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)

Glycoprotein G (thrombin-sensitive protein, thrombospondin) is a high-Mr calcium-sensitive protein secreted by activated platelets. We observed that this protein was precipitated with barium citrate, and this property was used to purify glycoprotein G. The simple and rapid purification procedure consisted of barium citrate adsorption followed by heparin-agarose affinity chromatography. Unlike other calcium-sensitive proteins that are precipitated by barium citrate, glycoprotein G does not contain gamma-carboxyglutamic acid. The ability of glycoprotein G to bind to both heparin and barium citrate is consistent with this protein possessing clusters of positive and negative charges.
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PMID:Quantitative adsorption of platelet glycoprotein G (thrombin-sensitive protein, thrombospondin) to barium citrate. 669 32

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions.
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PMID:Thrombospondin: synthesis and secretion by cells in culture. 675 43

Thrombospondin, the major glycoprotein released from alpha-granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin. Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 kdalton subunits, the possibility that GP-160 is thrombospondin was investigated. Tritiated GP-160 could be immunoisolated from [3H]leucine-labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin. Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems. Both proteins are disulfide-bonded trimers of acidic 160-kdalton subunits. A competitive radioimmunoassay for binding of 125I-thrombospondin to the rabbit antibodies indicated that 49 micrograms of thrombospondin antigen per 10(6) confluent endothelial cells accumulated in postculture medium over 24 h. Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin.
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PMID:Synthesis and secretion of thrombospondin by cultured human endothelial cells. 709 42

A high molecular weight glycoprotein, reported to be secreted by endothelial cells (Sage, H., Crouch, E., and Bornstein, P. (1979) Biochemistry 18, 5433-5442), has been purified to apparent homogeneity from culture medium of adult bovine aortic endothelial cells. Purification was achieved by ammonium sulfate fractionation and successive chromatography on gelatin-Sepharose, Sephacryl S-300, and hydroxylapatite. The glycoprotein was found to be a disulfide-linked oligomer with a subunit molecular weight of 190,000, as judged by its mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels. The endothelial cell-derived protein is distinct from high molecular weight serum glycoproteins such as fibronectin and alpha 2-macroglobulin. However, immunological and structural studies indicate that the Mr = 190,000 glycoprotein is either identical with or closely related to thrombospondin, a glycoprotein contained in platelet granules and released in response to thrombin-induced aggregation.
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PMID:Isolation and characterization of a glycoprotein secreted by aortic endothelial cells in culture. Apparent identity with platelet thrombospondin. 728 69

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

Thrombomodulin (TM) is an anionic (pI approximately 4) protein cofactor that promotes thrombin (THR) cleavage of protein C to generate activated protein C (APC), a potent anticoagulant. We find that the cationic platelet alpha-granule protein platelet factor 4 (PF4) stimulates 4-25-fold the cofactor activity of rabbit TM and two differentially glycanated versions of an extracellular domain human TM polypeptide in which the glycosaminoglycan (GAG) is either present (GAG+ TM) or absent (GAG- TM) with an ED50 of 3.3-10 micrograms/ml. No such stimulation occurs in response to beta-thromboglobulin or thrombospondin, or when protein C lacking its gamma-carboxyglutamic acid (Gla) domain is the substrate. Heparin and chondroitin sulfates A and E reverse PF4 stimulation. PF4 minimally affects the Kd for THR but decreases 30-fold (from 8.3 to 0.3 microM) the Km for protein C of APC generation by GAG+ TM. PF4 also strikingly transforms the [Ca2+] dependence profile of rabbit and GAG+ TM to resemble that of GAG- TM. A potential explanation for this is that PF4, like Ca2+, induces heparin-reversible alterations in native (but not Gla-domainless) protein C conformation as assessed by autofluorescence emission analysis. We conclude that PF4 stimulates TM APC generation by interacting electrostatically with both the TM GAG and the protein C Gla domain to enhance markedly the affinity of the THR.TM complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.
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PMID:Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis. 752 87

Platelets are activated by substances from the subendothelial matrix in endothelial lesions or by factors in the plasma coagulation cascade. Conversely, activated platelets are potent activators of this cascade. Only activated platelets express the adhesion molecules Gp53, GMP140 and thrombospondin on the plasma membrane. The postmortem activation status of platelets, therefore, can be determined immunoelectron microscopically by immunogold labeling of antibodies against these glycoproteins. Our studies revealed that the vast majority of these antigens were located within the granules postmortem, hence the platelets had not been activated. Thrombin-induced activation of platelets in vitro was only possible in the early postmortem interval, as demonstrated by labeling of the adhesion molecules on the plasma membrane. Later, such activation was no longer possible even though thrombin-induced fibrin formation gave the appearance of "coagulated blood". In forensic medicine, these findings can possibly be applied to distinguish intravital clotting from the postmortem coagulation phenomena and intravital hematomas from postmortem hematomas.
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PMID:The postmortem activation status of platelets. 753 69

The free thiols of platelet thrombospondin (TSP) were modified with thiol-specific spin labels and fluorescence probes. The conformational effects of thrombin complexation with TSP were monitored by thiol-specific spin labels covalently attached to TSP and active site specific spin labels on thrombin. The results provide evidence supporting speculations that the thiols of the three polypeptide chains in TSP are not conformationally identical. Studies on the effects of Ca2+ and temperature confirm that TSP exists in multiple conformations which are under dynamic equilibrium. The ESR spectra of spin-labeled TSP are sensitive to the proteolytic effects of thrombin in the presence and absence of calcium. Phenylsulfonyl fluoride spin labels specific for the active site of thrombin are excellent indicators of thrombin: TSP complex formation in the absence of calcium. The anticoagulant thrombin inhibitor hirudin competes with TSP for the same binding locus on thrombin (which includes the requirement of an intact anion exosite). The results suggest that the species observed here is the noncovalent complex formed during the first step of the TSP--thrombin interaction, showing also that thrombin activity is not essential for complex formation. ESR and fluorescence studies of thiol-labeled TSP indicate that the sulfhydryls are not affected in the noncovalent thrombin: TSP complex, although they must be playing a major role in the second step, i.e., formation of the covalent complex, through intermolecular thiol exchange.
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PMID:Electron spin resonance and fluorescence studies of the conformational environment of the thiol groups of thrombospondin: interactions with thrombin. 765 3

Vitronectin is a structurally labile molecule with a native, non-heparin binding form and a conformationally altered, heparin binding form. To understand the physiological significance of the two conformers of vitronectin, we examined the metabolism of both conformers by cultured human skin fibroblasts. Both native and altered vitronectin bound to confluent fibroblast monolayers. Both conformers of vitronectin competed equally well for the binding of altered vitronectin to the cell layer, suggesting that both conformers bound to the same site in the cell layer. In contrast, 125I-altered vitronectin, but not 125I-native vitronectin, was degraded to trichloroacetic acid-soluble radioactivity by the fibroblast monolayer. Degradation of vitronectin was saturable, sensitive to chloroquine, and occurred intracellularly, suggesting that vitronectin was degraded through a lysosomal pathway. Heparin and thrombospondin inhibited the degradation of altered vitronectin. The degradation of native vitronectin was induced by addition of gamma-thrombin which exposes vitronectin's cryptic heparin-binding domain. These studies suggest that the heparin-binding domain in vitronectin is required for the clearance of vitronectin from the matrix. In addition, these data demonstrate that the conformation of vitronectin regulates its half-life in the matrix. These studies provide the first evidence for a distinct function for the conformers of vitronectin.
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PMID:Receptor-mediated endocytosis of vitronectin is regulated by its conformational state. 768 28

CD36 (glycoprotein [GP] IV) is a membrane GP of 88 kD found on monocytes, endothelial cells, and platelets. It may serve as a receptor for collagen and is also able to bind thrombospondin (TSP), because a monoclonal antibody to CD36 inhibits TSP binding to thrombin-stimulated platelets. In the following study, we investigated the subcellular distribution of CD36 within normal resting platelets, thrombin-stimulated platelets, and in cultured megakaryocytes (MK) by an immunogold staining technique and electron microscopy. We used an affinity-purified monospecific polyclonal antibody showing a single major band of precipitation at 88 kD via immunoblot analysis. In normal platelets, ultrastructural observation detected immunolabeling for CD36, homogeneously distributed along the platelet plasma membrane and in the luminal side of the open canalicular system (OCS). Moreover, some labeling was found around the alpha-granules along the inner face of their limiting membrane. An average of 70% of granules were labeled. The granule-associated pool of CD36 was estimated at approximately 25% of the total cell content. To exclude the possibility of a cross-reaction with GPIIb-IIIa, platelets from a patient with type I Glanzmann's thrombasthenia (which completely lack GPIIb-IIIa) were studied and showed a similar subcellular distribution of CD36, including alpha-granule membrane labeling. In activated platelets, CD36 was shown to be redistributed to the OCS and pseudopods of the plasma membrane. Platelets from a patient with the Gray platelet syndrome expressed CD36 on their plasma membrane, and some immunolabeling was also found within small abnormal alpha-granules. In cultured MK, CD36 immunolabeling was detected in the Golgi saccules, associated vesicles, immature alpha-granules, and demarcation membranes. In conclusion, this study shows the existence of a significant intragranular pool of CD36 in platelets that may play a critical role in the surface expression of alpha-granule TSP during platelet activation.
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PMID:Ultrastructural demonstration of CD36 in the alpha-granule membrane of human platelets and megakaryocytes. 769 34


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