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)

We have used platelets as a model system to study the function of c-src in signal transduction and cell adhesion. Numerous proteins were found to be phosphorylated on tyrosine in response to thrombin-induced platelet activation and aggregation. Two phases of phosphorylation were observed, with the second phase, but not the first, being inhibited by blocking platelet aggregation with an Arg-Gly-Asp-Ser tetrapeptide. As a first step towards identifying those proteins phosphorylated on tyrosine and to determine the specific role of p60src during platelet activation, we looked for changes in p60src kinase activity and for associations of p60src with other tyrosine phosphoproteins. The data presented here demonstrate an increase in p60src kinase activity within 1 min of thrombin-induced activation. Furthermore, p60src transiently associates with a tyrosine phosphoprotein during platelet activation and aggregation. This tyrosine phosphoprotein, p80/85, is a previously characterized cytoskeletal substrate for v-src in transformed cells. The data presented here suggest a model in which p60src functions in platelets to link upstream events, such as cell-surface adhesive interactions, with changes in platelet shape and cytoskeletal organization.
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PMID:Platelet activation leads to increased c-src kinase activity and association of c-src with an 85-kDa tyrosine phosphoprotein. 128 3

Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells.
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PMID:Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar). 147 61

Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. We have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, we isolated and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the "estrogen receptor-related protein" is HSP27, and the three major 29-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.
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PMID:The 29-kDa proteins phosphorylated in thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein. 176 35

To determine the relationship between the expression of bone proteins and the formation of mineralized-tissue matrix, the biosynthesis of non-collagenous bone proteins was studied in cultures of fetal-rat calvarial cells, which form mineralized nodules of bone-like tissue in the presence of beta-glycerophosphate. The temporal pattern of protein synthesis in both mineralizing and non-mineralizing cultures was studied by metabolic labelling with [35S]methionine, 35SO4(2-) or 32PO4(3-) over a 5-day period. After a 24 h labelling period, the culture media were harvested and the cell layers extracted sequentially with aq. 0.5 M-NH3, followed by 4 M-guanidinium chloride (GdmCl), 0.5 M-EDTA and a second extraction with 4 M-GdmCl. Protein associated with collagenous bone matrix was analysed after digestion with bacterial collagenase. On the basis of [35S]methionine labelling, the major proteins extracted from the mineralizing matrix were secreted phosphoprotein-1 (SPP-1; osteopontin), bone sialoprotein (BSP) and a 14 kDa phosphoprotein. The presence of SPP-1 and BSP in the conditioned media of both mineralizing and non-mineralizing cultures and their incorporation into the mineralizing nodules indicated that these proteins associate with preformed mineral crystals. However, some BSP was also present in GdmCl extracts and, together with a 35 kDa sulphated protein, was released from a bacterial-collagenase digestion of the tissue residue in both non-mineralizing and mineralizing cultures. Two forms of sulphated SPP-1 were identified, a highly phosphorylated 44 kDa species being the predominant form in the mineralized matrix. The BSP was more highly sulphated but less phosphorylated than SPP-1. Bone SPARC (secreted protein, acid and rich in cysteine) protein (osteonectin) was present almost entirely in the conditioned media and did not incorporate 32PO4(3-) or 35SO4(2-). The SPP-1 and the 14 kDa protein were susceptible to thrombin digestion, the 44 kDa SPP-1 being specifically cleaved into 28 and 26 kDa fragments. The fragments were labelled uniformly with [35S]methionine, but the 28 kDa fragment incorporated more 35SO4(2-), but less 32PO4(3-), than the 26 kDa fragment. These studies demonstrate that SPP-1 and BSP are the major osteoblast-derived bone proteins to bind to the bone mineral. That BSP also binds to the collagenous bone matrix indicates a potential role for this protein in linking the hydroxyapatite with collagen.
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PMID:Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, osteopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture. 200 15

Trypsin causes rapid activation of intact platelets that mimics many actions of thrombin, including the stimulation of phospholipase C (PLC). We have examined the effects of thrombin and trypsin on PLC in a platelet membrane preparation using exogenous [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. Trypsin induced PIP2 breakdown, which was maximal at 20 micrograms/ml, but was reduced at higher concentrations. alpha- and gamma-Thrombins also stimulated PLC-induced hydrolysis of PIP2 in membranes. This effect was inhibited by leupeptin. Exogenous [3H]phosphatidylinositol 4-monophosphate (PIP) was hydrolyzed in response to both thrombin and trypsin in the same ratio as PIP2. Activation of membrane-bound PLC persisted after removal of thrombin and trypsin. The hydrolysis of [3H]phosphatidylinositol was not activated by alpha-thrombin and trypsin. We examined the question of whether calpain was involved in the observed PLC activation by thrombin and trypsin. Although dibucaine activated a Ca2(+)-dependent protease as judged by the hydrolysis of actin-binding protein and by the activation of phosphoprotein phosphatases, it failed to stimulate the generation of phosphatidic acid in 32P-prelabeled platelets. Moreover, when PLC was assayed in the membranes, the addition of Ca2(+)-activated neutral proteinases did not increase the rate of hydrolysis of either PIP or PIP2. Our results show that proteases such as trypsin and thrombin are able to stimulate membrane-bound PLC, but this activation does not seem to be related to calpain.
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PMID:Persistent activation of platelet membrane phospholipase C by proteolytic action of trypsin and thrombin. 229 26

We describe the isolation, lipid-binding properties and partial amino acid sequence of PS-p68, a novel 68 kDa phosphatidylserine-binding protein from human platelets. PS-p68 is an abundant constituent of platelets, accounting for 0.5-0.75% of total cell protein. It was purified from platelet cytosol by affinity chromatography. Amino acid sequence analysis yielded no similarity to identified proteins. In contrast with most known phospholipid-binding proteins, PS-p68 does not bind Ca2+ and does not require Ca2+ for its binding of phosphatidylserine. Phosphatidylserine binding to PS-p68 was inhibited by phosphatidic acid and by alkylphospholipids. PS-p68 was isolated as a major phosphoprotein from 32P-labelled platelets and was found to function as a protein kinase C substrate in vitro. However, treatment of intact platelets with phorbol 12-myristate 13-acetate, thrombin or carbacyclin did not increase PS-p68 phosphorylation. Platelets appear to be the only blood cells containing PS-p68, which was not detected in neutrophils, monocytes and lymphocytes.
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PMID:Purification and characterization of a major phosphatidylserine-binding phosphoprotein from human platelets. 239 65

Preincubation of human platelets with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) has been shown previously to attenuate the ability of agonists both to suppress formation of cAMP and to stimulate hydrolysis of phosphoinositides. In the present study, we have examined whether the attenuation caused by PMA can be attributed to the phosphorylation of the alpha subunit(s) of Gi, a GTP-binding regulatory protein implicated in several pathways of signal transduction. PMA was found to promote the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [gamma-32P]ATP and [32P]H3PO4, respectively. None of the phosphoproteins, however, was precipitated by either of two antisera containing antibodies differing in specificities for epitopes within Gi alpha, despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently described Gz alpha or both Gz alpha and Gi alpha, precipitated a 40-kDa phosphoprotein. Phosphorylation of this protein occurred not only in response to PMA, but to thrombin and the thromboxane A2 analog U46619. These data suggest that activators of protein kinase C lead to the phosphorylation within platelets of a select population of G alpha subunits. The identified phosphoprotein is not Gi alpha, but is similar or identical to Gz alpha. Because Gz alpha does not contain the consensus site for ADP-ribosylation by the Bordetella pertussis toxin islet-activating protein, the data also suggest that effects of PMA on processes otherwise sensitive to this toxin are not exerted at the level of G proteins responsible for transduction.
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PMID:Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets. 250 48

A wide variety of rodent and human tumor cells secrete antigenically related phosphoproteins with molecular weights (Mr) of approximately 58,000 (hamster), 62,000 (rat, mouse), 67,000 (human) (Senger, D.R. and Perruzzi, C.A. (1985) Cancer Res. 45, 5818-5823). Expression of these phosphoproteins is transformation-related; tumor cells produce at least 10-fold or more of this protein as compared to their normal or untransformed counterparts. N-terminal and internal sequences derived from the rat tumor-secreted phosphoprotein indicate that it is identical to rat osteopontin, a bone protein with an Arg-Gly-Asp cell-binding sequence (Oldberg, A., Franzen, A. and Heinegard, D. (1986) Proc. Natl. Acad. Sci. USA 83, 8819-8823). Antibody raised to the Mr 62,000 rat tumor-secreted phosphoprotein was found to bind Mr 75,000 and Mr 35,000 components of human milk, indicating that milk contains antigenically related proteins. The Mr 75,000 protein, which is present in human milk at concentrations ranging from 3 to 10 micrograms/ml, has been purified to homogeneity. The Mr 35,000 component is apparently derived from the Mr 75,000 protein by proteolytic cleavage, and this cleavage also occurs in vitro in the presence of thrombin. N-terminal and internal amino acid sequences were derived from the Mr 75,000 milk protein and found to be similar (12/21 residues) to N-terminal and internal sequences derived from the rat tumor-secreted phosphoprotein and osteopontin. Moreover, sequence derived from the N-terminus of the human milk protein is identical to that of human bone sialoprotein I (the likely human homolog of rat osteopontin) (Fisher, L.W., Hawkins, G.R., Tuross, N. and Termine, J.D. (1987) J. Biol. Chem. 262, 9702-9708).
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PMID:Purification of a human milk protein closely similar to tumor-secreted phosphoproteins and osteopontin. 273 58

Metabolic labeling has revealed that rat bone cell populations in culture synthesize several forms of the secreted phosphoprotein, SppI. Most cell populations produced two major [32PO4]-labeled forms that behaved anomolously on SDS-PAGE migrating at 60 kDa and 56 kDa on 10% gels and 55 kDa and 44 kDa on 15% gels. Minor forms of intermediate sizes were also resolved. In normal bone cells the 60 kDa form was predominant and was the only form produced by the clonal bone cell line, RCA 11, whereas the 56 kDa a form predominated in the transformed bone cell line, ROS 17/2.8. In all populations [35S]-methionine-labeling revealed SppIs at approximately 60 kDa but no 56 kDa form. Each form of SppI was specifically cleaved by thrombin which generated fragments of approximately 28 kDa. Transforming growth factor beta 1 increased SppI mRNA levels 3 to 6-fold within 24 h in the normal bone cells, but no increase occurred in the ROS 17/2.8 cells. The elevated expression of SppI was reflected in a selective increase in the synthesis of the [32PO4]-and [35S]-methionine-labeled 60 kDa SppIs.
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PMID:Multiple forms of SppI (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: regulation by TGF-beta. 276 41

Human fibrinogen, a phosphoprotein, was either left untreated or phosphorylated by protein kinase C. Then both were dephosphorylated by calf intestinal alkaline phosphatase. The dephosphorylated fibrinogen gave an increased fibre thickness during thrombin-induced gelation. Whole blood anticoagulated by heparin, EDTA or sodium citrate, contained dephosphorylating activity against 32P-labeled fibrinogen, although there were significant differences in activity among the three anticoagulants.
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PMID:Dephosphorylation of human fibrinogen, previously phosphorylated in vitro by protein kinase C, by whole blood or intestinal alkaline phosphatase. Effects on thrombin-induced gelation of in vitro dephosphorylated human fibrinogen. 292 98


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