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)

The importance of PLC activation in cell proliferation is evident from the fact that the hydrolysis of PtdIns(4,5)P2 is one of the early events that follow the interaction of many growth factors and mitogens with their respective receptors. However, the importance of PLC activation is not restricted to proliferation; it is one of the most common transmembrane signaling events elicited by receptors that regulate many other cellular processes, including differentiation, metabolism, secretion, contraction, and sensory perception. It is also clear that cell proliferation signaling does not always require PLC, as indicated by the fact that growth factors such as insulin and CSF-1 do not appear to elicit the hydrolysis of PtdIns(4,5)P2, even though the intracellular domains of their receptors carry a PTK domain and the receptors show topologies very similar to those of the PLC-activating growth factors PDGF, EGF, and FGF. The growth factor-dependent activation of PLC is initiated by the formation of a complex between the receptor PTK and PLC-gamma; the formation of this complex is mediated by a specific interaction between a tyrosine phosphate residue on the intracellular domain of PTK and the SH2 domain of PLC-gamma. The receptor PTK subsequently phosphorylates PLC-gamma, of which two distinct isozymes, PLC-gamma 1 and PLC-gamma 2, have been identified. Proliferation of T cells and B cells in response to the aggregation of their respective cell surface receptors is also accompanied by the activation of PLC-gamma isozymes at an early stage. Unlike growth factor receptors, the T cell and B cell receptors lack intrinsic PTK activity but associate with several non-receptor PTKs of the Src and Syk families. Although the specific kinases are not known, one or more of these enzymes phosphorylate and activate PLC-gamma 1 and PLC-gamma 2. Transduction of growth signals by G protein-coupled receptors such as those for thrombin or bombesin also requires PtdIns(4,5)P2 hydrolysis, which, in this instance, is mediated by PLC-beta isozymes. The PLC-beta subfamily consists of four distinct members: PLC-beta 1, PLC-beta 2, PLC-beta 3, and PLC-beta 4. Agonist interaction with specific G protein-coupled receptors causes the dissociation of Gq proteins into G alpha and G beta gamma subunits and the exchange of GDP bound to G alpha for GTP. The resulting GTP-bound G alpha subunit then activates PLC-beta isoforms by binding to the carboxyl-terminal region of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphoinositide-specific phospholipase C and mitogenic signaling. 749 69

A dysfibrinogenemia was attributable to a single amino acid substitution from glycine to cysteine at residue 15 of the B beta chain in a fibrinogen molecule designated as fibrinogen Fukuoka II. The fibrinogen Fukuoka II showed prolonged thrombin and reptilase times and impaired fibrinopeptide B release by thrombin, resulting in abolition of fibrin monomer repolymerization under physiological conditions. Repolymerization of the des-(B beta 1-42)-fibrin monomers, however, was not distinguished from the normal pattern of des-(B beta 1-42)-fibrin monomers, suggesting that no other abnormality existed in fibrinogen Fukuoka II. Although an additional cysteine was substituted at residue 15 of the B beta chain, fibrinogen Fukuoka II had no free sulfhydryl group within the molecule. Instead, fibrinogen Fukuoka II formed a disulfide bond with cysteine, albumin, another mutated B beta chain within the same molecule, or intermolecular dimeric fibrinogen Fukuoka II. The mutation in fibrinogen Fukuoka II was the same as that in fibrinogen Ise published previously (Yoshida, N., Wada, H., Morita, K., Hirata, H., Matsuda, M., Yamazumi, K., Asakura, S., and Shirakawa, S. (1991) Blood 77, 1958-1963). Fibrinogen Ise, however, has been described as having prolonged thrombin time but normal reptilase time. Reasons for the discrepancy were not clear. Analysis of the B beta 1-42 fragment showed that fibrinogen was heterogeneous at position 31 of the B beta chain with respect to proline or hydroxyproline.
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PMID:An abnormal fibrinogen Fukuoka II (Gly-B beta 15-->Cys) characterized by defective fibrin lateral association and mixed disulfide formation. 749 75

The Naka isoantigen is expressed on glycoprotein (GP) IV (CD36), a platelet membrane GP that has been identified as having a role in platelet interactions with collagen and thrombospondin and in binding Plasmodium falciparum-infected erythrocytes to endothelial cells and melanoma cells. We have studied normal platelets and Naka- platelets from two Japanese donors that have 1% of GPIV by concentration-dependent antibody binding and flow cytometry. We studied the adherence of normal and Naka- platelets to types I, III, and IV collagen in static and to type I collagen in flowing systems at high shear force. We have also studied aggregation of normal and Naka- platelets to type I collagen. Naka- platelets showed normal or increased aggregation to type I collagen and normal adhesion to types I, III, and IV collagen in the presence of Mg++ or EDTA. Platelet aggregation and adhesion were inhibited by the anti-alpha 2 beta 1 antibody 176D7 to the same extent in Naka- as in normal platelets. We also studied endogenous thrombospondin surface expression and found that thrombin-stimulated Naka- platelets expressed the same amount of thrombospondin as did normal platelets. From our studies with Naka- platelets, we cannot identify a definitive role for GPIV in platelet aggregation, in adhesion to types I, III, and IV collagen, or in endogenous thrombospondin binding to platelets.
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PMID:Platelet adhesion to collagen in individuals lacking glycoprotein IV. 751 49

Guinea pig bone marrow megakaryocytes were cultured on a type I rat tail collagen gel which stimulated proplatelet formation. Proplatelet formation was inhibited by monoclonal antibody LM609 to the alpha v beta 3 integrin (VnR), but not by monoclonal antibodies to the alpha 5, alpha 6, beta 1, or IIb beta 3(GPIIb-IIIa) integrin proteins. Megakaryocytes cultured on a plastic surface and stimulated with thrombin undergo a spreading and an adhesion reaction. This reaction is blocked in a dose-dependent manner by the tetrapeptide RGDS and by the monoclonal antibody PG2 to the GPIIb-IIIa integrin, but not by the monoclonal antibody LM609 to the VnR. Immunoprecipitation and affinity chromatography experiments demonstrate that guinea pig megakaryocytes have distinct GPIIb-IIIa and VnR integrins with similar electrophoretic mobility. Spreading was significantly inhibited in a dose-dependent fashion by drugs which elevate cellular cyclic AMP, including forskolin, dibutyryl cAMP, and isobutylmethylxanthine. In contrast to spreading, megakaryocyte proplatelet formation was stimulated by these agents in a dose-dependent manner. Megakaryocyte spreading was stimulated by the protein kinase C (PKC) activator phorbol myristate acetate (PMA) and inhibited by the PKC inhibitors Calphostin C and K5720 in a dose-dependent manner. PKC inhibitors did not inhibit megakaryocyte proplatelet formation. These results demonstrate that the closely related VnR and GPIIb-IIIa integrins regulate different aspects of megakaryocyte morphological change and appear to be associated with different second messenger systems.
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PMID:Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading. 753 14

The extracellular matrix proteins fibulin-1 (variants C and D) and fibulin-2 occur in basement membranes and in vessel walls and are thus potential candidates for cellular interactions. Recombinant forms of these proteins were obtained from stably transfected kidney cell clones and examined for cell-adhesion activity and binding to five different purified integrins. The two variants of mouse fibulin-1 were inactive in all these assays. Mouse fibulin-2, however, bound to alpha IIb beta 3 integrin almost as strongly as fibrinogen, while a lower activity was found for alpha V beta 3 and almost none for alpha 5 beta 1 integrin. Synthetic SVPRGDLDG peptide, corresponding to the single RGD site of mouse fibulin-2, was a strong antagonist of alpha IIb beta 3 integrin binding. Its affinity for alpha V beta 3 and alpha 5 beta 1 integrins was, however, 10- to 50-fold lower compared to GRGDS. Mouse fibulin-2 also promoted adhesion of thrombin-stimulated platelets and of some established cell lines which could be inhibited by RGD peptides. Human fibulin-2, in which the RGD sequence is changed to RSS, bound less strongly to alpha IIb beta 3 integrin and showed no cell-adhesion activity. Together these data suggest a potential role in hemostatic control for mouse fibulin-2 and possibly also for human fibulin-2.
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PMID:Integrin-binding and cell-adhesion studies of fibulins reveal a particular affinity for alpha IIb beta 3. 754 56

Basic fibroblast growth factor (bFGF; FGF-2) lacks a signal sequence and thus is not secreted by classical pathways. It has been speculated that one mode of bFGF release may be injury, either sublethal or lethal; and, transient disruption of the plasma membrane has been shown to release bFGF [Muthukrihnan et al. (1991): J Cell Physiol 148:1-16]. This observation has led to the concept of bFGF as a "wound hormone," involved in tissue integrity and repair. Findings of elevated bFGF following injury in vivo support this concept. Using an in vitro model, we have examined the regulation of bFGF gene expression following its release by sublethal injury. Analysis of bFGF protein by ELISA revealed that scraping subconfluent bovine aortic EC (BAE) released up to 80% of their bFGF. Following scraping, there was a 4- to 10-fold increase in the steady state level of bFGF mRNA, which reached a maximum at 2-3 h. There was a parallel increase in protein so that by 6 h after the scrape-induced release, bFGF levels were restored to those measured prior to scraping. Since bFGF has been reported to induce its own expression, we hypothesized that the released bFGF might be responsible for the increase in bFGF mRNA. However, inclusion of neutralizing antibodies against bFGF had a negligible effect on the scrape-induced increase in bFGF mRNA levels. Because of the important role of transforming growth factor type-beta 1 (TGF-beta 1), the plasminogen/plasminogen activator system, and thrombin in wound healing, we investigated their potential contributions to the increase in bFGF expression. Addition of anti-TGF-beta 1 antibodies, plasminogen activator inhibitor-1 (PAI-1), or the thrombin inhibitory combination of heparin and anti-thrombin III (AT III) to the cells at the time of scraping blocked about 50% of the increase in bFGF mRNA; the effects of these agents were not additive. The suppression of bFGF mRNA was associated with a proportional reduction in bFGF protein. Inclusion of the antagonists for 2 h at the time of scraping led to reduced cell proliferation, suggesting that cell-associated bFGF may be required for recovery and growth. Finally, studies to characterize the molecular mechanisms underlying the increased bFGF mRNA following sublethal injury revealed an increase in the transcriptional activation of bFGF gene. These results indicate that in spite of the fact that bFGF is not a secreted protein, levels of bFGF in the cell are tightly regulated. Furthermore, these findings suggest a role for bFGF in recovery from cell injury.
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PMID:Regulation of basic fibroblast growth factor (bFGF) gene and protein expression following its release from sublethally injured endothelial cells. 759 55

Cyclic mechanical strain (1 Hz) causes a mitogenic response in neonatal rat vascular smooth muscle cells due to production and secretion of PDGF. In this study, the mechanism for sensing mechanical strain was investigated. Silicone elastomer strain plates were coated at varying densities with elastin, laminin, type I collagen, fibronectin, or vitronectin. Strain was applied by cyclic application of a vacuum under the dishes. Cells adhered, spread, and proliferated on each matrix protein, but the mitogenic response to strain was matrix dependent. Strain increased DNA synthesis in cells on collagen, fibronectin, or vitronectin, but not in cells on elastin or laminin. When strain was applied on matrices containing both laminin and vitronectin, the mitogenic response to strain depended upon the vitronectin content of the matrix. Fibronectin, in soluble form (0-50 micrograms/ml), and the integrin binding peptide GRGDTP (100 micrograms/ml) both blocked the mitogenic response to mechanical strain in cells grown on immobilized collagen. Neither soluble laminin nor the inactive peptide GRGESP blocked the response to strain. GRGDTP did not alter the mitogenic response to exogenous PDGF or alpha-thrombin but did prevent the secretion of PDGF in response to strain. Furthermore, GRGDTP, but not GRGESP, prevented strain-induced expression of a PDGF-A chain promoter 890 bp-chloramphenicol acetyltransferase construct that was transiently transfected into vascular smooth muscle cells. Finally, the response to strain was abrogated by antibodies to both beta 3 and alpha v beta 5 integrins but not by an antibody to beta 1 integrins. Thus interaction between integrins and specific matrix proteins is responsible for sensing mechanical strain in vascular smooth muscle cells.
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PMID:Mechanical strain of rat vascular smooth muscle cells is sensed by specific extracellular matrix/integrin interactions. 759 24

Fibrin forms the cohesive network of hemostatic plugs and thrombi, and it also provides the temporary matrix for initial support of healing and revascularization. Because cell proliferation is needed for revascularization after vessel injury, we have characterized structural requirements of fibrin needed to support cell proliferation on fibrin in vitro. Proliferation of cultured human endothelial cells and fibroblasts was measured by 3H-thymidine incorporation on fibrin surfaces varying in structure. Fibrin prepared with thrombin and lacking both fibrinopeptides A and B (desAB fibrin) supported proliferation of both endothelial cells and fibroblasts. In contrast, fibrin prepared with reptilase, which cleaves only fibrinopeptide A, supported significantly less proliferation. Also, fibrin prepared by thrombin treatment of fibrinogen lacking residues beta 1-42 supported only a low level of proliferation. Therefore, fibrinopeptide B cleavage and exposure of beta 15-42 enhanced proliferation of cells on fibrin. Specific proteolytic inhibitors were used to eliminate the potential mitogenic effects of residual fibrin-bound thrombin. Additional controls showed that neither catalytically inactive thrombin nor addition of the thrombin receptor-activating peptide (SFLLRNPNDKYEPF [SFLL]) stimulated proliferation on desA fibrin. The results indicate that cell proliferation on fibrin is enhanced by fibrinopeptide B cleavage and exposure of the amino terminus of the fibrin beta chain. They also show that specific structural features of the temporary fibrin matrix formed at sites of injury may modulate the proliferative response of vascular cells.
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PMID:Cell proliferation on fibrin: modulation by fibrinopeptide cleavage. 765 10

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.
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PMID:Immunolocalization of beta 1 integrins in platelets and platelet-derived microvesicles. 768 92

The permeability of bovine pulmonary artery endothelial (CPAE) monolayers to Evans blue-labelled albumin (Evans blue-albumin) has been measured in vitro. Thrombin caused a concentration-dependent increase in Evans blue-albumin clearance across endothelial monolayers. Isoprenaline inhibited thrombin-induced Evans blue-albumin clearance in a concentration-dependent manner (EC50 21 nM). This effect was mimicked by the selective beta 2-adrenoceptor agonists salbutamol (EC50 64 nM) and salmeterol (EC50 2.7 nM), but not by the selective beta 1-adrenoceptor agonist, RO-363 ((1-[3',4'-dihydroxyphenoxy]-2-hydroxy-[3",4"- dimethoxyphenethylamino]-propane)oxalate), nor by the selective beta 3-adrenoceptor agonist, CL-316,243 (disodium (R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3- benzodioxole-2,2-dicarboxylate). Isoprenaline, salbutamol and salmeterol, but not RO-363 or CL-316,243 produced small, but significant reductions in Evans blue-albumin clearance across unstimulated endothelial monolayers. Inhibition of the response to thrombin by isoprenaline was antagonised by the selective beta 2-adrenoceptor antagonist, ICI-118,551 ((erythro-DL-1(7-methylindan-4- yloxy)3-isopropylaminobutan-2-ol), pKB 8.4). Salmeterol also inhibited hydrogen peroxide-stimulated Evans blue-albumin clearance. Hence, the widely used beta 2-adrenoceptor agonists, salbutamol and salmeterol, are able to reduce endothelial permeability at nanomolar concentrations.
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PMID:Beta 2-adrenoceptors mediate a reduction in endothelial permeability in vitro. 776 83


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