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PMID:Haemostatic function changes in Singapore women using Norplant implants: a four year review. 1228 17

Phospholipase D (PLD) is present in human placental tissue. Since purinergic receptor agonists activate PLD in many different cell types, we evaluated the purinergic activation of the enzyme in cultured trophoblasts from the placenta. We found that P(2) receptor agonists stimulate PLD. The preferred ligand for P(2X7) (P(2Z)) receptor subtype, BzBz-ATP (10(-3)M ), induced the enzyme more than ten times over basal (unstimulated) activity, while ATP caused a much smaller increase. ATPgammaS, ADP and UTP were even less effective, compared to BzBz-ATP or ATP. AMP and alpha,beta-methyl-ATP, a P(2X) agonist that is uniquely inactive on the P(2X7) subtype, had no effect. This represents the first suggestion of the presence of the P(2X7) type of receptor in human trophoblasts that was directly confirmed by immunoblot detection. The action of BzBz-ATP was dependent upon the presence of calcium in the culture medium and was inhibited by high (5m M ) Mg(++) concentration. P(2X7) receptor subtype specific antagonists, ATP-2',3'-dialdehyde (o-ATP), CBB and the broad specificity P(2) inhibitor PPADS inhibited the effect of BzBz-ATP. Pertussis toxin treatment did not inhibit the effect. Down-regulation of cPKC/nPKC isoforms by prolonged PMA treatment (36 h, 10(-7)M ) prevented the stimulation of PLD by P(2) agonists or the calcium ionophore A-23187. PLA(2) inhibitors did not block the effect of BzBz-ATP. The possibility for a calcium influx related interdependence of PLC and PLD was evaluated. For PLC activation, UTP and ATP surpassed BzBz-ATP, while ionophore did not elevate PLC (assessed by IP(3) measurements). This suggested the predominance of a P(2Y2) receptor in the whole cell in gross activation of PLC. PLD was affected with a reversed order of potency. These results and the dependence of PLD on PKC activity implies that a restricted, membrane localized calcium flux activates PKC and in turn, mediates the P(2X7) dependent stimulation of PLD. This may have implications for physiologic regulation of trophoblast function.
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PMID:Regulation of phospholipase D in human placental trophoblasts by the P(2) purinergic receptor. 1236 78

P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP, regulating several physiologic responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. P2 receptor characterization has been hampered by the lack of selective antagonists. The aim of the current study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and in endothelial cells (EC). Smooth muscle cells were obtained from human mammary artery and EC from human umbilical vein. Using real-time PCR, the authors established quantitative mRNA assays. Protein expression was studied using Western blotting with recently developed antibodies. The P2X1 receptor was highly specific for human SMC, while the P2X4 was the highest expressed receptor in EC. The P2Y2 receptor was present in both SMC and EC. UTP-mediated effects in these cells are likely to be mediated by P2Y2 and not P2Y4 receptors since the latter had considerably lower expression. The P2Y6 receptor was expressed in both SMC and EC. The P2Y1 and surprisingly the P2Y11 receptors were the most abundantly expressed P2Y receptors in the endothelium. Overall, Western blotting confirmed the mRNA findings in most aspects, and most interestingly, indicated oligomerization of the P2Y1 receptor that may be important for its function. In conclusion, P2X1, P2Y2, and P2Y6 are the most expressed P2 receptors in SMC and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and are most likely mediating release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development against the large P2 receptor family.
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PMID:P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. 1245 17

In a previous report we showed that Lachesis muta crude venom displays potent indirect hemolytic activity and myotoxicity when injected into mice. Then, a phospholipase A(2) (PLA(2)) (LM-PLA(2)-I) responsible for these activities was isolated. More recently, a catalytically active isoenzyme (LM-PLA(2)-II) with molecular mass of 18 kDa and isoeletric point at pH 5.4 was isolated from the same snake venom. LM-PLA(2)-II inhibited ADP- and collagen-induced platelet aggregation as well as induced a potent paw edema reaction in rats. Here we show that LM-PLA(2)-II induced myotoxic effects both in vitro characterized by an increase on the rate of creatine kinase (CK) release from isolated mice extensor digitorum longus (EDL) muscles and in vivo by increasing plasma CK activity of injected mice. Histological analysis showed an intense damage in muscle cells injected with LM-PLA(2)-II. It was also shown that exogenous lysophosphatidylcholine (lyso-pc) behaved as a typical myotoxin damaging muscle cells, producing myonecrosis characterized by local infiltration of inflammatory cells similarly to that observed for LM-PLA(2)-II. Hemorrhage and lethal effects were not observed neither with LM-PLA(2)-II nor lyso-pc. As previously observed for other biological activities, pretreatment of LM-PLA(2)-II with p-bromophenacyl bromide (p-BPB) or acetic anhydride abolished all the enzyme's actions. The data confirms that biological activities displayed by LM-PLA(2)-II, including the myotoxic effects reported here, are all dependent on its enzymatic activity where the product formed (lyso-pc) may play an important function on such myotoxicity.
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PMID:Myotoxicity induced by an acidic Asp-49 phospholipase A(2) isolated from Lachesis muta snake venom. Comparison with lysophosphatidylcholine. 1281 42


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