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)

Analysis of platelet membrane proteins and glycoproteins by SDS polyacrylamide gel electrophoresis was carried out before and after treatment with thrombin. Extended incubation with thrombin (in the presence of EDTA or adenosine, which inhibit aggregation) produced extensive changes in the bands observed. With incubation times of a few minutes however, the changes were restricted to a glycopeptide, GP IV (approx. 90,000 Daltons) and one or two polypeptides of low molecular weight, in particular polypeptide 16 (approx. 23,000 Daltons). At 0--3 degrees C only polypeptide 16 was still hydrolyzed. Chymotrypsin, which does not activate platelets, attacked glycopeptides I, II, III but no changes were apparent in GP IV and polypeptide 16. When chymotrypsin-treated platelets were further incubated with thrombin, only GP IV and one to two low molecular weight polypeptides, especially polypeptide 16, were affected. As polypeptide 16 appears to be an integral membrane component it is possible that it, either by itself or in combination with GP IV, represents the primary thrombin substrate involved in platelet activation. Aggregated IgG, which also activates platelets, does not modify the membrane glycoproteins but does change the low molecular weight region in particular band 16.
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PMID:Effects of thrombin, chymotrypsin and aggregated gamma-globulins on the proteins of the human platelet membrane. 7 80

The effects of bolus intravenous injections of various serine proteases (thrombin, trypsin, plasmin, neutrophil elastase and chymotrypsin) on arterial blood pressure were evaluated in anesthetized, normotensive rats. The activity to intravenous trypsin was also studied in anesthetized, normotensive dogs. In the rat, both thrombin (0.33-10 nmol/kg) and trypsin (4.2-420 nmol/kg) produced pronounced vasodepressor responses. The activity on blood pressure was observed immediately following injection of either protease, and both the magnitude and duration of the responses were dose dependent. Plasmin (37-350 nmol/kg) and neutrophil elastase (91-910 nmol/kg) also induced dose-dependent hypotension but at much higher dose levels. In addition, the magnitude of the blood pressure responses after plasmin and neutrophil elastase was less than those produced by thrombin and trypsin. Chymotrypsin, on the other hand, had a more diverse blood pressure profile. The protease induced a modest decrease in pressure at doses of 40 and 120 nmol/kg, a pressor response after 400 and 1,200 nmol/kg and at the highest dose tested (4,000 nmol/kg) profound hypotension. In the dog, trypsin produced a dose-dependent vasodepressor response similar to that observed in the rat. The doses of proteases producing alterations of blood pressure in the rat correlated inversely with the ability of rat serum or plasma to completely inhibit those proteases. The pharmacology of the trypsin or thrombin blood pressure response suggests the requirement of specific active enzymes to mediate the vasodepression induced by both proteases.
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PMID:Acute blood pressure effects of selected serine proteases in normotensive rats and dogs. 177 Nov 72

A new cell line (LC-1/sq) of human lung squamous-cell carcinoma was established from a surgically resected specimen of primary lung cancer. Upon continuous propagation in serum-free culture medium, it secreted trypsin inhibitors into the conditioned medium. The major fraction of the trypsin inhibitor (T1-1) was purified to apparent homogeneity by anion-exchange and gel-filtration high-performance liquid chromatography (HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transblotting to Immobilon. T1-1 effectively inhibited trypsin. Chymotrypsin, plasmin and kallikrein were inhibited to a lesser extent, but urokinase-type plasminogen activator, elastase, thrombin and papain were not inhibited. The activity of T1-1 was acid-stable and heat-resistant, and its molecular weight was 115 kDa by SDS-PAGE. It exhibited single NH2-terminal sequence, and its first 20 NH2-terminal amino-acid residues were identical with those of protease nexin-II (PN-II)/amyloid beta-protein precursor (APP). These characteristics of T1-1 suggest that the major trypsin inhibitor secreted by LC-1/sq is indistinguishable from PN-II/APP. LC-1/sq is the first lung squamous carcinoma cell line that secretes functionally active trypsin inhibitor, PN-II/APP, in vitro and is useful for studying its biological significance in malignant tumor.
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PMID:Establishment of a new human cancer cell line secreting protease nexin-II/amyloid beta protein precursor derived from squamous-cell carcinoma of lung. 191 42

We have obtained evidence that the ligand-recognition region of the integrin beta-subunit, platelet glycoprotein IIIa (GPIIIa), is discontinuous. Receptor function can be localized to residues near the N-terminus and to the central region of the polypeptide chain. The epitope recognized by our monoclonal antibody, CS-1, which substantially inhibits fibrin(ogen) binding to ADP- and thrombin-stimulated platelets [Ramsamooj, Doellgast & Hantgan (1990) Thromb. Res. 58, 577-592], is contained within residues 349-422 of GPIIIa. This sequence is adjacent to a proteinase-resistant domain of GPIIIa which is linked by disulphide bond(s) to an N-terminal segment near to the putative Arg-Gly-Asp recognition site [D'Souza, Ginsberg, Burke, Lam & Plow (1988) Science 242, 91-93]. Limited trypsin digestion of purified platelet GPIIIa yielded a mixture of two-chain molecules comprised of an N-terminal fragment disulphide-bonded to one of four fragments, which began at residues 299, 303, 353 or 423. Tryptic cleavage of the 300-422 segment correlated with loss of immunoreactivity with anti-GPIIIa monoclonal antibody, CS-1. Chymotrypsin cleavage of GPIIIa resulted in an N-terminal 19 kDa fragment joined by at least one intrachain cystine residue to a 46 kDa polypeptide beginning at residue 349. Partial reduction with dithiothreitol released the larger chymotryptic fragment with its epitope for CS-1 intact. These results have enabled us to localize the epitope recognized by our inhibitory monoclonal antibody, CS-1, to residues 349-422 of GPIIIa. Our data are consistent with a structure in which both the N-terminal and central regions of GPIIIa, which may be in close proximity in the functional GPIIb-IIIa complex, participate in ligand binding.
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PMID:Evidence that the central region of glycoprotein IIIa participates in integrin receptor function. 206 10

The inhibition of six serine proteinases by a tumour-associated trypsin inhibitor (TATI) was studied using synthetic peptide substrates. Physiological concentrations of TATI inhibited the amidolytic activities of trypsin, plasmin, urokinase and tissue plasminogen activator (tPA). Chymotrypsin, kallikrein and thrombin were also inhibited, but by much higher concentrations of TATI. The ability of TATI to inhibit trypsin, plasmin, urokinase and tPA suggests that it has a role in proteolytic processes in vivo involving these enzymes.
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PMID:Reaction of a tumour-associated trypsin inhibitor with serine proteinases associated with coagulation and tumour invasion. 246 2

The proteolytic digestion of GPIIIa on intact platelets by chymotrypsin, thrombin, plasmin, trypsin, and staphylococcal V8 protease was monitored in immunoblot studies employing three different antibodies to GPIIIa, one of which was made against a 13-residue synthetic peptide containing the amino terminus of GPIIIa. Chymotrypsin, plasmin, and trypsin gave similar patterns, from which it could be inferred that the major products after extensive digestion were two-chain molecules composed of amino-terminal fragments of Mr approximately 17,000-18,000 disulfide bonded to carboxyl-terminal remnants of Mr approximately 58,000-70,000. These patterns suggest that GPIIIa contains a large disulfide-bonded loop of at least 325 amino acids that is susceptible to proteolytic cleavage, and that the 4 cysteine residues between residues 177 and 273 bond with each other. Such a structure can also account for the presence of the PIA1 epitope, which has recently been localized to a polymorphism at position 33 on these late digestion products. Thrombin did not proteolyze GPIIIa even at 2.5 units/ml. Still to be resolved is whether the minor immunoreactive GPIIIa bands found on normal platelets are related to in vivo or in vitro proteolysis and whether GPIIIa proteolysis plays a role in chymotrypsin-induced exposure of the GPIIb/IIIa receptor.
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PMID:Evidence that platelet glycoprotein IIIa has a large disulfide-bonded loop that is susceptible to proteolytic cleavage. 252 61

The glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet-sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin-binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five-fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme-to-substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30-kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin-cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin-susceptible site, which has been observed in the long connecting arm, which is particularly Ca-stabilized. Heparin-binding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low-affinity binding of the large fragments but not the high-affinity binding of the small fragments. Two-dimensional gel electrophoresis of the chymotryptic heparin-binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin-binding capacity of both high and low-affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The structure of endothelial cell thrombospondin. Characterization of the heparin-binding domains. 282 10

The authors report the effects of four proteases (trypsin, plasmin, chymotrypsin and thrombin) on human heart adenylate cyclase (HHAC) activity. Trypsin and plasmin inhibited HHAC at concentrations higher than 0.3 and 1 microgram/ml, respectively. Chymotrypsin had a biphasic effect, with a stimulation (from 0.5 to 10 micrograms/ml) followed by an inhibition at higher concentrations. Maximal stimulation was obtained at 3 micrograms/ml and averaged 67.2 +/- 5.4%. Thrombin had no significant effect at concentrations up to 1 mg/ml. These data indicate that proteases might regulate HHAC and therefore influence cardiac function.
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PMID:The effects of trypsin, plasmin, chymotrypsin and thrombin on human heart adenylate cyclase activity. 295 13

Human platelets that have undergone the release reaction do not deaggregate readily. We examined conditions under which washed human platelets can be deaggregated after they have undergone an extensive release reaction induced by thrombin (1 or 5 U/ml). To make fibrinogen receptors unavailable, either CP/CPK (or apyrase) was used to remove released ADP, or PGE1 was used to increase cAMP. Chymotrypsin was used to digest proteins that might link platelets, and heparin to interact with released proteins and interfere with their binding to platelets and to each other. Individually, none of these caused deaggregation; heparin did not inhibit the effect of thrombin because no antithrombin III was present. Platelets exposed to thrombin (1 U/ml) which was neutralized at 90 sec by hirudin, could be deaggregated by combinations of CP/CPK (or apyrase) and chymotrypsin, or PGE1 and chymotrypsin. When a higher concentration of thrombin was used (5 U/ml) these combinations caused platelets to deaggregate only when heparin was added before thrombin induced the release reaction. Thus, when extensive release occurs three mechanisms may come into play to link human platelets: one that requires the fibrinogen receptor; a heparin-sensitive reaction that may involve the binding of released proteins; and a linkage that can be disrupted only by proteolysis, providing the other two mechanisms are also inhibited.
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PMID:Deaggregation of human platelets aggregated by thrombin. 298 9

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.
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PMID:Lipoprotein lipases from cow, guinea-pig and man. Structural characterization and identification of protease-sensitive internal regions. 353 11


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