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)

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aalpha, Bbeta and gamma fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 microg/ml, with total secretion of subunits approaching 700 microg/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bbeta and gamma chains were rate limiting. Both the Bbeta and gamma chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for gamma chains over Bbeta chains. Also, the subunit complexes gamma2, Aalphagamma2 and the individual subunits Aalpha, Bbeta and gamma were found as secretion products. When the Bbeta was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of Bbeta chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.
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PMID:Secretion of recombinant human fibrinogen by the murine mammary gland. 1558 68

Diet can be one of the most important factors that influence risks for atherothrombotic diseases. Hesperetin included in grapefruits and oranges is one candidate that may benefit the cardiovascular system. Here, we investigated antiplatelet activity of hesperetin in vitro. In addition, possible antiplatelet mechanism was also investigated. Hesperetin concentration-dependently inhibited washed rabbit platelet aggregation induced by collagen and arachidonic acid, with IC50 of 20.5+/-3.5 and 69.2+/-5.1 microM, respectively, while has little effect on thrombin- or U46619-, a thromboxane (TX) A2 mimic, mediated platelet aggregation, suggesting that hesperetin may selectively inhibit collagen- and arachidonic acid-mediated signal transduction. In accordance with these findings, hesperetin revealed blocking of the collagen-mediated phospholipase (PL) C-gamma2 phosphorylation, and caused concentration-dependent decreases of cytosolic calcium mobilization, arachidonic acid liberation and serotonin secretion. In addition, hesperetin inhibited arachidonic acid-mediated platelet aggregation by interfering with cyclooxygenase-1 activity as established by the measurement of arachidonic acid-mediated TXA2 and prostaglandin D2 formations as well as cyclooxygenase-1 and TXA2 synthase activity assays. Taken together, the present results provide a cellular mechanism for the antiplatelet activity of hesperetin through inhibition of PLC-gamma2 phosphorylation and cyclooxygenase-1 activity, which may contribute to the beneficial effects of grapefruits and oranges on cardiovascular system.
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PMID:Antiplatelet activity of hesperetin, a bioflavonoid, is mainly mediated by inhibition of PLC-gamma2 phosphorylation and cyclooxygenase-1 activity. 1709 6

To determine the significance of the gamma2 calcium-binding site in fibrin polymerization, we synthesized the fibrinogen variant, gammaD298,301A. We expected these two alanine substitutions to prevent calcium binding in the gamma2 site. We examined the influence of calcium on the polymerization of gammaD298,301A fibrinogen, evaluated its plasmin susceptibility, and solved 2.7 and 2.4 A crystal structures of the variant with the peptide ligands Gly-Pro-Arg-Pro-amide (GPRP) and Gly-His-Arg-Pro-amide (GHRP), respectively. We found that thrombin-catalyzed polymerization of gammaD298,301A fibrinogen was modestly impaired, whereas batroxobin-catalyzed polymerization was significantly impaired relative to normal fibrinogen. Notably, the influence of calcium on polymerization was the same for the variant and for normal fibrinogen. Fibrinogen gammaD298,301A was more susceptible to plasmin proteolysis in the presence of GPRP. This finding suggests structural changes in the near-by "a" polymerization site. Comparisons of the structures revealed minor conformational changes in the gamma294-301 loop that are likely responsible for the weakened "a" site. When considered altogether, the data suggest that the gamma2 calcium-binding site does not significantly modulate polymerization. We cannot, however, rule out the possibility that the weakened "a" polymerization site masks an important role for the gamma2 calcium-binding site in normal polymerization. Somewhat unexpectedly, the structure data showed that GPRP bound to the "b" site and induced the same local conformational changes as GHRP to this site. This structure shows that "A:b" interactions can occur and suggests that these may participate in normal polymerization.
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PMID:Probing the gamma2 calcium-binding site: studies with gammaD298,301A fibrinogen reveal changes in the gamma294-301 loop that alter the integrity of the "a" polymerization site. 1741 Oct 74

Subendothelial collagen supports platelet adhesion, activation, and thrombus growth at sites of vascular damage. Previous studies have shown that chalcones possess antiplatelet activity, but their mechanism of action is not fully understood. In this study, a recently synthesized chalcone, 2'-ethoxy-5'-methoxy-2-(5-methylthienyl)chalcone (EMMTC), was used to investigate chalcone effects on platelet aggregation and adhesion. We found that EMMTC potently inhibited collagen-induced platelet aggregation with an IC(50) of 1.01 micromol/l. In contrast, it did not inhibit thrombin- and ADP-induced platelet aggregation. EMMTC could inhibit arachidonic acid (AA)-induced platelet aggregation and collagen- and AA-induced thromboxane B(2) (TXB(2)) formation, suggesting that cyclooxygenase and/or thromboxane synthase were affected during this process. Moreover, EMMTC suppressed collagen-induced protein tyrosine phosphorylation, including phospholipase C-gamma2 (PLC-gamma2), phosphatidylinositol-3 kinase (PI-3K), Src, Fyn and LAT. Strikingly, EMMTC also blocked platelet adhesion to immobilized collagen and convulxin (a snake venom-derived protein that activates platelet glycoprotein VI receptor). The attenuation of phosphorylation of focal adhesion kinase (FAK) was observed during adhesion. Taken together, our results presented here demonstrate that the chalcone derivative EMMTC affects collagen-induced protein tyrosine phosphorylation and TXB(2) formation and functionally blocks collagen-induced platelet aggregation and adhesion.
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PMID:2'-Ethoxy-5'-methoxy-2-(5-methylthienyl)chalcone inhibits collagen-induced protein tyrosine phosphorylation and thromboxane formation during platelet aggregation and adhesion. 1969 Apr 43


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