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)

Twenty-eight male Sprague Dawley rats were divided into two groups: a control group (C) of 15 animals and a streptozotocin-induced diabetes group mildly balanced by insulin (D) of 13 animals. After 15 weeks, plasma and high-density lipoprotein (HDL) lipids were determined in each group. Apoprotein A-I concentration was evaluated in HDL fractions. The capacity of the HDL fraction to inhibit thrombin and ADP-induced aggregation of normal platelets was determined for each rat, and in an additional experiment the relation dose-effect of HDL was established. The effect of HDL of the two groups on the stabilization of prostacyclin was compared by aggregation bioassay. After 15 weeks, HDL cholesterol (free + esterified). After 15 weeks, HDL cholesterol (free + esterified) tended to increase in group D compared with group C (P < 0.08). By contrast, apoprotein A-I was very significantly decreased in HDL-D compared with HDL-C (P < 0.001). These alterations were accompanied by a significantly decreased capacity of HDL (60 micrograms/ml platelet suspension) to inhibit ADP-induced aggregation (P< 0.0001) in group D compared with group C. Furthermore, HDL-D incubated 45 or 90 min with prostacyclin showed a significantly decreased capacity to stabilize prostacyclin compared with HDL-C (P<0.04; P <0.03, respectively). These alterations in HDL could be involved in thrombosis and atheromatous complications associated with this disease.
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PMID:Decreased capacity to inhibit platelet hyperactivity and to stabilize prostacyclin of high-density lipoproteins in experimental diabetes. 859 Jul 86

Calcium-dependent conformational states of calmodulin (CaM) were probed by thrombin to determine quantitative differences in the susceptibility of two bonds: Arg37-Ser38 (R37-S38, near site I in the N-terminal domain) and Arg106-His107 (R106-H107, near site III in the C-terminal domain). Quantitative thrombin footprinting of a discontinuous equilibrium calcium titration of wild-type calmodulin showed that the R37-S38 bond of the apoprotein was cleaved at a barely detectable level while the R106-H107 bond was maximally susceptible. Calcium binding to sites III and IV monotonically protected R106-H107 from proteolysis; concomitantly, the susceptibility of R37-S38 increased. However, calcium binding to sites I and II protected R37-S38 from cleavage, yielding a peaked biphasic profile composed of equal and opposite transitions. Both bonds were fully protected when calmodulin was saturated with calcium. Susceptibility profiles resolved from the fractional abundance of primary cleavage products (peptides 1-37, 38-148, 1-106, 107-148) were interpreted as directly reflecting calcium-induced conformational changes in whole calmodulin; free energies of calcium binding and cooperativity were estimated. Secondary cleavage was never observed; both R37 and R106 were sites of thrombinolysis in whole calmodulin only. In studies of E140Q-CaM (having a mutation in site IV), the susceptibility of R37-S38 decreased monotonically. Thus, the biphasic character of cleavage of R37 in helix B was not intrinsic to that domain but depended on propagation of effects of calcium-induced changes in the C-terminal domain. The observed patterns of susceptibility indicated that partially saturated wild-type calmodulin adopts at least one intermediate conformation whose structure is determined by calcium-mediated interactions between the domains.
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PMID:Calcium-induced interactions of calmodulin domains revealed by quantitative thrombin footprinting of Arg37 and Arg106. 860 32

We have previously shown that plasma HDL-E, a minor subclass of high-density lipoproteins (HDL) containing apolipoprotein (apo) E, has a potent anti-platelet effect and implicated apoE as the active constituent. Recently, apoE complexes with phospholipids (DMPC) were reported to inhibit thrombin-induced aggregation by sequestering platelet membrane cholesterol. Here we demonstrate that platelet cholesterol depletion is an improbable explanation for the suppressive effect of apoE:DMPC on ADP-mediated platelet aggregation; only 0.5% of cholesterol was released prior to addition of ADP to initiate aggregation while lactoferrin, which does not accept cellular cholesterol, was also inhibitory. Previous studies have shown that apoE and lactoferrin are both bound by platelets but whether this provides the initial stimulus for suppression of aggregation remains to be established.
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PMID:Inhibition of ADP-induced platelet aggregation by apoE is not mediated by membrane cholesterol depletion. 905 55

Alzheimer's amyloid beta-protein (A beta) is a modified, pathogenic form of a constitutive host protein, soluble amyloid beta-protein (sA beta). Both are conformational isomers encoded by the gene for the beta-amyloid precursor protein (APP), located on chromosome 21. sA beta and A beta have identical sequence but are thought to differ in their secondary structure and physicochemical properties, hence they are conformational isomers. sA beta is easily degraded, while A beta is particularly resistant. A beta has a high beta-pleated sheet content, while sA beta is thought to be more random-coil and/or alpha-helical. A beta, unlike sA beta, adopts an amyloidogenic conformation, forms aggregates and gives rise to fibrils. Most early-onset forms of Alzheimer's disease (AD) have been linked to mutations of the presenilin 1, presenilin 2 or APP genes, located on chromosomes 14, 1 and 21, respectively. Their relationship to amyloidogenesis is being investigated. On the other hand, the major risk factor for the most common form, sporadic and familial late-onset AD, is the presence of the apoE epsilon 4 allele. Recent studies have shown that a 10 kDa C-terminal fragment of apoE is complexed to A beta in neuritic plaques and that apoE isoforms can modulate amyloid formation in vitro. Moreover, thrombin cleavage of apoE generates a similar C-terminal fragment that can form amyloid-like fibrils. Thus neuritic plaques may contain both A beta and apoE amyloid fibrils. AD can be neuropathologically defined by the presence of several interacting proteins that can adopt an amyloidogenic conformation. This has led us to hypothesize that in AD, amyloidosis may be reactive rather than causative.
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PMID:Apolipoprotein E and amyloidogenesis. 891 8

A 22 kDa fragment of apoE containing a putative cytotoxi domain was identified in postmortem human brain tissue and fresh CSF. This fragment is apparently equivalent to the major apoE thrombin cleavage product. In vitro toxicity assays demonstrate that the corresponding fragment derived from recombinantly expressed human apoE is toxic to primary neurons in culture and that the E4-derived fragment is significantly more toxic than the fragment derived from the E3 isoform. These results suggest that proteolytic fragments of apoE may play a direct role in the pathology associated with AD and other diseases in which apoE has been implicated.
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PMID:A thrombin cleavage fragment of apolipoprotein E exhibits isoform-specific neurotoxicity. 898 17

We have previously shown that plasma HDL-E, a minor subclass of high-density lipoproteins (HDL) containing apolipoprotein (apo) E, has a potent anti-platelet effect and implicated apoE as the active constituent. Recently, apoE complexes with phospholipids (DMPC) were reported to inhibit thrombin-induced aggregation by sequestering platelet membrane cholesterol. Here we demonstrate that platelet cholesterol depletion is an improbable explanation for the suppressive effect of apoE:DMPC on ADP-mediated platelet aggregation; only 0.5% of cholesterol was released prior to addition of ADP to initiate aggregation while lactoferrin, which does not accept cellular cholesterol, was also inhibitory. Previous studies have shown that apoE and lactoferrin are both bound by platelets but whether this provides the initial stimulus for suppression of aggregation remains to be established.
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PMID:Inhibition of ADP-induced platelet aggregation by apoE is not mediated by membrane cholesterol depletion. 861 Feb 78

The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1-292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.
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PMID:Human low density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli. 932 68

Apolipoprotein (apo) E plays an important role in lipid metabolism, and the major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different metabolic effects. Apolipoprotein E4 is associated with a higher risk of both heart disease and Alzheimer's disease (AD). Patients homozygous for apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2 may be protective against AD. Structure/function studies have proved to be a useful tool in understanding how the different apoE isoforms result in different pathological consequences. As these studies continue, it is essential to have a reliable method to produce large quantities of apoE and mutants of apoE. We describe here a method of apoE production in Escherichia coli strain BL21(DE3). The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter and a fusion partner (thioredoxin). The T7 promoter results in high expression of an easily purified His-tagged fusion protein. A thrombin recognition site was positioned in the expression vector so that only two novel amino acids (Gly-Ser) are added to the amino terminus of apoE following the removal of thioredoxin. Approximately 20 mg of apoE is obtained from a 1-liter culture. The major isoforms of apoE produced with this system were extensively characterized for their ability to bind the low-density lipoprotein (LDL) receptor, for their characteristic lipid association preferences, and for their stability as measured by guanidine denaturation. The recombinant proteins behaved identically to plasma-derived apoE isoforms.
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PMID:Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli. 1041 18

Thioredoxin fusion expression vectors for two carboxyl-terminal fragments of human apolipoprotein (apo) E (residues 223-272 and 223-299) were generated from an apoE cDNA with the objective of obtaining structural information on this functionally important region of apoE by X-ray crystallography. A thrombin cleavage recognition site was positioned at the fusion junction to release the apoE fragments from the fusion protein. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. After gel filtration and ion exchange chromatography, yields of each fragment were approximately 14 mg/L. Both fragments bind to the phospholipid dimyristoylphosphatidylcholine in a manner similar to that of the 216-299 fragment of apoE isolated from plasma, which represents the major lipid-binding region of the protein. Orthorhombic crystals of the apoE 223-272 fragment that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0-7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 35.17 A, b = 38.95 A, and c = 133.27 A.
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PMID:Carboxyl-terminal domain of human apolipoprotein E: expression, purification, and crystallization. 1054 75

We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE(-/-)) x LDL receptor-deficient (LDLR(-/-)) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE(-/-) x LDLR(-/-) mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 micromol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol x kg x (-1)min(-1)) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.
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PMID:Thrombin inhibitor reduces myocardial infarction in apoE-/- x LDLR-/- mice. 1503 Nov 24


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