Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptor-activating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, thrombin and TRAP induced different sequences of activation causing distinctive morphological and biochemical changes. Platelets exposed to thrombin showed centralized organelles encircled by constricted microtubule coils and granules secreting their contents through narrow channels of the open canalicular system. In contrast, activation by TRAP induced swelling of the open canalicular system with organelles remaining randomly dispersed and microtubules peripherally distributed. Compared to thrombin activation, TRAP induced higher rates of actin polymerization; increased association of actin-binding protein, myosin, and alpha-actinin; and higher association of tyrosine-phosphorylated proteins with the insoluble cytoskeletal fraction. Secretion of intragranule substances, measured as expression of P-selectin and lysosomal integral membrane protein at the surface level, were similar for both agonists at equivalent concentrations. Our biochemical observations indicate that TRAP causes more intense changes in signaling through tyrosine phosphorylation of proteins associated with the cytoskeletal fraction than thrombin. However, as derived from ultrastructural observations, TRAP seems to be less efficient in triggering cytoskeletal assembly and internal contraction in an organized manner in contrast with the natural protease.
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PMID:TRAP induces more intense tyrosine phosphorylation than thrombin with differential ultrastructural features. 1205 26

We have previously shown that lysates from HL-60 myeloid leukemia cells or from peripheral blood monocytes are able to degrade alpha-actinin to form a 31-kDa amino-terminal fragment with monocyte/macrophage maturation promoting activity. In contrast, intact alpha-actinin, which is a 100-kDa actin-binding protein, has no differentiating activity. The aim of this study was to investigate the enzyme responsible for the degradation of alpha-actinin to form this fragment, named mactinin. The ability of cell lysates to degrade [125I]alpha-actinin in the presence of various enzyme inhibitors, including inhibitors of metalloproteinases, cysteine proteinases, and serine proteases, was measured. Phenylmethylsulfonyl fluoride (PMSF) was the only inhibitor able to prevent formation of mactinin by cell lysate degradation of alpha-actinin, suggesting that a serine protease is responsible for the digestion. Of the various serine proteases tested (thrombin, plasmin, and urokinase), only urokinase was able to produce a 31-kDa band. The urokinase-generated 31-kDa band promoted maturation in HL-60 cells. Amiloride, a specific inhibitor of urokinase, inhibited production of the 31-kDa alpha-actinin fragment by HL-60 cell lysates. For in vivo tests, inflammatory fluid (from bronchoalvelolar lavage) was collected from uPA (urokinase) knockout mice and their wild-type counterparts after intratracheal challenge with Pneumocystis carinii. Although most (6 of 8) wild-type mice had mactinin in their inflammatory fluid samples, none (0 of 8) of the uPA knockout mice had mactinin present (P<0.01). These results demonstrate that urokinase is necessary and sufficient for the formation of the monocyte/macrophage maturation promoting fragment, mactinin, in vitro and in vivo. These findings support the role of urokinase in the regulation of monocyte/macrophage functions, such as that occurring in inflammatory reactions.
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PMID:Urokinase is required for the formation of mactinin, an alpha-actinin fragment that promotes monocyte/macrophage maturation. 1218 60

Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.
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PMID:The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin. 1507 Sep

A growing body of literature indicates that chronic morphine exposure alters the expression and function of cytoskeletal proteins in addition to the well established interactions between mu opioid receptors and G proteins. In the present study, we hypothesized that chronic morphine alters the expression and functional effects of G alpha12, a G protein that regulates downstream cytoskeletal proteins via its control of RhoA. Our results showed that chronic morphine treatment decreased the expression of G alpha i2 (64%) and G alpha i3 (60%), had no effect of G alpha o, and increased G alpha12 (66%) expression in Chinese hamster ovary (CHO) cells expressing the cloned human mu opioid receptors (hMOR-CHO cells) but not in cells expressing a mutant mu opioid receptor that do not develop morphine tolerance and dependence (T394A-CHO cells). Morphine treatment had no significant effect on PAR-1 thrombin receptor-activated G protein activity, as measured by thrombin-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate binding. Chronic morphine treatment significantly enhanced thrombin-stimulated RhoA activity and thrombin-stimulated expression of alpha-actinin, a cytoskeletal anchoring protein, in hMOR-CHO cells. Proteomic analysis of two-dimensional gel spots prepared from hMOR-CHO cells showed that morphine treatment affected the expression of a number of proteins associated with morphological changes. Up-regulation of G alpha12 and alpha-actinin by chronic morphine was also observed in mouse brain. Viewed collectively, these findings indicate, for the first time, that chronic morphine enhances the G alpha12-associated signaling system, which is involved in regulating cellular morphology and growth, supporting other findings that chronic morphine may alter cellular morphology, in addition to cellular function.
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PMID:Chronic morphine up-regulates G alpha12 and cytoskeletal proteins in Chinese hamster ovary cells expressing the cloned mu opioid receptor. 1598 28

Reactive oxygen species have an important role in the control of platelet activity. Superoxide anion (O(2)(-)) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO(-)) that is formed from the reaction between O(2)(-) and nitric oxide (NO). There are conflicting data on ONOO(-) effects in platelets because it presents pro- or anti-aggregatory actions. 3-morpholinosydnonimine (SIN-1) co-generates NO and O(2)(-), yielding ONOO(-). Therefore, the present study aimed to investigate the mechanisms involved in the inhibition of human platelet adhesion by SIN-1. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8)platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001-100 microM) concentration-dependently inhibited adhesion, which was accompanied by marked increases in the cyclic GMP levels. In non-activated platelets, the soluble guanylate cyclase inhibitor ODQ prevented the SIN-1-induced cGMP elevations and adhesion inhibition. In thrombin-activated platelets, ODQ fully prevented the SIN-1-induced cGMP elevations, but only partly prevented the adhesion inhibition. The O(2)(-) and ONOO(-) scavengers superoxide dismutase (SOD) and -(-)epigallocatechin gallate, respectively, had minimal effects in non-activated platelets. The inhibition of activated platelets by SIN-1 was reversed by SOD and partly reduced by ECG. Western blot analysis of SIN-1-treated platelets showed a single 105 kDa-nitrated band. Nanospray LC-MS-MS identified the protein containing 3-nitrotyrosine residues as human alpha-actinin-1-cytoskeletal isoform. Our data show that platelet adhesion inhibition by SIN-1 in activated platelets involves cGMP-independent mechanism through O(2)(-) generation. Superoxide anion signaling pathway includes ONOO(-) formation and alpha-actinin nitration.
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PMID:The role of superoxide anion in the inhibitory effect of SIN-1 in thrombin-activated human platelet adhesion. 1989 7


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