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)

This study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se-methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms.
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PMID:Platelet size and age determine platelet function independently. 672 54

A pathway for the synthesis of membrane phosphatidylcholine involving the N-methylation of phosphatidylethanolamine has been detected in several types of mammalian cells. Furthermore, it has been implicated in the coupling of agonist binding to cell response. We examined whether human platelets exhibit this synthetic pathway and whether platelet agonists influence its activity. When washed platelets were incubated with 0.15 microM L-[methyl-3H]methionine at 37 degrees C, they incorporated methyl-3H into their phospholipids linearly at the rate of 1 pmole/10(9) platelets/hr. When incubated with 20 microM radiolabeled methionine, they incorporated about 15 pmole/10(9) platelets/hr. The radioactivity was found predominantly in phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Thrombin caused an immediate (within 15 sec) and sustained (up to 30 min) decrease in the rate and extent of N-methylation of platelet phospholipids. This was accounted for by a decrease in synthesis of methylated phospholipids rather than an increase in their degradation. This thrombin effect correlated with serotonin release and could be dissociated from platelet aggregation and prostaglandin synthesis. Thrombin also decreased the synthesis of phosphatidylcholine when choline was used as the radiolabeled substrate. Other agonists such as epinephrine, adenosine diphosphate (ADP), or A23187 also decreased phospholipid methylation under conditions in which they stimulated serotonin release. These data demonstrate that platelets are capable of synthesizing phosphatidylcholine from phosphatidylethanolamine by N-methylation and that agonists perturb this pathway as they induce platelet secretion. The precise role of phospholipid methylation in either resting or stimulated platelets remains to be established.
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PMID:Inhibition of platelet phospholipid methylation during platelet secretion. 677 78

Heparin accelerates the rate of inhibition of thrombin by antithrombin III. Reduction of one of the three antithrombin disulfide bonds with dithiothreitol under mild conditions abolishes this rate-enhancing effect without affecting the rate of reaction in the absence of heparin. Alkylation of mildly reduced antithrombin III with [3H]iodacetic acid followed by digestion with cyanogen bromide yielded two major labeled peptides. The smaller peptide, containing Cys-422, was identified as extending from Gly-414 to the C-terminus, Lys-424. Our data are consistent with the larger labeled peptide being the one extending from Glu-104 to Met-243 and containing Cys-239. Cys-422 has been shown by others to be linked to Cys-239. These data indicate that the sensitive disulfide bond in antithrombin III extends between Cys-239 and Cys-422; the site at which thrombin cleaves the antithrombin III is between these two half-cystines.
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PMID:Localization of the disulfide bond in human antithrombin III required for heparin-accelerated thrombin inactivation. 683 Feb 63

A computer graphic molecular display system has been used to construct a three-dimensional model of the B chain of bovine thrombin. The model is derived from the bovine alpha-chymotrypsin structure as determined by X-ray crystallographic studies. The amino acid sequence of bovine thrombin has been substituted for that of alpha-chymotrypsin, preserving the beta-barrel structure and maximizing homology of the amino acid sequence of the two proteins. With the exception of an area in the vicinity of the specificity binding pocket, most of the changes observed in thrombin occur on the surface of the molecule. The most notable changes observed in the model are the increases on the surface of positively charged (arginine and lysine) and negatively charged (glutamate and aspartate) residues. A glutamate replaces methionine 192 near the entrance to the specificity binding pocket. The nature of this site was further altered by the substitution of an aspartate for serine 189 and an alanine for serine 190. The structure of the resulting specificity binding pocket is consistent with that of serine proteases, which have trypsin-like substrate specificity. The computer graphics molecular display system has been used to insert models of synthetic thrombin inhibitors into the active site of the thrombin B chain model. With the model, it has been possible to correlate the interaction of thrombin with the observed binding constants of two inhibitors of trypsin-like serine proteases, p-amidinophenylmethylsulfonylfluoride (Ki = 1.27 x 10(-6) M) and m-[m-(trifluoromethyl)phenoxypropoxy]benzamidine (KD = 2.9 x 10(-6) M).
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PMID:A computer-generated three-dimensional model of the B chain of bovine alpha-thrombin. 694 67

The amino-terminal 173 residues of the murine histocompatibility antigen H-2Kb have been assigned by using radiochemical methodology. The complete sequence of an 86 residue glycopeptide (CN-Ib), which is one of the five major CNBr fragments of Kb, was determined by analysis of peptides obtained from digests using thrombin and V8 staphylococcal protease. Complete sequences were obtained for the three large thrombic peptides, and these were aligned by using peptides from the V8 protease digest. Alignment of the CNBr fragments was carried out by using [35S]Met-labeled peptides from a tryptic digest of the papain-cleaved H-2Kb molecule. Positive identification was possible for all the common amino acids except Asp (Asp) which was indirectly assigned and which is designated in italics. The sequence obtained in our studies was Gly-Pro-His-Ser-Leu-Arg-Tyr-Phe-Val-Thr-Ala-Val-Ser-Arg-Pro-Gly-Leu-Gly-Glu-Pro-Arg-Tyr-Met-Glu-Val-Gly-Tyr-Val-Asp-Asp-Thr-Glu-Phe-Val-Arg-Phe-Asp-Ser-Asp-Ala-Glu-Asn-Pro-Arg-Tyr-Glu-Pro-Arg-Ala-Arg-Trp-Met-Glu-Gln-Glu-Gly-Pro-Glu-Tyr-Trp-Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly-Asn-Glu-Gln-Ser-Phe-Arg-Val-Asp-Leu-Arg-Thr-Leu-Leu-Gly-Tyr-Tyr-(Asn)-Gln-Ser-Lys-Gly-Gly-Ser-His-Thr-Ile-Gln-Val-Ile-Ser-Gly-Cys-Glu-Val-Gly-Ser-Asp-Gly-Arg-Leu-Leu-Arg-Gly-Tyr-Gln-Gln-Tyr-Ala-Tyr-Asp-Gly-Cys-Asp-Tyr-Ile-Ala-Leu-Asn-Glu-Asp-Leu-Lys-Thr-Trp-Thr-Ala-Ala-Asp-Met-Ala-Ala-Leu-Ile-Thr-Lys-His-Lys-Trp-Glu-Gln-Ala-Gly-Glu-Ala-Glu-Arg-Leu-Arg-Ala-Tyr-Leu-Glu-Gly-Thr-Cys-Val-Glu-Trp-Leu-Arg-Arg-Tyr-Leu-Lys. These data represent the longest reported amino acid sequence determined by utilizing radiochemical methodology and provide the first extensive information on the primary structure of murine histocompatibility antigens.
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PMID:Primary structure of murine major histocompatibility complex alloantigens: amino acid sequence of the amino-terminal one hundred and seventy-three residues of the H-2Kb glycoprotein. 698 68

The association rate constants for the interaction of alpha-1-proteinase inhibitor, oxidized alpha-1-proteinase inhibitor, and alpha-1-antichymotrypsin with several mammalian serine proteinases have been determined. The results indicate that leukocyte elastase reacts more rapidly with alpha-1-proteinase inhibitor than any other proteinase tested, while leukocyte cathepsin G shows the strongest association with alpha-1-antichymotrypsin. Oxidation of the critical methionine residue of alpha-1-proteinase inhibitor reduces the association with leukocyte elastase by a factor of more than 2000 and also lowers the association with all of the other enzymes tested with the exception of chymotrypsin. Significantly, oxidation completely abolishes any interaction of alpha-1-proteinase inhibitor with porcine elastase, human plasmin or human thrombin. These data support previous results (Johnson, D., and Travis, J. (1979) J. Biol. Chem. 254, 4022-4026) which indicated that oxidation of human alpha-1-proteinase inhibitor in vivo could reduce the effectiveness of this inhibitor in controlling proteolysis. In the lung, in particular, oxidizing agents of both chemical and biological sources could, indirectly, augment elastolysis in this tissue, resulting in the development of pulmonary emphysema.
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PMID:Kinetics of association of serine proteinases with native and oxidized alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin. 698 30

Human platelets are capable of synthesizing their major membrane phospholipid, phosphatidylcholine, by a methylation pathway. This involves the sequential transfer of methyl groups from S-adenosyl-L-methionine (AdoMet) to phosphatidylethanolamine, and in the process, AdoMet is converted to S-adenosylhomocysteine (AdoHcy). The activity of this methylation pathway is decreased upon stimulation of platelets by various agonists. We inhibited methylation reactions pharmacologically to see whether this inhibition plays any role in the process of platelet activation. Two inhibitors of AdoHcy hydrolase, 3-deaza-adenosine and 3-deaza-(+/-)aristeromycin (500 microM each), were effective in increasing platelets levels of AdoHcy and preventing turnover of AdoMet. Also, these compounds were equipotent in inhibiting platelet phospholipid methylation. However, while 3-deaza-adenosine potentiated platelet aggregation and 14C-serotonin release induced by epinephrine or adenosine diphosphate (ADP) (p less than 0.01), 3-deaza-aristeromycin had no such effect. Neither compound affected platelet responses to thrombin or collagen. Inhibition of methylation reactions was not the only biochemical effect of 3-deaza-adenosine since it also blunted significantly the elevation of platelet cyclic adenosine monophosphate (AMP) levels induced by prostaglandin E1 (p less than 0.02). Therefore, these studies demonstrate that inhibition of platelet phospholipid methylation, per se, has no discernable effect on the function of human platelets. The methylation pathway, though active in platelets, does not appear to be primarily involved in membrane events responsible for platelet activation.
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PMID:The effect of pharmacologic inhibition of phospholipid methylation on human platelet function. 707 19

The NH2-terminal 98 amino acid residues of the murine histocompatibility antigen H-2Db have been assigned using radiochemical methodology. This represents the first extensive, continuous sequence information for a histocompatibility antigen encoded by the H-2D locus and allows comparison with the recently determined amino acid sequence of the H-2Kb molecule. The amino acid sequence was obtained from the sequences of three CNBr peptides, CN-E, CN-D, and CN-B, which comprise residues 1-5, 6-52, and 53-98, respectively. The amino acid sequence of CN-E was determined directly while the sequences of CN-D and CN-B were determined by NH2-terminal sequence analyses and sequence determinations of peptides produced by thrombin, staphylococcal V8 protease, and trypsin cleavage. Alignment of the CNBr peptides was accomplished by NH2-terminal sequence analysis of the H-2Db papain fragment (CN-E to CN-D) and by analyzing peptides from a tryptic digest of the intact H-2Db molecule. Positive identification was possible for all amino acids except Asp and Asn-86 which were indirectly assigned (in italics). The sequence obtained was Gly-Pro-His-Ser-Met-Arg-Tyr-Phe-Glu-Thr-Ala-Val-Ser-Arg-Pro-Gly-Leu-Glu-Glu-Pro -Arg-Tyr-Ile-Ser-Val-Gly-Tyr-Val-Asp-Asn-Lys-Glu-Phe-Val-Arg-Phe-Asp-Ser-Asp-Ala-Glu-Asn-Pro-Arg-Tyr-Glu-Pro-Arg-Ala-Pro-Trp-Met-Glu-Gln-Glu-Gly-Pro-Glu-Tyr-T rp-Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly-Gln-Glu-Gln-Trp-Phe-Arg-Val-Ser-Leu-Arg-Asn-Leu-Leu-Gly-Tyr-Tyr-Asn-Gln-Ser-Ala-Gly-Gly-Ser-His-Thr-Leu-Gln-Gln-Met.
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PMID:Primary structure of murine major histocompatibility complex alloantigens. Amino acid sequence of the NH2-terminal ninety-eight residues of the H-2Db glycoprotein. 722 4

Endothelial cell dysfunction is a key factor in oxidative stress-related pathology. Disruption of Ca2+ homeostasis is thought to be responsible for much of the endothelial cell dysfunction in oxidative stress. The expression of molecular chaperones (MC), which stabilize protein structures in normal and in stress conditions, reflects the Ca(2+)-dependent and -independent stress effects in the different cell compartments. By two-dimensional (2-D) gel electrophoresis combined with immunoblotting or microsequencing, we have identified 12 major MC in human umbilical vein endothelial cells (HUVEC): (i) the endoplasmic reticulum-located MC GRP78, GRP94, protein disulfide isomerase, and calreticulin; (ii) the mitochondrial MC HSP65 and GRP75; and (iii) the cytosolic/nuclear MC HSP27, HSC70, HSP70, HSP90, cyclophilin, and ubiquitin. To differentiate oxidative stress- and Ca(2+)-mediated effects, HUVEC were exposed to 1) xanthine oxidase plus hypoxanthine to generate oxidative stress, 2) ionomycin plus ethylene glycol-bis(beta-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA) to deplete intracellular Ca2+ stores, or 3) thrombin to increase cytosolic Ca2+. De novo protein synthesis after exposure was quantified by the incorporation of [35S]methionine. Image processing with the MELANIE system was used to create and compare the 2-D maps of [35S]methionine-labeled proteins under conditions 1)-3) with those of the controls. In a total of 24 2-D gels, 9 different MC were detected in at least 5 out 6 experimental replicates and were subjected to numeric analysis. The statistics showed a > 10% increase in GRP78 (p < 0.05), HSP27, cyclophilin, and ubiquitin after oxidative stress.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of oxidative stress and Ca2+ agonists on molecular chaperones in human umbilical vein endothelial cells. 749 68

The role of thrombin's catalytic groove in the interaction with serpin has been investigated by comparing the association rate constant (kon) of several mutated thrombins with various serpins. The results indicated that Glu192, located three residues prior to the catalytic serine, and the major insertion in the sequence of thrombin compared with trypsin (residues Tyr60A-Trp60D) play an important role in modulating thrombin's interactions with serpins. Replacement of Glu192 by glutamine increased by 3 orders of magnitude the kon value with alpha 1-antitrypsin (which has a P1 methionine) but did not markedly alter the kon value with serpins containing a P1 arginine. The des-PPW thrombin mutant (lacking residues Pro60B, Pro60C, and Trp60D) exhibited a similar kon value as thrombin with protease nexin-1 but a kon value 2 orders of magnitude lower with antithrombin III. Thus, the 60-loop insertion of thrombin appears critical for its interaction with antithrombin III but dispensable for the formation of a complex with protease nexin-1. Heparin increased markedly the kon values for antithrombin III and protease nexin-1 with all thrombin variants tested, but a more dramatic effect was observed with a thrombin mutant (des-ETW) lacking residues Glu146, Thr147, and Trp148 (on the opposite side of the catalytic site relative to the 60-loop insertion). At the optimum concentration, heparin increased the kon value of the des-ETW--antithrombin III interaction by nearly 5 orders of magnitude, considerably more than for thrombin, suggesting that heparin is able to compensate in part for the adverse effects of the des-ETW mutation on the structure of thrombin.
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PMID:Identification of thrombin residues that modulate its interactions with antithrombin III and alpha 1-antitrypsin. 754 66


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