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 presence of specific Factor VIII/von Willebrand factor (FVIII/vWF) binding sites on human platelets has been demonstrated by using 125I-FVIII/vWF and washed human platelets. Binding is ristocetin-dependent and increases in proportion to the concentration of ristocetin from 0.2 to 1 mg/ml. Binding of 125I-FVIII/vWF to platelets can be competitively inhibited by unlabeled human or bovine FVIII/vWF, but not by human thrombin, fibrinogen, alpha 2-macroglobulin, equine collagen, or a lectin of Ricinus communis. Scatchard analysis of binding data indicated that the dissociation constant of FVIII/vWF receptors is 0.45--0.5 nM. There are 31,000 binding sites per platelet at 1 mg/ml of ristocetin concentration. The optimal pH range for binding is from 7.0 to 7.5. At a concentration of 2 mM, EGTA inhibits 86% of the binding; however, 20 mM of Ca++, Mg++, or EDTA have little effect. Binding sites for FVIII/vWF were found only on platelets, and no significant binding was detected with human erythrocytes or polymorphonuclear leukocytes.
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PMID:Demonstration and characterization of specific binding sites for factor VIII/von Willebrand factor on human platelets. 10 91

Washed human platelets were solubilized and the proteins were separated by preparative gel electrophoresis in the presence of sodium dodecyl sulphate. The gel was cut into slices and the effect of the eluted proteins on the clotting of fibrinogen by thrombin was evaluated. The isolate from only one gel slice strongly inhibited the clotting of fibrinogen. The prolongation of the clotting time was dependent on the concentration of the protein and reached a plateau around 5 microgram. Gel electrophoresis of this isolate showed a prominent glycoprotein with an apparent Mr=150 000. Gel filtration studies with [125I]thrombin showed that the protein isolate bound a significant amount of thrombin which could be displaced with unlabelled thrombin. Another preparation from the same gel or purified gamma-globulin did not bind thrombin or prolong the clotting time of fibrinogen. Glycoprotein I was isolated from human platelets by affinity chromatography on lectin-Sepharose columns. The isolated glycoprotein prolonged the clotting of fibrinogen and bound [125I]thrombin which could be displaced by unlabelled thrombin. It is proposed that the high affinity receptor of thrombin on human platelets is glycoprotein I. In addition, the antithrombin activity of intact platelets is due to binding of thrombin to this glycoprotein.
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PMID:Thrombin receptors of human platelets: thrombin binding and antithrombin properties of glycoprotein I. 46 56

Wheat germ agglutinin induced aggregation and secretion of fresh platelets. Aggregation, but not secretion of serotonin by platelets in plasma, by the lectin was inhibited by 5 mM EDTA. Further, the lectin-induced stimulation of fresh platelets was blocked by prostaglandin E1. Thus, this lectin stimulates platelets by a mechanism which closely mimics thrombin activation and is independent of intercellular crosslinking. Lentil lectin did not stimulate platelets. Each platelet contained about 6 . 10(-5) binding sites for the lectins with an apparent dissociation constant of 3.0 . 10(-7) M. Wheat germ agglutinin, which binds mainly to glycoprotein I (Mr 150 000), increased the subsequent binding of thrombin to fixed platelets while lentil lectin was without effect. It appears that thrombin and wheat germ agglutinin bind to independent but interacting sites. Wheat germ agglutinin, but neither thrombin nor lentil lectin, inhibited the agglutination of platelets by ristocetin. Further, rat platelets were not aggregated by either ristocetin or wheat germ agglutinin. It appears that the interaction sites of ristocetin and wheat germ agglutinin on platelets are overlapping.
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PMID:Interaction of lectins with human platelets. Effects on platelet stimulation by thrombin and ristocetin. 47 55

Throbin-activated human platelets cause agglutination of trypsinized, formalinized bovine erythrocytes. This lectin activity of stimulated platelets was blocked by galactosamine, glucosamine, mannosamine, lysine, and arginine, but not by N-acetylated sugars, other neutral sugars, or other amino acids. Inhibitors of the thrombin-induced lectin activity also blocked thrombin-induced platelet aggregation. It appears that a membrane surface component that has lectin activity mediates platelet aggregation.
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PMID:Thrombin-induced platelet aggregation is mediated by a platelet plasma membrane-bound lectin. 66 8

We report a technique for the isolation of plasma membranes from gel-filtered platelets exposed to thrombin, using 125I-labeled lentil lectin as an external marker. Labeled cells not exposed to thrombin could be lysed on a gradient of glycerol. Those cells incubated with thrombin (without external Ca2+) were made more susceptible to breakage on a gradient of glycerol-EDTA, and homogenized with a zero-clearance homogenizer. Lysates were spun on gradients of sodium diatrizoate. The membranes obtained from such gradients have been examined by electron microscopy and by assays for enzymes and 125I label. Membranes from platelets incubated without and with thrombin were found to be enriched as follows: lectin marker, 8- and 9-fold, respectively; phosphodiesterase, 9- and 12-fold; acid phosphatase, 2.5 and 2-fold. There is thus a particularly close correlation of lectin marker with phosphodiesterase, an enzyme characteristic of normal purified membranes. Monitoring for 125I-labeled lentil lectin appears to be a useful procedure for following platelet membranes during isolations from relatively small quantities of blood.
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PMID:Isolation of membranes from normal and thrombin-treated gel-filtered platelets using a lectin marker. 81 77

A forty-kilodalton (40-kDa) protein was extracted from alveolar bone of young adult rabbit with 0.5 M EDTA after extraction with 4 M GuHCl, and purified by gel-filtration, anion-exchange and hydroxyapatite columns using a high-pressure liquid chromatography system under denaturing conditions. The purified 40-kDa protein was not susceptible to bacterial collagenase and thrombin, but was cleaved by cyanogen bromide. The protein was stained blue with Stains-all. Among various lectins, concanavalin A and lentil lectin agglutinin bound to this protein, but peanut agglutinin, Ricinus communis agglutinin, phytohemagglutinin-E and wheatgerm lectin agglutinin did not. Lectin binding assays showed that the protein is a glycoprotein containing large amounts of mannose and/or glucose residues, but is not a fragment of proteoglycan. The amino acid composition of the protein shows a characteristically high content of acidic amino acids. Therefore, the mineral-binding 40-kDa glycoprotein is considered to be osteonectin/secreted protein acidic and rich in cysteine (SPARC), in terms of similarities to bovine and porcine osteonectins with regard to molecular weight and contents of glycoses and amino acids.
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PMID:Characterization of mineral-binding 40-kDa glycoprotein extracted from young adult rabbit alveolar bone. 132 44

Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been a failure to isolate and propagate human glomerular capillary endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogeneous monolayers of primate (baboon and human) glomerular capillary endothelial cells. Using selective media and growth factors, the following criteria were identified to optimize the isolation and proliferation of glomerular endothelial cells: (1) collagenase treatment of isolated glomeruli; (2) requirement for 20% serum, endothelial cell growth factor and heparin; (3) requirement of fibronectin as surface matrix; and (4) isolation from donors less than 60 years old, as premature senescence was directly proportional to the age of the human kidney donor. Under these conditions, primary cultures with an endothelial cell composition greater than 70% were reproducibly obtained. Homogeneous endothelial monolayers were developed from 20 of 23 human kidneys, and maintained for 5 to 10 passages, depending on the age of the kidney donor. Purification to homogeneity was achieved by patch cloning or by fluorescence-activated cell sorting. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence, expressed factor VIII-related antigen, angiotensin converting enzyme activity, and endocytosed acetylated low-density lipoproteins. Electron microscopy revealed the presence of intracellular Weibel-Palade bodies and caveolae and microvillous projections on the luminal surface. Glomerular cells also stained positive for Ulex europaeus, a lectin characteristic of human endothelial cells. In addition, preliminary results indicate that human glomerular endothelial cells increase intracellular cyGMP in response to alpha-human 5 to 28 atrial natriuretic peptide and intracellular free calcium in response to thrombin.
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PMID:Culture of endothelial cells from baboon and human glomeruli. 150 7

Two glycoforms of a soluble mutant of recombinant human thrombomodulin (rec.TM) were used to identify critical N- and O-linked glycans of the endothelial cell thrombin receptor. While N-linked glycans were not found to be involved in any function of rec.TM, an acidic chondroitin sulphate-like glycosaminoglycan (CSGAG) was found to be critical for all the direct anticoagulant functions of rec.TM, including inhibition of thrombin-mediated platelet aggregation. A glycoform of rec.TM lacking CSGAG had very poor anticoagulant activity. Furthermore, the glycoform of rec.TM possessing CSGAG showed strong inhibition by and had high affinity for poly-cationic basic proteins, whereas the CSGAG-deficient rec.TM did not. Monoclonal antibody binding as well as lectin mapping of rec.TM with agglutinins identified sialic acid containing O-linked glycans in both glycoforms additional to the CSGAG in high molecular weight rec.TM These findings define important molecular interactions modulating the anticoagulant function of TM, which appear to be critically regulated by CSGAG, and also showed that the overall post-translational glycosylation pattern of the two glycoforms was very similar except for the presence of CSGAG. The possibility exists that differently expressed glycoforms of TM may be crucial for the expression of endothelial cell-related anticoagulant potential in different vascular beds.
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PMID:Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin. 165 91

Monosaccharide binding competition, lectin affinity chromatography, and glycosylation inhibitors have been used to determine if glycosylation plays a role in thrombin-receptor interactions. Mannose appeared to specifically inhibit thrombin binding to mouse embryo (ME) and hamster fibroblasts. Concanavalin A bound to antibody-purified receptor fractions, and was used as an affinity ligand to purify receptor fractions that retained thrombin binding activity. Cells treated with tunicamycin (6.25 ng/ml) for 24 h lost approximately 35% of their high-affinity thrombin binding sites, yet binding of receptor monoclonal antibody TR-9 was not affected, indicating that the receptor was present in the membrane, but unable to bind thrombin. Thus thrombin receptor glycosylation may be directly involved in thrombin binding.
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PMID:Glycosylation of high-affinity thrombin receptors appears necessary for thrombin binding. 165 72

GMP-140 is a membrane glycoprotein located in secretory granules of platelets and endothelium. When these cells are activated by agonists such as thrombin, GMP-140 is rapidly translocated to the plasma membrane. GMP-140, along with ELAM-1 and the peripheral lymph node homing receptor, defines the selectin family of structurally related molecules that regulate interactions of leukocytes with the blood vessel wall. Each of these molecules contains an N-terminal lectin-like domain, followed by an EGF-like region, a series of consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. The genomic structures of the selectins suggest that they arose by duplication and modification of exons encoding specific structural domains. GMP-140 is a receptor for neutrophils and monocytes when it is expressed on activated platelets and endothelium. This property facilitates rapid adhesion of leukocytes to endothelium at regions of tissue injury as well as platelet-leukocyte interactions at sites of inflammation and hemorrhage. Like other leukocyte adhesion molecules, GMP-140 may also participate in pathologic inflammation, thrombosis, and tumor metastasis. Confirmation of such pathologic roles may lead to design of new drugs that block adhesive receptor function in human disease.
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PMID:GMP-140: a receptor for neutrophils and monocytes on activated platelets and endothelium. 171 28


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