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)

A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100-solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde-fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard-Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.
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PMID:A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen. 380 72

We compared the effects of phorbol 12-myristate 13-acetate (PMA) and thrombin with those of nonlytic concentrations of reactive oxygen species (ROS) generated by hypoxanthine (HX)-xanthine oxidase (XO) on the adhesion properties of human umbilical cord vein endothelial cells (HUVEC) to resting polymorphonuclear neutrophils (PMN). PMN adherence to HX-XO-treated HUVEC was increased approximately twofold to 2.5-fold relative to untreated HUVEC, both immediately and after 2 hours. It was not additive to that induced by PMA or thrombin stimulation of HUVEC. ROS-induced adherence was not due to platelet-activating factor (PAF) or P-selectin expression, as it was neither antagonized by BN52021 (PAF receptor antagonist) nor inhibited by anti-P-selectin monoclonal antibody (MoAb), contrary to the increased adhesion of PMA- and thrombin-stimulated HUVEC. PMN preincubated with mannose-6-P or N-acetylneuraminic acid (sialic acid), but not mannose or galactose-6-P, showed reduced adherence to ROS-treated HUVEC, suggesting that carbohydrate molecules were expressed on the latter and served as the ligand for the PMN L-selectin. Intercellular adhesion molecule (ICAM-1), constitutively present on the surface of resting HUVEC, was involved in the PMN adherence to ROS-treated HUVEC, since this adherence was inhibited by anti-ICAM-1, anti-CD11a, anti-CD11b, and anti-CD18 MoAbs. A non-CD18, non-ICAM-1-dependent mechanism is also involved in this adherence, since effects of these MoAbs were not additive; moreover, combinations of anti-CD18 and anti-ICAM-1 MoAbs with mannose-6-P and sialic acid completely inhibited PMN adherence. The increased binding of PMN to HX-XO-exposed HUVEC observed here involved IC-AM-1, but was independent of its upregulation, and another non-ICAM-1-dependent mechanism, in which carbohydrates expressed on HUVEC recognize L-selectin on PMN.
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PMID:Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable upregulation. 751 10

TNF is a strong secretagogue for surface-contacting neutrophils. During inflammation, endothelium offers the first substrate for neutrophil adherence and for modulation of the toxic response of neutrophils to soluble agonists such as TNF. In this in vitro study, evidence is presented that endothelium participates actively in TNF-induced neutrophil respiratory burst activity by expressing platelet-activating factor (PAF) in response to initial neutrophil H2O2 release. Three findings are shown that favor such a mechanism. First, PAF receptor antagonists reduced H2O2 release by TNF-activated neutrophils placed on endothelium approximately by 50%, whereas H2O2 responses by neutrophils placed on serum-coated polystyrene remained intact. Second, preincubation of HUVEC with known PAF-inducing agents PMA, H2O2, and thrombin, followed by fixation, enhanced neutrophil H2O2 release in response to TNF. H2O2 release by these neutrophils was sensitive to the presence of PAF receptor antagonists, whereas H2O2-release from neutrophils placed on fixed nonactivated endothelial cells was not. Finally, replacing endothelium by monolayers of human renal cortical epithelial cells and human fibroblasts, cells that are known to produce less PAF than endothelial cells, reduced the effect of PAF receptor antagonists. P-selectin expression and IL-8 release, two other ways by which endothelial cells might influence H2O2-release by TNF preincubated neutrophils, were examined in parallel, and were found not to influence TNF-induced neutrophil H2O2-release. We conclude that during neutrophil-endothelial interaction in inflammation, endothelium modulates the toxic response of neutrophils to TNF. Endothelial cell-associated PAF, but not endothelial cell IL-8 release and P-selectin expression, is likely to participate in TNF-induced neutrophil respiratory burst activity.
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PMID:Endothelial cell associated platelet-activating factor (PAF), a costimulatory intermediate in TNF-alpha-induced H2O2 release by adherent neutrophil leukocytes. 752 2

Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS.
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PMID:Platelet activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis. 754 96

The effect of a new PAF (platelet activating factor; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoryl-choline) receptor antagonist, PCA-4248 (2-phenylthio)ethyl-5-metoxycarbonyl-2,4,6-trimethyl-1, 4-dihydropyridine-3-carboxylate), on phosphoinositide turnover evoked by PAF was investigated. PAF treatment resulted in an increased 32P incorporation into phosphoinositides and phosphatidic acid in rabbit platelets. Treatment with PCA-4248 abolished both effects in a dose-dependent manner, 10 microM being the most effective dose. In thrombin stimulated platelets, phosphoinositide turnover was not influenced by PCA-4248. In addition, PAF caused a rapid and significant increase in protein phosphorylation. Thus, PAF treatment resulted in a marked phosphorylation of two proteins of 47 kDa and 20 kDa. Treatment with PCA-4248 resulted in an inhibition of these actions. Serotonin secretion evoked by PAF was also inhibited by PCA-4248. It is concluded that PCA-4248 antagonizes the PAF effects by acting as a competitive antagonist at the PAF receptor level as evidenced from binding studies.
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PMID:PCA-4248, a PAF receptor antagonist, inhibits PAF-induced phosphoinositide turnover. 758 12

Platelet-activating factor (PAF) is known to modulate polymorphonuclear leukocyte (PMN) adhesion to endothelial cells cultured under static conditions and activated by thrombin. In contrast, there are no data on the role of PAF in PMN adhesion to cells exposed to flow conditions and activated by stimuli other than thrombin. Here we used the PAF receptor antagonist L-659,989 to evaluate PMN adhesion to human umbilical vein endothelial cells (HUVEC) in basal conditions or upon challenge with thrombin or tumor necrosis factor-alpha (TNF-alpha). Experiments were performed under dynamic flow using a parallel-plate flow chamber and a computer-based image analysis system. Rolling and adhesion of PMNs to endothelial cells significantly increased upon stimulation with thrombin. Thrombin-stimulated HUVEC also synthesized higher amounts of PAF than untreated cells. Pretreatment of PMNs with L-659,989 significantly reduced their rolling and adhesion to thrombin-activated HUVEC. Stimulation of HUVEC with TNF-alpha significantly increased the number of rolling and adherent PMNs as compared with untreated cells. Adhesion of PMNs to and migration across TNF-alpha-stimulated HUVEC were reduced by L-659,989, whereas cell rolling was unchanged. We conclude that PAF mediates leukocyte interaction under flow conditions with HUVEC activated by inflammatory stimuli.
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PMID:PAF mediates neutrophil adhesion to thrombin or TNF-stimulated endothelial cells under shear stress. 763 59

Whether or not two alkylglycerols could initiate a functional response in human platelets or modify responses induced by platelet activating factor (PAF) was evaluated. It was found that 1-100 microM 1-O-hexadecyl-2-metoxy-glycero-3-phosphatidylcholine (Et-16-OCH3) induced platelet aggregation but 1-O-hexadecyl-sn-glycerol (chimyl alcohol; CA) did not. Et-16-OCH3-induced platelet aggregation was abolished by pretreatment with the PAF receptor antagonist WEB 2086. While CA had no effect on platelet aggregation induced by PAF, pretreatment with Et-16-OCH3 (0.1 microM or higher) significantly inhibited platelet aggregation induced by PAF, but had no effect on aggregation caused by ADP, thrombin or phorbol myristate acetate (PMA). A receptor binding study using radiolabelled [3H]WEB 2086 showed that Et-16-OCH3 exerts its actions through interaction with the PAF receptor. Moreover, Et-16-OCH3 inhibited neutrophil chemiluminescence responses induced by PAF, but not reactions to PMA or a formyl peptide. Finally, 1 microM Et-16-OCH3 induced a rise in the intracellular calcium concentration in platelets equal to that induced by PAF and also had an calcium ionophore-like effect at 100 microM. Thus, this study shows that Et-16-OCH3 is both a potent inducer of platelet aggregation and an inhibitor of PAF-induced platelet aggregation and neutrophil chemiluminescence, through interaction with the PAF receptor.
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PMID:1-O-hexadecyl-2-metoxy-glycero-3-phosphatidylcholine--a methoxy ether lipid inhibiting platelet activating factor-induced platelet aggregation and neutrophil oxidative metabolism. 778 98

We have previously demonstrated that leukotriene B4 (LTB4) induces in vitro a transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HUVEC) for neutrophils (PMN). The magnitude of this response is intermediate of that conferred by thrombin and by platelet-activating factor (PAF). This report shows that the LTB4 response was neither related to HUVEC expression of PAF (because it could not be blocked by the PAF receptor antagonist WEB-2086), nor to access to LTB4 receptors on neutrophils (as shown by LTB4 receptor desensitization experiments). However, it could be partly blocked by treating HUVEC with an LTB4 receptor antagonist (SC-41930). LTB4 evoked a rise of intracellular calcium concentrations, [Ca2+]i, in the HUVEC, and the hyperadhesive HUVEC response to LTB4 was abrogated by buffering of [Ca2+]i by Quin-2. The response was not inhibited by treating HUVEC with pertussis toxin before LTB4. Neutrophils showed no signs of activation when adhering to LTB4-treated HUVEC because they did not i) release lactoferrin, or ii) react with an increase of [Ca2+]i, and iii) they bound equally well to the stimulated endothelial cells after having been treated with pertussis toxin so that up-regulation of PMN adhesion to LTB4 was abolished. LTB4-treated HUVEC did not shed factors that modulated neutrophil adherence or chemotaxis. Thus, LTB4 promotes HUVEC hyperadhesiveness for PMN, and the transduction mechanism involves calcium ions, may depend on a surface receptor for LTB4, but does not involve pertussis toxin-sensitive G proteins or PMN activation.
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PMID:Signal transduction mechanisms for leukotriene B4 induced hyperadhesiveness of endothelial cells for neutrophils. 825 96

The effects of CIS-19 (cis-2-(3,4-dimethoxyphenyl)-6-isopropoxy-7-methoxyl-1-(N-methylforma mido)-1,2, 3,4-tetrahydronaphthalene) was determined in vitro in rabbit platelets and in vivo in rats and guinea-pigs. CIS-19 inhibited in a selective and concentration-dependent manner the aggregation and ATP release reaction of rabbit platelets induced by PAF (4 nM). The IC50 values of CIS-19 on PAF-induced aggregation of washed platelets and platelet-rich plasma were 11.3 +/- 2.7 and 16.8 +/- 3.0 microM respectively. BN52021 also inhibited PAF-induced aggregation of washed platelets with an IC50 value of 11.7 +/- 2.8 microM. CIS-19 inhibited [3H]PAF (4 nM) binding to washed rabbit platelets with an IC50 value of 1.5 +/- 0.2 microM. The concentration-response curve of PAF-induced aggregation of washed platelets was shifted rightwards by CIS-19 with pA2 and pA10 values of 7.1 (6.8-7.3 for 95% confidence limit) and 6.1 (5.8-6.2) respectively. The thromboxane B2 formation of washed platelets caused by AA, collagen or thrombin was not affected by CIS-19 of concentrations below 400 microM. CIS-19 (25 microM) completely blocked PAF-induced, but not collagen- or thrombin-induced [3H]inositol monophosphate formation of washed platelets. When CIS-19 (2.5 and 5 mg/kg) was injected i.v. into the femoral vein, it did not affect the blood pressure of rats, but antagonized PAF (2.5 micrograms/kg, i.v.)-induced hypotensive shock either preventively or curatively. CIS-19 (2.5 and 5 mg/kg) also blocked PAF (50 ng/kg)-induced, but not AA (50 micrograms/kg)-induced, bronchoconstriction in guinea-pigs. It is concluded that CIS-19 is an effective PAF receptor antagonist not only in vitro, but also in vivo.
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PMID:CIS-19, a novel platelet activating factor receptor antagonist: in vitro and in vivo studies. 838 Mar 43

Infusion of the thrombolytic agents streptokinase (SK, 666 units/kg per minute for 60 minutes) and tissue-type plasminogen activator (t-PA, 10 micrograms/kg per minute for 15 minutes) in rabbits induced a significant hypotension and decrease in platelet count that were completely prevented by treatment with platelet-activating factor (PAF) receptor antagonists SDZ 63-675 and WEB 2170. PAF synthesis by vascular tissue was suggested by its extraction from blood-free heart and aorta of rabbits treated in vivo with SK or t-PA but not of control rabbits. In contrast, PAF was not detected in peripheral blood. Ex vivo studies on platelet aggregation response to ADP and PAF performed on platelet-rich plasma obtained before and after SK and t-PA infusion demonstrated an early hyperaggregable phase, abrogated by PAF receptor antagonists and followed by reduced sensitivity of platelets to PAF. The ED50 values for the aggregation of washed rabbit platelets induced by PAF but not thrombin were significantly increased at 60 and 120 minutes after SK and t-PA infusion, suggesting a specific desensitization of platelets to PAF. In contrast to PAF receptor antagonists, aspirin did not significantly modify the hypotension and the platelet hyperaggregability induced by SK or t-PA or the platelet hypoaggregability induced by t-PA. Thrombocytopenia induced by t-PA, but not by SK, was partially prevented by aspirin. The effect of SK, t-PA, and plasmin on the aggregation of washed platelets from untreated rabbits and from humans was also studied. Whereas SK and t-PA were inactive, plasmin induced dose-dependent platelet aggregation that was inhibited by platelet pretreatment with PAF receptor antagonists. In conclusion, the effect of PAF receptor antagonists observed in the present experimental model suggests that the hypotension and activation of platelets induced by SK and t-PA infusion are mediated by PAF.
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PMID:Role of platelet-activating factor in hypotension and platelet activation induced by infusion of thrombolytic agents in rabbits. 838 25


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