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)

Thrombomodulin (TM) is a constituent glycoprotein of endothelial cell membrane, and soluble TM is present also in plasma and urine. It was revealed by experiments using cultured HUVEC in vitro that TM is released from endothelial cell membrane not with monensin, thrombin, fibroblast growth factor, interleukin-1 or endotoxin, but with H2O2 or endotoxin-treated granulocytes. And the release was suppressed by the coexistence of gabexate mesilate or superoxide dismutase. It was suggested that soluble TM was released from endothelial cell membrane by its injury and digested to multiple molecular forms by endogenous and granulocytic protease(s). TM level in circulation is increased in cases of SLE, MCLS, diabetic angiopathy. It was increased in cases of overt DIC and decreased to the normal level when the patient was recovered from DIC. TM level in circulation was also increased in cases of decompensated liver cirrhosis and markedly in cases of renal insufficiency. It was concluded that plasma TM is a parameter reflecting endothelial injury due to inflammation or metabolic disorders of vascular system. But the interpretation of increased plasma TM was difficult when renal insufficiency was complicated.
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PMID:[Soluble thrombomodulin: a specific parameter of endothelial injury]. 185 Dec 35

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet-fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.
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PMID:Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton. 189 46

Calcium-activated neutral proteinase (calpain) has been shown to cleave proteins involved in the maintenance of cell structure. In human platelets, substrates of calpain include glycoprotein Ib (GPIb), actin-binding protein (ABP), and talin. GPIb-ABP complexes can be isolated in detergent extracts and are thought to represent membrane-cytoskeleton attachment sites. It has been hypothesized that the hydrolysis of GPIb-ABP by calpain is regulated by the extent of binding of this proteinase to the plasma membrane-cytoskeleton interface with platelet activation. Recently, another calpain substrate (talin) has been shown to redistribute from the cytoplasm to the plasma membrane-cytoskeleton interface as the result of thrombin stimulation. To investigate the intracellular distribution of calpain I, we employed the monoclonal antibody B27D8, specific for the heavy chain (catalytic subunit) of calpain I. Indirect immunofluorescent staining of resting human platelets revealed undetectable surface antigen. Permeabilization with Triton X-100, however, revealed a diffuse intracellular antigen consistent with a cytosolic distribution. To determine whether this antigen distribution reflected the proenzyme or the activated form of calpain I and to assess the degree of hydrolysis of ABP, GPIb, and talin, we employed B27D8 and murine monoclonal antibodies against ABP (1B3 and 3D1), GPIb (LJIb10), and rabbit polyclonal antibodies against talin (A2 and B11) in a quantitative immunotransblot assay. Examination of resting platelets revealed that calpain I existed as the 85-kd proenzyme form and that ABP, GPIb, and talin existed in their native intact forms. When platelets were aggregated with thrombin, autoproteolysis of calpain I occurred within the 30 seconds required to completely solubilize platelet aggregates in sodium dodecyl sulfate-containing buffer and not as a direct result of thrombin-induced activation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activation of calpain I and hydrolysis of calpain substrates (actin-binding protein, glycoprotein Ib, and talin) are not a function of thrombin-induced platelet aggregation. 190 52

A fibrinogenase from Vipera lebetina venom was isolated by gel filtration in a Superose 12 column prep grade HR 16/50 and by ion-exchange in a Mono Q HR 5/5 column. The purified enzyme, which was obtained with a yield of 8 mg from 60 mg of crude venom, is a glycoprotein having an isoelectric point of 5.9 +/- 0.1 and a mol. wt of 26,000 +/- 1000 as estimated by SDS-PAGE. The biochemical characterization of the enzyme revealed that it hydrolyzes readily the B beta chain of fibrinogen and the A alpha chain as well as fibrin and casein. Over a pH range from 4 to 11 the enzyme was not inactivated by a 20 min treatment at 90 degrees C. The isolated fibrinogenase is inhibited by ethylenediamine tetraacetic acid, dithiothreitol and L-cysteine but not by phenylmethylsulfonyl fluoride. On the other hand, it is activated by Ca2+ and Mg2+. Purified fibrinogenase up to a dose of 100 micrograms/mouse shows no toxicity and has no hemorrhagic activity.
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PMID:Purification and characterization of a fibrinogenase from Vipera lebetina (desert adder) venom. 192 82

We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20-40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10-70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.
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PMID:Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets. 193 78

Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin and chymotrypsin. Inhibition occurs when the protease attacks the reactive site peptide bond in HCII (Leu444-Ser445) and becomes trapped as a covalent 1:1 complex. Dermatan sulfate and heparin increase the rate of inhibition of thrombin, but not of chymotrypsin, greater than 1000-fold. The N-terminal portion of HCII contains two acidic repeats (Glu56-Asp-Asp-Asp-Tyr-Leu-Asp and Glu69-Asp-Asp-Asp-Tyr-Ile-Asp) that may bind to anion-binding exosite I of thrombin to facilitate covalent complex formation. To examine the importance of the acidic domain, we have constructed a series of 5' deletions in the HCII cDNA and expressed the recombinant HCII (rHCII) in Escherichia coli. Apparent second-order rate constants (k2) for inhibition of alpha-thrombin and chymotrypsin by each variant were determined. Deletion of amino acid residues 1-74 had no effect on the rate of inhibition of alpha-thrombin or chymotrypsin in the absence of a glycosaminoglycan. Similarly, the rate of inhibition of alpha-thrombin in the presence of a glycosaminoglycan was unaffected by deletion of residues 1-52. However, deletion of residues 1-67 (first acidic repeat) or 1-74 (first and second acidic repeats) greatly decreased the rate of inhibition of alpha-thrombin in the presence of heparin, dermatan sulfate, or a dermatan sulfate hexasaccharide that comprises the minimum high-affinity binding site for HCII. Deletion of one or both of the acidic repeats increased the apparent affinity of rHCII for heparin-Sepharose, suggesting that the acidic domain may interact with the glycosaminoglycan-binding site of native rHCII. The stimulatory effect of glycosaminoglycans on native rHCII was decreased by a C-terminal hirudin peptide which binds to anion-binding exosite I of alpha-thrombin. Furthermore, the ability of native rHCII to inhibit gamma-thrombin, which lacks the binding site for hirudin, was stimulated weakly by glycosaminoglycans. These results support a model in which the stimulatory effect of glycosaminoglycans on the inhibition of alpha-thrombin is mediated, in part, by the N-terminal acidic domain of HCII.
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PMID:The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans. 193 83

To characterize the interaction between thrombospondin and human platelets, thrombospondin was purified from the supernatant of thrombin-activated human platelets, labeled with iodine 125, and allowed to interact with the washed platelets. With concentrations of 10 to 50 micrograms/ml, only minute amounts of 125I-labeled thrombospondin bound to resting platelets or to platelets activated by adenosine diphosphate. In contrast, when platelets were stimulated with thrombin, binding increased fivefold to sixfold in a time-dependent and 125I-labeled thrombospondin concentration-dependent manner. Binding of 125I-labeled thrombospondin to thrombin-activated platelets required the presence of divalent cations, proceeded concomitantly with platelet release, and at a concentration of 1 nmol/L thrombin, reached a maximum of 2200 +/- 260 molecules of 125I-labeled thrombospondin bound per platelet. After its binding to platelets, 125I-labeled thrombospondin was not internalized, because up to 85% of the 125I-labeled thrombospondin was dissociated from the cell surface by adding ethylenediaminetetraacetic acid. Using various experimental approaches, including studies with severe type I thrombasthenic platelets, we further demonstrated that the interaction of 125I-labeled thrombospondin with thrombin-stimulated platelets occurred as a fibrinogen- and fibrin-independent process, and that the glycoprotein IIb-IIIa complex did not function as a physiologic plasma membrane receptor for 125I-labeled thrombospondin. Last, about 60% of the 125I-labeled thrombospondin molecules bound to the platelet surface were found to be associated with the platelet cytoskeleton recovered from platelets solubilized with Triton X-100. On Western blot analysis, this cytoskeletal fraction lacked detectable glycoprotein IV, the putative platelet receptor for thrombospondin. These results suggest that on the surface of thrombin-activated platelets, a fraction of 125I-labeled thrombospondin does not associate with glycoprotein IV but instead with other plasma membrane components that have yet to be identified.
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PMID:Characterization of the binding of thrombospondin to human platelets and its association with the platelet cytoskeleton. 194 May 85

Several studies have suggested that the glycoprotein (GP) IIb-IIIa complex, which serves as the platelet fibrinogen receptor, also plays a role in the regulation of Ca2+ influx across the platelet plasma membrane. To examine this possibility further, we have compared Ca2+ transport in platelets and human erythroleukemia (HEL) cells, a megakaryoblastic cell line which synthesizes GP IIb-IIIa complexes that appear to be identical to those found on platelets. As with platelets, the results show the presence in unstimulated HEL cells of a rapidly exchangeable cytosolic Ca2+ pool that is in equilibrium with an intracellular sequestered Ca2+ pool and with extracellular Ca2+. Allowing for differences in cell size, the rate constants for Ca2+ exchange in HEL cells were similar to those in platelets. As in platelets, thrombin caused an increase in cytosolic Ca2+ that was due partly to enhanced Ca2+ influx and partly to the mobilization of internal Ca2+ stores. Incubation of the HEL cells with EDTA at 37 degrees C irreversibly altered the GP IIb-IIIa complex as evidenced by decreased binding of a complex-specific monoclonal antibody. In platelets this was accompanied by a 40% decrease in the rate of Ca2+ influx. However, in HEL cells there was neither a diminution in Ca2+ influx nor a reduction in the magnitude of the increase in cytosolic Ca2+ caused by thrombin. These results show that the parameters of Ca2+ distribution and movement are similar in HEL cells and platelets and that in HEL cells, as in platelets, the GP IIb-IIIa complex can be altered by removing Ca2+. However, unlike platelets, dissociation of the HEL cell IIb-IIIa complex has no discernible effect on plasma membrane Ca2+ transport. This suggests that earlier observations in platelets correlating changes in the rate of Ca2+ influx with changes in the number of intact IIb-IIIa complexes reflect an indirect, rather than a direct, role of these proteins in Ca2+ transport.
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PMID:Role of the glycoprotein IIb-IIIa complex in plasma membrane Ca2+ transport: a comparison of results obtained with platelets and human erythroleukemia cells. 195 76

Three spatially distant surface loops were found to mediate the interaction of the coagulation protein factor X with the leukocyte integrin Mac-1. This interacting region, which by computational modeling defines a three-dimensional macromotif in the catalytic domain, was also recognized by glycoprotein C (gC), a factor X receptor expressed on herpes simplex virus (HSV)-infected endothelial cells. Peptidyl mimicry of each loop inhibited factor X binding to Mac-1 and gC, blocked monocyte generation of thrombin, and prevented monocyte adhesion to HSV-infected endothelium. These data link the ligand recognition of Mac-1 to established mechanisms of receptor-mediated vascular injury.
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PMID:Structurally homologous ligand binding of integrin Mac-1 and viral glycoprotein C receptors. 195 71

A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (10(3)/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on Fc gamma RII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.
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PMID:The murine monoclonal antibody, 14A2.H1, identifies a novel platelet surface antigen. 195 84


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