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)

The effects of Ca-antagonists on the thrombin-induced mobilization of radiolabeled arachidonate preincorporated into rat platelets as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products were analyzed in the presence of either Ca2+ or Ca2+-substitutes, Sr2+ and Ba2+. Results indicate that following thrombin stimulation (0.2 U/ml) in the presence of Ca2+, nitrendipine (Nit), Cd2+ or Mn2+ reduced the release of arachidonate and the biosynthesis of thromboxane B2. Inhibition of arachidonic acid release and metabolism were also obtained by both Nit and Cd2+ in the presence of Sr2+ and Ba2+. Results from studies with a semi-purified phospholipase A2 fraction prepared from rat platelets indicated that the activity was almost unaffected by Nit or Cd2+. From these findings, we concluded that inhibition of platelet-induced release and metabolism of arachidonic acid by the Ca-antagonists tested require intact platelets. These data support the hypothesis of an interaction of these agents at an unknown surface membrane level.
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PMID:Effects of organic and inorganic Ca antagonists on rat platelet arachidonic acid metabolism in the presence of Ca2+, Sr2+ and Ba2+. 249 42

The addition of arachidonic acid induced a rapid release of 45Ca2+ from human platelet membrane vesicles which accumulated 45Ca2+ in the presence of ATP. Docosahexaenoic acid, eicosapentaenoic acid, linolenic acid and linoleic acid were less active than arachidonic acid. In contrast, oleic acid, myristic acid and palmitic acid were without effect. The thromboxane A2 analogue induced no 45Ca2+ release. The cyclooxygenase/lipoxygenase inhibitor failed to suppress arachidonic acid-induced 45Ca2+ release at the concentration which inhibited the production of lipid peroxides. These data indicate that the activity of arachidonic acid may be due to fatty acid itself and not to its metabolites. The combination of arachidonic acid and inositol 1,4,5-trisphosphate (IP3) resulted in a greater 45Ca2+ release from platelet membrane vesicles than either compound alone. When the intracellular free Ca2+ concentration ([Ca2+]i) was measured using fura-2, the thrombin-induced [Ca2+]i increase was reduced in platelets which had been treated with a phospholipase A2 inhibitor, ONO-RS-082 (2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid). These results provide evidence that arachidonic acid alone may cause Ca2+ increase and also may induce an additional Ca2+ mobilization to IP3-induced Ca2+ release in human platelets.
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PMID:Evidence of Ca2+ mobilizing action of arachidonic acid in human platelets. 249 58

CPIB possesses antiplatelet as well as hypolipidemic activities. Two cyclic CPIB analogs, 6-phenylchroman-2-carboxylic acid (PCCA) and 6-cyclohexylchroman-2-carboxylic acid (CHCCA) were also found to be antagonists of the prostaglandin (PG)-dependent pathway of human platelet activation. PCCA and CHCCA were inhibitors of aggregation (AGG) and secretion (SEC) induced by ADP or epinephrine (E) [second waves only] and arachidonic acid (AA) with IC50 values ranging from 2.3-8.7 microM for PCCA and 3.7-12.1 microM for CHCCA. Neither compound antagonized the proaggregatory effects of the thromboxane A2 (TXA2) receptor agonist, U46619. CPIB blocked ADP and E-induced AGG and SEC (IC50's greater than 1200 microM) but not AA- or U46619-induced responses. Only CPIB blocked thrombin-induced AA release. Data showed that PCCA and CHCCA inhibited AA-induced malondialdehyde formation (IC50's = 9.3 and 11.3 microM, respectively) and thrombin-induced production of prostaglandin E2, prostaglandin F2 alpha, and TXB2 with IC50's ranging from 2.9 to 13.4 microM. PCCA and CHCCA were at least 200- to 500-fold more potent than CPIB as inhibitors of the PG-dependent pathway of human platelet activation. We conclude that PCCA and CHCCA act by inhibiting platelet cyclooxygenase activity whereas CPIB blocks the activity of phospholipase A2. Hypolipidemic PCCA and CHCCA represent a potent class of cyclooxygenase inhibitors which may be more useful than CPIB for treatment of atherosclerotic and thrombotic disorders.
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PMID:Potent antiplatelet effects of cyclic clofibric acid (CPIB) analogs on human platelets. 250 6

The endothelium produces many substances which have the potential to alter hemostasis. We examined the influence of a substance(s) released by cultured bovine aortic endothelial cells on very early events (less than 5 sec) in platelet function, using a new quenched-flow approach. Contributions of the endothelial cell surface to alterations in platelet function were avoided by separating the endothelium and platelets. Superfusates from endothelial cells were allowed to interact with platelets for several seconds, followed by challenge with ADP to induce aggregation. Single-particle counting provided a sensitive index of aggregation kinetics. In this system, unstimulated endothelial-cell superfusate had no effect on aggregation induced by ADP. Stimulation of the endothelial cells with bradykinin produced an unstable factor(s) that potentiated ADP-induced platelet aggregation, was not affected by hemoglobin or cyclooxygenase inhibitions, and had no platelet aggregatory activity on its own. Nitric oxide inhibited aggregation and its effect was blocked by hemoglobin. The identity of the potentiating factor is unknown, but it does not appear to be platelet activating factor, thrombin, ADP, thromboxane, nitric oxide, or endothelium-derived relaxing factor.
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PMID:Endothelium-dependent potentiation of human platelet aggregation. 250 2

The techniques we developed to propagate HTM cells in serial cell culture have provided an opportunity to investigate the spectrum of endogenous PGs and other eicosanoids that are produced by these cells. PGE2 and PGF2 alpha were the major cyclooxygenase products detected by both radioimmunoassay and thin-layer chromatography. A small amount of 6-keto PGF1 alpha was also detected, indicating that these cells are able to produce prostacyclin. The observation of a substantial increase in the proportion of PGF2 alpha relative to PGE2 at later time periods after a media change suggests a metabolic conversion of PGE2 to PGF2 alpha by these cells. Bradykinin, thrombin, platelet activating factor, and serum were found to be effective stimulators of PG production by HTM cells, whereas calcium ionophore produced only a minor effect. Using high pressure liquid chromatography, elution profiles of radiolabeled metabolites of AA suggested the presence of certain lipoxygenase products, including LTB4, 12-HETE, 15-HETE, and a small amount of 5-HETE in HTM cells. The formation of these products was inhibited by both DEX and BW 755c, reinforcing the view that metabolic conversions of AA through the lipoxygenase pathway were possible in the trabecular meshwork. We also examined the effects of glucocorticoids on specific protein synthesis in the HTM cells, using 35S-methionine labeling and SDS-PAGE techniques. Short-term (1 day) DEX treatment revealed a major induction of a protein band at approximately 30 kDa. Longer treatments (1 to 3 weeks) resulted in major inductions at approximately 55 kDa inside the cells, with the presence of secreted forms (probably glycoproteins) between 55 and 72 kDa. The short-term DEX effect on protein synthesis a phospholipase inhibitor regulating eicosanoid production within the HTM. The longer-term induction may, on the other hand, be related more directly to the development of steroid glaucoma, based on our findings that the inductions of these proteins correlate with the observed time course and dose-dependence topical glucocorticoid effects on IOP. Continued in vitro and in vivo evaluations of the eicosanoid pathways in cultured HTM cells obtained from normal and glaucomatous human eyes may help to delineate their relationship to IOP regulation and the pathogenesis and treatment of glaucoma. Glucocorticoid-induced proteins may be key participants in the regulation of phospholipase activity and hence may represent a major control mechanism of the AA cascade.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Eicosanoid production and glucocorticoid regulatory mechanisms in cultured human trabecular meshwork cells. 250 21

Acquired platelet storage pool disease has been shown to be associated with reduced platelet thromboxane synthesis. However, the mechanisms for this dysfunction are incompletely understood. The present experiments were designed to evaluate some of the possible defects which may account for impaired thromboxane formation in human platelets previously exposed to thrombin in vitro. Washed platelets pretreated with 0.5 U/ml thrombin for 20 sec and subsequently recovered as single degranulated platelets were incapable of forming normal amounts of thromboxane upon a second stimulation with thrombin (as compared to Tyrode-pretreated control platelets). In contrast, thrombin-degranulated platelets released additional amounts of thromboxane in response to arachidonate, or collagen, indicating that short-time exposure to thrombin does not irreversibly inactivate platelet cyclooxygenase or thromboxane synthetase. Upon incubation of the thrombin-pretreated platelets in autologous plasma in the presence of 14C-arachidonate, the label became associated with the platelets to the same extent as with control platelets. However, the platelets did not recover their ability to synthesize normal amounts of thromboxane upon restimulation with thrombin, and only about half of the label was lost from the thrombin-pretreated platelets as compared to control platelets in response to thrombin. The ability of collagen to cause loss of 14C-arachidonate and formation of thromboxane was the same regardless of whether or not the platelets had been pretreated with thrombin. Incubation of platelets in plasma in the presence of added arachidonate for 90 min resulted in complete refractoriness to a second stimulation with thrombin but not with collagen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Failure of preactivated human blood platelets to restore defective thromboxane synthesis despite prolonged incubation in plasma. 251 74

Human platelets were incubated alone or in whole blood with specific agonists such as thrombin or collagen, and 12-hydroxy-heptadecatrienoic (HHT) and 12-hydroxy-eicosatetraenoic (12-HETE) acids were measured by HPLC as indices of platelet cyclooxygenase and lipoxygenase activities, respectively. We found that both arachidonic acid metabolites are significantly formed at concentrations of thrombin insufficient to provoke platelet aggregation. The ratio HHT/12-HETE varied with increasing concentrations of thrombin, with an increase in the absence and a decrease in the presence of albumin in the incubation. When platelets were stimulated in whole blood, this ratio favoured HHT and the addition of albumin to isolated platelets had the same effect. The formation of oxygenated products of 12-HETE by leukocyte LTB w-hydroxylase and 5-lipoxygenase in unstimulated and stimulated leukocytes, respectively, was also investigated. We failed to detect any significant amounts of these products in whole blood incubated with relatively high concentrations of collagen in the presence or absence of the chemotactic peptide FMLP. We conclude that, although 12-HETE is a good substrate for leukocyte oxygenases when incubated at high concentration with the cells alone, its oxygenation is unlikely to occur in whole blood, making 12-HETE and/or HHT potential markers of platelet activation in vivo, provided they are not substantially degraded during passage of the blood through various organs.
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PMID:Metabolism of endogenous arachidonic acid in weakly activated platelets. Absence of leukocyte cooperative products in whole blood. 251 43

Human platelets were prelabelled with [14C]arachidonate and stimulated with thrombin or methyl mercury. [14C]PGH2 and the more stable of the other [14C]eicosanoids formed were rapidly extracted with organic solvent cooled to -30 degrees C and analyzed by radio-TLC. TXB2 and PGH2 were also quantified by radioimmunoassay, the latter as its index metabolite PGE2. PGH2 reached its peak concentration of 12 nmol/l after 20-30 s when it amounted to approximately 2/3 of the TXB2 concentration. In the presence of the thromboxane synthase inhibitor dazoxiben, PGH2 peaked after 60 s and afterwards declined in favour of PGE2, PGD2 and PGF2 alpha. Thirty seconds after stimulation with thrombin 1 IU/ml or methyl mercury 20 mumol/l, PGH2 amounted to 35 or 28% of the cyclooxygenase products in the absence and to 66 or 63% in the presence of dazoxiben, respectively. The platelet-activating potency of PGH2 was evaluated with purified PGH2 in platelets pretreated with acetylsalicylic acid. The EC50 values of PGH2 were 0.69 and 19 nmol/l for shape change and aggregation, respectively. U 46619 produced the same effects at 4.1 and 23 nmol/l. PGH2-induced [3H]serotonin release did not exceed 25%, whereas U 46619 was able to induce approximately 50% [3H]serotonin release. Dazoxiben enhanced the aggregation induced by PGH2. Human serum albumin inhibited the aggregating effect of PGH2, suppressed the enhancing effect of dazoxiben and shifted the metabolism of PGH2 to the inhibitory PGD2. The TXA2/PGH2 receptor antagonist daltroban suppressed the agonistic effects of endogenous or added PGH2, demonstrating that the TXA2/PGH2 receptor was its site of action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transient concentrations and agonist potency of PGH2 in platelet activation by endogenous arachidonate. 251 34

Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet-vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta-thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA-treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.
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PMID:Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets. 252 23

The exposure of fibrinogen receptors is an early event in agonist-induced platelet activation. Previous measurements of fibrinogen binding or aggregation in platelet-rich plasma or washed platelets have failed to define whether the initial response to epinephrine results solely from a direct effect of this agonist. To address this problem, we have measured fibrinogen receptor exposure on platelets in whole blood by using flow cytometry and a fluorescein isothiocyanate-labeled monoclonal antibody specific for the activated fibrinogen receptor (FITC-PAC1). We also measured platelet-bound fibrinogen with an antifibrinogen monoclonal antibody (FITC-9F9) as well as platelet aggregation in whole blood. In blood anticoagulated with citrate and in the presence of a cyclooxygenase inhibitor, epinephrine (0.1 to 100 mumol/L) caused significant FITC-PAC1 binding (P less than .001) that was maximal at 10 mumol/L epinephrine. The maximal epinephrine response was one third of that observed with 10 mumol/L adenosine diphosphate (ADP) and was eliminated by yohimbine, an alpha 2-adrenergic antagonist. Incubation of the blood with apyrase or phosphoenolpyruvate plus pyruvate kinase to remove extracellular ADP resulted in a 40% to 50% reduction in the epinephrine response. Despite this, FITC-PAC1 binding was still significant at epinephrine greater than or equal to 1 mumol/L (P less than .05). No reduction in epinephrine-induced FITC-PAC1 binding was observed in the presence of ATP alpha S, an ADP receptor antagonist; cinanserin, a serotonin antagonist; or WEB-2086, a platelet activating factor antagonist. Furthermore, addition of the thrombin inhibitors hirudin or leupeptin to citrated blood had no effect on the extent of the epinephrine response. Blood anticoagulated with hirudin also demonstrated an epinephrine response, even in the presence of apyrase. Similar results were obtained when FITC-9F9 was used to detect fibrinogen binding or when aggregation was assessed by a decrease in the number of single platelets. We conclude that epinephrine itself can induce fibrinogen receptor exposure, fibrinogen binding, and aggregation. This primary response is independent of synergistic interaction of epinephrine with traces of ADP, serotonin, platelet activating factor, or thrombin. However, such synergistic interaction with ADP present in whole blood may enhance the responses induced by epinephrine.
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PMID:Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the absence of other excitatory agonists. 253 42


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