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 N(alpha)-arylsulfonyl-L-arginine ethyl- and propyl esters were synthesized and the kinetics of their hydrolysis by thrombin was studied. The values of kcat and Km were shown to depend on the structure of the leaving group and to decrease in the line: OCH3 greater than OC2H5 greater than OC3H7. Using methanol as an additional nucleophile, the kinetic parameters - k2, k3 and Ks - were measured for both thrombin- and trypsin-catalysed reactions. A similarity of two enzymes at the stage of Michaelis complex formation was revealed: the Ks values for both enzymes were practically identical (18.10(-5)M). The differences between thrombin and trypsin were observed at the stages of chemical conversion of substrates and were especially well-pronounced at the stage of acylation. It was shown that the k2 values for thrombin were lower than that for trypsin and the k2/k3 ratio of TAME hydrolysis by trypsin was equal to 21, while that for thrombin was 4.5. This finding is indicative of an essential role of the acylation step in thrombin-catalysed hydrolysis of the esters under study.
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PMID:[Determination of the kinetic parameters of individual stages of M(alpha)-arylsulfonyl-L-arginine ester hydrolysis by thrombin and trypsin]. 65 99

A neutral mixture of chloroform and methanol was compared to an acidic mixture of these solvents for the extraction of diacylglycerol from platelets labelled with 3H-arachidonic acid. Using a neutral solvent we found that thrombin caused a rapid increase in the radioactivity of diacylglycerol. With an acidic solvent there was 10 times more background radioactive diacylglycerol, but no increase was detected after stimulation with thrombin. Acidic extraction, but not neutral extraction, caused a small percentage of phosphatidylinositol and phosphatidylcholine to hydrolyse and form diacylglycerol. The extent of hydrolysis accounted for the greater amount of radioactive diacylglycerol found after acidic extraction of radiolabelled platelets. In addition, when platelets were extracted by the acidic solvent a modified form of hydroxy-heptadecatrienoic acid appeared, and thin-layer chromatography in two dimensions was required to separate it from diacylglycerol. It is therefore important to use a neutral extraction method when studying diacylglycerol in platelets.
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PMID:Interference caused by acid extraction in the study of diacylglycerol in platelets. 189 58

The release of free fatty acids from rat platelets, triggered by thrombin stimulation, was monitored by high-performance liquid chromatography (HPLC) after precolumn derivatization with monodansylcadaverine (MDC). A rapid filtration procedure was devised for the precise determination of free fatty acids released from aggregated platelets, instead of the conventional method using a stop solution or enzyme reactions. The fatty acids thus collected were derivatized with MDC in the presence of diethyl phosphorocyanidate (DEPC). The simultaneous separation of MDC derivatives of fatty acids was achieved on a reversed-phase TSKgel ODS-80TM column within 60 min by linear gradient elution, using 0.2 M Tris-HCl buffer (pH 7.8)-methanol (50:50, v/v) and acetonitrile. The MDC derivatives were detected with excitation and emission wavelengths of 340 and 518 nm, respectively. The amounts of liberated fatty acids were in the range from 45.0 pmol for myristoleic acid (C14:1) to 395.0 pmol for palmitic acid (C16:0) per 1.9 x 10(7) platelets.
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PMID:Rapid determination by high-performance liquid chromatography of free fatty acids released from rat platelets after derivatization with monodansylcadaverine. 228 73

Stimulation of human or rabbit platelets with thrombin in the presence of fibrinogen caused a large decrease, compared with unstimulated controls, in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2) that could be extracted with acidified chloroform/methanol (60% at 60 s). In contrast, stimulation in the absence of added fibrinogen increased the amount of PIP2. The decrease was specific for PIP2, because similar decreases could not be demonstrated for other phosphoinositides or phospholipids. The interaction of polymerizing fibrin with stimulated platelets was required for the decrease in PIP2, since polymerized fibrin formed by reptilase did not cause the decrease in the amount of extractable PIP2, and inhibition by glycyl-L-prolyl-L-arginyl-L-proline of polymerization of fibrin formed by the action of thrombin prevented the large decrease in extractable PIP2. The decrease in extractable PIP2 could not be explained by increased degradation of PIP2, since sufficient degradation products were not formed. Thus, when platelets are stimulated with thrombin in the presence of fibrinogen, an association of polymerizing fibrin with the stimulated platelets occurs that leads to decreased extractability of PIP2. This may mean that PIP2 forms a specific association with platelet proteins that are involved in clot retraction.
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PMID:Phosphatidylinositol 4,5-bisphosphate is selectively retained by platelet-fibrin clots formed by thrombin. 282 29

We have studied the in vitro exposure to various oxysterols on isolated rat platelets. The oxysterols (1-20 uM) were incubated either dissolved in methanol or as albumin-bound complexes. Aggregation (analyzed by turbidimetry) was measured after stimulation by thrombin or ADP. Serotonin secretion (analyzed by voltammetry) was measured after thombin stimulation. We found that platelet aggregation and serotonin secretion could be either not significantly affected (7B-hydroxy cholesterol) or potentiated (22S-hydroxy cholesterol, 3,5,6-hydroxy cholestan triol) or inhibited (25-hydroxy- and 7-oxo cholesterol), after in vitro incubation with different oxysterols. Our data indicate that a modulation of the platelet behavior occurs after in vitro incubation with different oxysterols, some derivatives acting as inhibitors and others as potentiators. These results provide new interesting information regarding the role of these sterols in cell membrane structure and function in relation to pathology.
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PMID:Biological effects of oxysterols on platelet function. 340 82

We studied the effect of the methanol extract of garlic bulbs (EOG) and of three pure components isolated from it (F1, F2, F3), on human platelet aggregation induced by ADP, epinephrine, collagen, thrombin, arachidonate, PAF, and the ionophore A-23187. Incubation of PRP with EOG, either in methanol or in homologous PPP, inhibits platelet aggregation induced by all of the above mentioned agonists. F1, F2, and F3 also inhibit platelet aggregation, however, F3 was about four times more potent. Addition of EOG or F3 to platelets that have already been irreversibly aggregated by 10 microM ADP, induces rapid deaggregation. Inhibition of aggregation was still present after three hours. The inhibitory effect persisted even after the treated platelets were Gel-Filtered (GFP) or separated from plasma through a metrizamide gradient and resuspended in new homologous PPP. Thrombin-induced release of ATP from GFP was inhibited by 75-80% after EOG or F3 treatment. Incorporation of [3-H]-arachidonate by intact platelets was decreased by 50-60% in treated platelets. However, platelets incubated with the inhibitors after incorporation of radiolabeled arachidonate, although did not aggregate, produced, after thrombin activation similar amounts of radiolabeled TXB2 and lipoxygenase products as the controls. Electron microscopy of inhibited platelets, in the presence of thrombin, showed no degranulation but an increase of spherical forms. Our results suggest that the effects described might be mediate by a perturbation of the physicochemical properties of the plasma membrane rather than by affecting arachidonate or calcium metabolism in the cells. Chemical structures of F1, F2 and F3 have been provisionally assigned: F1 is diallytrisulfide, F2 is 2-vinyl-1,3-dithiene, and F3 is most probably allyl 1,5-hexadienyltrisulfide.
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PMID:Effects of garlic extract and of three pure components isolated from it on human platelet aggregation, arachidonate metabolism, release reaction and platelet ultrastructure. 641 74

Dipeptidyl argininal (arginine aldehyde) affinity resins of general formula R-(X-Y-argininal) (where R = resin matrix and X, Y = amino acids of varied structure) are synthesized in a solid-phase procedure in which the dipeptide (-X-Y-) is first attached to the resin, followed by the joining of the Y amino acid to argininal semicarbazone, and decomposition of the semicarbazone in a methanol/acetic acid/formaldehyde reagent. An R-(Gly-Gly-argininal) resin binds urokinase tightly, but does not bind thrombin. However, thrombin binds strongly to R-(Phe-Pro-argininal), whereas urokinase does not bind. Accordingly, the X-Y-argininal ligands selectively bind proteinases of identical primary binding site specificity to arginine, but different secondary site specificity in -X-Y-. The selectivity is due to an amplification of peptide binding specificity caused by the transition-state analog properties of the ligands. While the affinity constants between peptide aldehyde and proteinase approach those of antibody-antigen interactions, the elution with semicarbazide (aldehyde-trapping reagent) buffers easily remove tightly bound proteinases without proteinase inhibitors or denaturation. Conditions for the binding and elution of proteinases, methods of regeneration and other characteristics of the resins are described.
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PMID:Transition-state affinity chromatography of trypsin-like proteinases with dipeptidyl argininal ligands. 662 59

The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On the methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants (ka) were derived for the interaction with trypsin (1.8 x 10(6) M-1 s-1), thrombin (1.2 x 10(5) M-1 s-1), urokinase plasminogen activator (4.0 x 10(4) M-1 s-1), plasmin (1.3 x 10(6) M-1 s-1), and activated protein C (7.5 x 10(3) M-1 s-1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.
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PMID:Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris. 754 63

Although the exact function of cholesteryl sulfate (CS) is unknown, it is present in low concentration in lipoproteins, in red blood cells and spermatozoa. In the present study, we investigated whether CS is present in blood platelets and its possible biological involvement in platelet function. Extensively washed platelets were prepared from rat and human blood. After lipid extraction and thin layer chromatography (TLC) on silica gel, a compound with the same mobility as authentic CS was isolated and identified by two different methods: (1) without hydrolysis, negative ion fast atom bombardment combined with tandem mass spectrometry (MS/MS); (2) after acidic hydrolysis, identification of cholesterol (Chol) by TLC and gas chromatography-MS. CS concentrations measured using beta-sitosteryl sulfate as internal standard in normal rat or human platelets were in the range of 164-512 pmol/10(9) platelets. This represented less than 1% of cell Chol. Biological effects of CS on platelet function were studied in vitro. CS incubated with rat platelets either as methanol solution or as albumin-bound complex potentiated the ADP- or thrombin-induced aggregation and serotonin secretion. The results of platelet sterol analysis indicated that CS was incorporated into platelet membrane and did not significantly change the platelet cholesterol composition. The potentiating effect of CS on platelet-induced aggregation and secretion was not obtained with cholesterol, cholesteryl acetate or estrone. In contrast, an inhibitory effect of estrone sulfate was observed. These results indicate that both the sulfate group and the cholesterol moiety are involved in the pro-aggregant property of CS. In addition, platelet mediators seem to be implicated in the mechanism since the thrombin-induced production of thromboxane B2, the stable end-product of arachidonic acid metabolism, was also enhanced in the presence of CS. These results suggest a new role for CS which may be involved in the modulation of platelet function.
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PMID:Occurrence and biological effects of cholesteryl sulfate on blood platelets. 854 37

1. The ability of several serum fractions to increase the cytosolic calcium concentration ([Ca2+]i) was tested in rat cerebellar cells maintained in primary culture. 2. Serum filtered through an ultrafiltration membrane with 3000 Da molecular mass cut-off (filtered serum, FS) selectively stimulated neurons whereas dialysed serum (DS) selectively stimulated glia. 3. The effects of FS were due to glutamate as they were reproduced by N-methyl-D-aspartate (NMDA), blocked by NMDA receptor antagonists and prevented by enzymatic removal of glutamate. 4. The effects of DS on glia were not reproduced by platelet-activating factor, thrombin or bradykinin. They were not lost on heating or extraction with diethyl ether. They were reproduced by a methanol-chloroform-HCl extract from DS and by several commercial fraction V plasma albumins. 5. These [Ca2+]i-increasing factors present in blood could contribute to brain damage during ischaemia if they reached the brain interstitium on disruption of the blood-brain barrier.
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PMID:Two different constituents of plasma increase cytosolic calcium selectively in neurons or glia of primary rat cerebellar cultures. 868 58


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