Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies showed that homocysteine, a thrombo-atherogenic and atherogenic agent, inhibits an endothelial thrombomodulin-protein C anticoagulant pathway. We examined whether homocysteine might affect another endothelial anticoagulant mechanism; i.e., heparin-like glycosaminoglycan-antithrombin III interactions. Incubations of porcine aortic endothelial cell cultures with homocysteine reduced the amount of antithrombin III bound to the cell surface in a dose- and time-dependent fashion. The inhibitory effect was observed at a homocysteine concentration as low as 0.1 mM, and the maximal suppression occurred at 1 mM of homocysteine after 24 h. In contrast with a marked reduction in the maximal antithrombin III binding capacity (approximately 30% of control), the radioactivity of [35S]sulfate incorporated into heparan sulfate on the cell surface was minimally (< 15%) reduced. The cells remained viable after homocysteine treatment. Although neither net negative charge nor proportion in total glycosaminoglycans of cell surface heparan sulfate was altered by homocysteine treatment, a substantial reduction in antithrombin III binding capacity of heparan sulfate isolated from homocysteine-treated endothelial cells was found using both affinity chromatography and dot blot assay techniques. The antithrombin III binding activity of endothelial cells decreased after preincubation with 1 mM homocysteine, cysteine, or 2-mercaptoethanol; no reduction in binding activity was observed after preincubation with the same concentration of methionine, alanine, or valine. This sulfhydryl effect may be caused by generation of hydrogen peroxide, as incubation of catalase, but not superoxide dismutase, with homocysteine-treated endothelial cells prevented this reduction, whereas copper augmented the inhibitory effects of the metabolite. Thus, our data suggest that the inhibited expression of anticoagulant heparan sulfate may contribute to the thrombogenic property resulting from the homocysteine-induced endothelial cell perturbation, mediated by generation of hydrogen peroxide through alteration of the redox potential.
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PMID:Homocysteine, a thrombogenic agent, suppresses anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells. 837 90

Surfactant protein C (SP-C) is a 3.7-kDa hydrophobic peptide isolated from organic extracts of pulmonary surfactant which is secreted by alveolar type II cells after synthesis and posttranslational processing of a 21-kDa proSP-C peptide (SP-C21). Previously characterized epitope-specific proSP-C antisera were used to study early proteolytic steps of proSP-C processing by adult rat type II cells. Western blotting and immunocytochemistry using anti-NPROSP-C (epitope = Met10-Glu23) each demonstrated marked attenuation of proSP-C protein expression by culture on plastic. Processing was therefore studied by metabolic labeling of freshly isolated type II cells maintained in suspension in serum-free media. With the use of anti-NPROSP-C, immunoprecipitation of cell lysates continuously labeled for 4 h with [35S]methionine demonstrated radiolabeled bands of M(r) 21, 16, and 10-6,000 while anti-CTERMSP-C (epitope = Ser149-Ser166) failed to detect 35S-bands of M(r) < 16,000. Pulse-chase studies demonstrated synthesis of 35S-proSP-C21 with a time-dependent dependent appearance of 16-kDa and 10- to 6-kDa forms which was blocked by addition of brefeldin A. SP-C precursors were not detected in the media. Quantitative analysis of the major bands by direct beta-counting indicated a precursor-product relationship between SP-C21 and SP-C16. These results demonstrate the utility of freshly isolated type II cells for characterization of SP-C synthetic pathways and show that early proSP-C processing events include synthesis of a 21-kDa primary translation product followed by extensive intracellular proteolysis of the proSP-C COOH-terminal in subcellular compartments of type II cells which are distal to the trans-Golgi network.
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PMID:Synthesis and processing of hydrophobic surfactant protein C by isolated rat type II cells. 857 36

Protein C Nagoya, an elongated variant of the human protein C, is retained and degraded within the cells in which it is produced (Yamamoto et al, J Clin Invest 90:2439, 1992). To determine the subcellular localization of the protein C Nagoya, the recombinant protein C bearing this mutation was expressed in Chinese hamster ovary (CHO) cells. The mutant protein C was not secreted from the cells and remained susceptible to endo-beta-N-acetylglucosaminidase H (endo H). Immunoelectron microscopy indicated that protein C Nagoya was retained in the endoplasmic reticulum (ER), whereas wild-type protein C was observed in both the ER and the Golgi apparatus. Metabolic radiolabeling with [35S] methionine in combination with chemical cross-linking showed that the protein C Nagoya existed in the ER as a complex with 78-kD glucose-regulated protein (GRP78) and 94-kD glucose-regulated protein (GRP94). Because both GRP78 and GRP94 associate to a far lesser degree with wild-type protein C than with protein C Nagoya, our data suggest that both stress proteins function as molecular chaperones and work in concert with the folding and assembly of protein C. These findings extend our understanding the molecular pathogenesis of protein C deficiency.
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PMID:Protein C Nagoya, an elongated mutant of protein C, is retained within the endoplasmic reticulum and is associated with GRP78 and GRP94. 863 75

Myocardial infarct size has been measured after 1 hr of mechanical occlusion of the circumflex coronary artery and 5 hr of reperfusion in control dogs infused with saline, and in dogs infused with activated protein C (aPC) (1mg/kg/hr i.v.). Infusion of aPC during reperfusion produced a sustained doubling of activated partial thromboplastin time and no change in thrombin time at a final plasma parent drug concentration of 1.25 +/- 0.11 mug/ml. aPC infusion did not alter systolic arterial pressure, cardiac rate or the rate pressure product when compared to time-related alterations observed in control dogs. ST-segment deviation and the intensity and duration of cardiac arrhythmias associated with reperfusion of ischemic myocardium also were similar between groups. Resultant infarct sizes were 34.8 +/- 3.9 and 33.2 +/- 6.2% of the left ventricular mass placed at risk of necrosis in control and aPC-treated dogs. respectively. aPC infusion was associated with a small reduction in leukocytosis in response to myocardial ischemic injury, but did not alter the localization of leukocytes within ischemic and infarcted myocardium. In vitro concentrations of aPC (0.3, 1 and 3 mug/ml), comparable to the plasma concentration that inhibited blood coagulation in dogs, did not alter superoxide production or CD11b/CD18-mediated adhesion of chemotactic factor f-Met-Leu-Phe-stimulated neutrophils. Present data indicate that aPC lacks cardioprotectant activity at an infusion rate inhibiting coagulation. Apart from inhibition of thrombin generation, no evidence of an anti-inflammatory effect of aPC was observed.
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PMID:Evaluation of activated protein C on canine infarct size in a nonthrombotic model of myocardial reperfusion injury. 878 41

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.
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PMID:The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern. 881 72

Little is known about the the signalling pathways driving the adenoma-to-carcinoma sequence in human colonic epithelial cells. Accumulation and activation of the src tyrosine kinase in colon cancer suggest a potential role of this oncogene in this early progression. Therefore, we introduced either activated src (m-src), polyoma-MT alone or combined with normal c-src in the adenoma PC/AA/C1 cell line (PC) to define the function and phenotypic transformations induced by these oncogenes in familial adenomatous polyposis (FAP) colonic epithelial cells. Functional expression of these oncoproteins induced the adenoma-to-carcinoma conversion, overexpression of the hepatocyte growth factor (HGF) receptor Met, but failed to confer invasiveness in vivo and in vitro, or to produce alterations in cell proliferation and differentiation. In contrast, PC-msrc cells became susceptible to the HGF-induced invasion of collagen gels and exhibited sustained activation of the pp60src tyrosine kinase and Tyr phosphorylation of the 120-kDa E-cadherin, which was further increased by HGF Transcripts of HGF were clearly identified by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot in the parental and transformed PC cells, suggesting an autocrine mechanism. Taken together, the data indicate that: (1) experimental activation of src and PyMT pathways directly induces tumorigenicity and Met upregulation in a colon adenoma cell line; (2) HGF-activated Met and src cooperate in inducing invasion; (3) in view of the molecular associations between catenins and cadherin or the tumour-suppressor gene product APC, the cell adhesion molecule E-cadherin may constitute a downstream effector of src and Met.
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PMID:Progression of familial adenomatous polyposis (FAP) colonic cells after transfer of the src or polyoma middle T oncogenes: cooperation between src and HGF/Met in invasion. 901 33

Residue 192 (chymotrypsin numbering system) in thrombin, activated protein C, and factor Xa contributes to the specificity of these enzymes toward their substrates and inhibitors. A Glu192-->Gln mutation in both thrombin and activated protein C yielded enzymes that reacted better with some, but not all, of their natural substrates and inhibitors. To determine whether the specificity change is due to productive interactions of Gln192 with substrates and inhibitors or elimination of repulsive electrostatic interactions, we prepared forms of thrombin, des-(1-45)-factor Xa and activated des-(1-45)-protein C with Glu, Gln, or Met at position 192 and compared their activities toward inhibitors and substrates. All mutants had nearly normal amidolytic activity. The Glu192-->Gln and Glu 192-->Met mutations of thrombin and activated des-(1-45)-protein C increased the second-order rate constant (k2) of inhibition by alpha 1-antitrypsin about 700-fold and 170-fold for thrombin, and 185-fold and 150-fold for activated des-(1-45)-protein C, respectively. [E192]faxtor Xa, but not [M192]factor Xa, was resistant to inhibition by alpha 1-antitrypsin. Glu-->Gln or Glu-->Met mutants of both thrombin and activated des-(1-45)-protein C were effectively inhibited by tissue factor pathway inhibitor (K1 < 200 nM) and, except for [M192]thrombin, by bovine pancreatic trypsin inhibitor (K1 < 60 nM). With respect to substrate cleavage, Glu192-->Gln and Glu192-->Met mutations of activated des-(1-45)-protein C both inactivated factor Va 2-3-fold faster than activated des-(1-45)-protein C. Thrombin and [M192]thrombin activated protein C at similar slow rates compared to rapid activation by [Q192]thrombin. The Gln192-->Met mutants of des-(1-45)-factor Xa activated prethrombin 1.8-11-fold slower than wild-type enzyme. With thrombomodulin or factor Va present, these differences in protein C and prethrombin 1 activation rates were decreased to about 2-fold. We conclude that residue 192 contribution to enzyme specificity is achieved by both productive and repulsive interactions and that the magnitude and nature of the participation varies among enzymes, substrates and inhibitors.
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PMID:Molecular basis of residue 192 participation in determination of coagulation protease specificity. 902 71

Hyperhomocysteinemia is a condition which, in the absence of kidney disease, indicates a disrupted sulfur amino acid metabolism, either because of vitamin (folate, B12 and B6) deficiency or a genetic defect. Epidemiological evidence suggests that mild hyperhomocysteinemia is associated with increased risk of arteriosclerotic disease and stroke. The relationship between hyperhomocysteinemia and thrombosis has been investigated in 10 studies involving a total of 1200 patients and 1200 controls. Eight of these studies demonstrated positive association with odds ratios that ranged from 2 to 13. This association was enhanced by including a methionine loading test. There is some evidence which suggests that hyperhomocysteinemia and APC resistance have a synergistic effect on the onset of thrombotic disease. Studies on the mechanism that underlies the relationship between thrombosis and hyperhomocysteinemia used non-physiologically high levels of homocysteine, rendering the data doubtful as to their patho-physiological relevance.
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PMID:Hyperhomocysteinemia and thrombosis: acquired conditions. 919 9

The link between vascular disease and elevated homocysteine levels has been recognized for more than 30 years, and association with moderately elevated levels has been suspected for 20 years. Homocysteine is a sulfhydryl-containing amino acid that is formed by the demethylation of methionine. It is normally catalysed to cystathionine by cystathionine beta-synthase a pyridoxal phosphate-dependent enzyme. Homocysteine is also remethylated to methionine by methionine synthase, a vitamin B12 dependent enzyme and by methylenetetrahydrofolate reductase. Environmental factors such as folate, or vitamin B12, or vitamin B6 deficiencies and genetic defects such as cystathionine beta-synthase or abnormality of methylene-tetrahydrofolate reductase or some vitamin B12 metabolism defects may contribute to increasing plasma homocysteine levels. Normal fasting levels of homocysteine lie within the range 6-16 mumol/l. Apart from differences in assay methods, age, sex and nutritional status may affect the plasma levels. Though it is now well known that homocysteine is an independent risk factor for premature vascular disease, the pathogenesis of homocysteine-induced vascular damage is, for the most part, unknown. It may be multifactorial, including direct homocysteine damage to the endothelium, an enhanced low-density lipoprotein peroxidation, an increase of platelet thromboxane A2, or a decrease of protein C activation.
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PMID:[Deregulation of homocysteine metabolism and consequences for the vascular system]. 923 30

To assess whether certain abnormalities of the sulfated amino acid metabolism are associated with the occurrence of thromboembolic events in patients with inherited thrombophilic conditions, the levels of homocyst(e)ine, before or after methionine load, and the presence of the Ala223Val substitution in the 5,10-methylenetetrahydrofolate reductase (MTHFR) were evaluated in 119 subjects with a congenital single thrombophilic condition (type I deficiency of antithrombin n = 10, protein C n = 24, protein S n = 16; activated protein C resistance due to factor V Leiden mutation n = 69). Sixty-three subjects had experienced at least one documented thrombotic event, while the remaining 56 subjects were still free from any thrombotic symptom. Our results show that (1) high homocyst(e)ine levels, either in fasting condition or after methionine load, were not more frequent in subjects with inherited thrombophilic alterations (14.4%) than in normal control subjects (10% by definition) and (2) the frequency of hyperhomocyst(e)inemia was similar in thrombophilic subjects, who already have (14.3%) or have not (14.6%) experienced thrombotic events. As regards the MTHFR mutation, the homozygous condition was present in 23.2% of the thrombophilic patients versus 17.5% in the control subjects, a nonsignificant difference. The mutation was slightly more frequent in those thrombophilic subjects who had suffered a thrombotic episode (25.5%) versus those with no thrombosis (20.8%), with odds ratios of 1.61 (confidence interval (CI) = 0.58-4.52) and 1.24 (CI = 0.42-3.43), respectively. These differences were also nonsignificant. It is concluded that in subjects with inherited thrombophilias, a condition of hyperhomocyst(e)inemia "per se" is not a factor increasing the risk of thrombosis. The risk enhancement conferred by the MTHFR mutation, if any, seems to be slight or limited, and its significance could be ascertained only in a large multicenter trial.
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PMID:Hyperhomocyst(e)inemia and a common methylenetetrahydrofolate reductase mutation (Ala223Val MTHFR) in patients with inherited thrombophilic coagulation defects. 940 77


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