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)

Bovine thrombin was insolubilized by attachment to cyanogen bromide-activated Sepharose (Sepharose-thrombin) or to activated (Affi-Gel 10) agarose containing a 10 A long arm (Affi-Gel-thrombin). Coupling in both instances approximated 7,000 units of thrombin per ml packed gel as determined by 125I-thrombin incorporation. The thrombin beads hydrolyzed the synthetic tripeptide Bz-Phe-Val-Arg-pNA (S-2160) at different rates, with the Sepharose-thrombin more active (220 esterase units per ml) than Affi-Gel thrombin (20.4 units per ml). The Km was significantly higher for the insolubilized thrombins (2 X 10(-3) M) than uncoupled thrombin (Km = 8 X 10(-5) M). The Sepharose-thrombin activated factor VIII significantly more rapidly than Affi-Gel-thrombin. Neither matrix-bound thrombin clotted a fibrinogen solution or liberated significant amounts of fibrinopeptides over 48 hr. This data indicates that a proteolysis of factor VIII, rather than a complex with thrombin, is the method of activation of factor VIII and that factor VIII is more accessible to the action of immobilized thrombin than is fibrinogen.
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PMID:The action of immobilized thrombin on factor VIII, fibrinogen and a synthetic tripeptide. 56 72

Factor XII was purified approximately 14 000-fold from bovine plasma by ammonium sulfate fractionation followed by heparin-agarose, DEAE-Sephadex, CM-cellulose, arginine-agarose, and benzamidine-agarose column chromatography. By this method, about 15 mg of protein was purified from 15 L of plasma with an overall yield of 18%. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal analysis. Bovine factor XII is a glycoprotein with a mol wt of 74 000 as determined by sedimentation equilibrium centrifugation. It contains 13.5% carbohydrate including 3.4% hexose, 4.7% N-acetylhexosamine, and 5.4% N-acetylneuraminic acid. Factor XII is a single polypeptide chain with an NH2-terminal sequence of Thr-Pro-Pro-Trp-Lys-Gly-Pro-?-Lys-His. This sequence is homologous to the reactive-site regions of a number of protease inhibitors. The amino acid sequence of a carboxyl-terminal fragments prepared by cyanogen bromide digestion was found to be Leu-Cys-Ala-Gly-Phe-Leu-Glu-Gly-Gly-Thr-Asp-Ala-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro-Leu-Val-Cys-Glu-Asp-Glu. This sequence is homologous with the active site of a number of plasma serine proteases including thrombin, factor IXa, factor Xa, and plasmin. These data indicate that bovine factor XII is a precursor to a serine enzyme with an inhibitor sequence and a catalytic site located in the same single polypeptide chain.
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PMID:Isolation and characterization of bovine factor XII (Hageman factor). 86 Dec 10

The amino-acid sequence of the heavy chain of bovine blood coagulation factor X1 (Stuart factor) isolated before and after activation has been determined. Sequence analysis was performed on fragments obtained by cleavage with cyanogen bromide and by tryptic digestion. Comparison of the complete sequence with those of other hepatic and pancreatic serine proteases demonstrates homology of the heavy chain of activated factor X1 (factor X1a) with the B chain of bovine thrombin as well as with bovine trypsin, chymotrypsins A and B, and porcine elastase. The activation peptide cleaved near the amino terminus by a protease from Russell's viper venom differs in both size and sequence from those of other serine proteases. With three exceptions, all of the residues which are important in the catalytic functions of trypsin and chymotrypsin occur in corresponding loci in the heavy chain of factor Xa. These finding suggest that the three-dimensional structure of the heavy chain is similar to that of the pancreatic serine proteases and that these enzymes have evolved from a common ancestral gene.
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PMID:Bovine factor X1 (Stuart factor): amino-acid sequence of heavey chain. 105 93

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

1. In bovine aortic endothelial cells (BAEC), thrombin (1 mu ml-1), bradykinin (1-10 nM) and adenosine triphosphate (ATP) (0.3 microM-100 microM) each induced a biphasic elevation of cytosolic calcium ([Ca2+]i), consisting of an initial transient followed by a sustained plateau phase. 2. Pretreatment of BAEC with 4 beta-phorbol 12-myristate 13-acetate (PMA; 100 nM) reduced the magnitude of the initial transient elevation of [Ca2+]i, induced by thrombin (1 mu ml-1), low concentrations of bradykinin (1 nM) or ATP (0.3 microM, 3 microM), but not by higher concentrations of the latter two agonists. Addition of PMA (100 nM) during the plateau phase of the increase in [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (10 nM) or ATP (30 microM) resulted in a fall in [Ca2+]i. 3. The inhibitory effects of PMA (100 nM) were inhibited by staurosporine (100 nM) but not mimicked by the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD; 100 nM). Furthermore, staurosporine (100 nM) increased [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by thrombin or bradykinin. In contrast, staurosporine (100 nM) reduced [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by ATP (30 microM). 4. Pretreatment with forskolin (10 microM) had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (1 nM and 10 nM) or ATP (30 microM). In contrast, forskolin (10 microM) and isoprenaline (10 microM) each induced biphasic elevations of [Ca21]i when added during the plateau phase of the increase in [Ca2+]i induced by the three agonists. Furthermore, in the presence of the inhibitor of calcium influx, nickel chloride (4mM), these biphasic elevations were reduced to monophasic transient elevations. 5. 8 Bromo cyclic GMP (30 microM), a membrane-permeant analogue of guanosine 3': 5'-cyclic monophosphate (cyclic GMP), had no effect on the magnitude of the initial transient elevation of [Ca21]i induced by thrombin (1 u ml 1), bradykinin (10 nM) or ATP (3 microM). Furthermore, 8 bromo cyclic GMP (30 microM) and sodium nitroprusside (1 microM), had no effect when added during the plateau phase of the increase in [Ca2+]i induced by the three agonists. 6. NG nitro-L-arginine (50,microM), an inhibitor of nitric oxide synthase, had no effect on the magnitude of the initial transient elevation of [Ca21]i induced by thrombin (1 uml- ), bradykinin (1 nM) or ATP (3,microM), and had no effect on the plateau phase of the increase in [Ca2+]i induced by these agents. 7. These findings suggest that while activation of protein kinase C inhibits and elevation of adenosine 3': 5'-cyclic monophosphate (cyclic AMP) augments calcium mobilisation in bovine aortic endothelial cells, elevation of cyclic GMP appears to have no effect.
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PMID:Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP. 166 33

1. Thrombin caused a tonic contractile response in rabbit aortic strips which showed tachyphylaxis. 2. Thrombin-induced contraction was partially dependent upon extracellular calcium. 3. Contractile response by lower concentrations of thrombin was suppressed by the endothelium. This endothelial effect was blocked by methylene blue, hemoglobin, bromophenacyl bromide or removal of extracellular calcium but not by indomethacin, nordihydroguaiaretic acid or nifedipine. 4. Cyclic GMP levels were not different between the thrombin-stimulated and control strips. 5. Thrombin could not stimulate prostacyclin release from the aortic strips. 6. These results suggest that thrombin possesses a contractile action in rabbit aortic smooth muscle which is attenuated by endothelium-derived relaxing factor (EDRF) spontaneously released from the endothelium during the contraction.
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PMID:Inhibitory effect of the endothelium on thrombin-induced contraction in rabbit aorta. 166

Platelets have abundant tyrosine kinase activities, and activation of platelets results in the increased tyrosine phosphorylation of numerous protein substrates. The stimulation of tyrosine phosphorylation elicited by thrombin can be completely inhibited by preincubation with 10nm prostacyclin (PGI2), 1 microM PGD2, or 1mM N2,2'-O-dibutyryl-cAMP. In contrast, incubation of platelets with agents that increase cGMP (sodium nitroprusside or with 1mM 8-Bromo-cGMP) was without effect. The inhibition by prostacyclin was dose dependent, with an IC50 of approximately 3nM, corresponding to the dose range necessary to inhibit other platelet activation processes. These results demonstrate a novel pathway by which agents which raise cAMP may inhibit platelet signal transduction and differential mechanism of action between compounds which raise cAMP and those which elevate cGMP.
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PMID:Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets. 169 64

Antithrombin (AT) Vicenza has been previously identified as a functionally abnormal antithrombin associated with familial thrombosis (Finazzi et al, 1985). It binds normally to heparin, but loses its affinity following interaction with thrombin: it is a poor inhibitor of thrombin. AT Vicenza was isolated from plasma by heparin-Sepharose and thrombin-Sepharose chromatography, fragmented with cyanogen bromide (CNBr) and its tryptic peptides were analysed by fast atom bombardment mass spectrometry mapping. An abnormal peptide mass 1112 was identified. Edman degradation confirmed a substitution of Ala to Pro in the sequence Ala 383-Arg 393. Polymerase chain reaction amplification of exon 6 of the gene followed by genomic sequencing, localized the mutation to codon 384, GCA to CCA. The same mutation has recently been reported in AT Charleville (Mohlo-Sabatier et al, 1989). Sodium dodecyl-sulphate polyacrylamide gel electrophoresis of AT Vicenza (/Charleville) under non-reducing conditions revealed an apparent increase in mol. wt following interaction with thrombin: under reducing conditions the mol. wt was less than that of normal AT. This indicated cleavage and unfolding of the molecule. The site of cleavage was determined by incubation of AT Vicenza (/Charleville) with thrombin-Sepharose, reduction and S-carboxymethylation and reverse phase FPLC. A peptide was identified with the NH2-terminal sequence beginning Ser-Leu-Asn, demonstrating the cleavage had occurred at the reactive site of the variant. It is concluded that the Ala 384 to Pro substitution transforms AT Vicenza (/Charleville) from an inhibitor into a substrate.
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PMID:Antithrombin Vicenza, Ala 384 to Pro (GCA to CCA) mutation, transforming the inhibitor into a substrate. 199 1

Antithrombin III (AT) binds to cultured cells mainly as a complex with thrombin or other serine proteases rather than in its free form. This implies that, upon complex formation, a new determinant appears on the AT molecule which is recognized by the cells. Fragmentation of AT by cyanogen bromide exposes this determinant and an 8-kDa fragment is recognized by cultured cells. The binding of this fragment to cultured cells is inhibited by antithrombin-III-thrombin (AT-T) complex, but not by free AT. The putative cellular binding domain of AT-T is located over amino acid residues 253-314 of AT, in the large loop close to the carboxy-terminus of the molecule. The cell-associated AT-T is internalized and degraded, forming the thrombin-modified AT (ATM). This process is inhibited by lysosomal degradation inhibitors, such as chloroquine or benzamidine. Thus, the appearance of ATM in cells is not a result of its binding to the cell surface, but probably a result of lysosomal degradation of cell-associated AT-T. Another degradation product, with a molecular mass of 43 kDa, appears in cells along with the appearance of ATM. Hirudin, a specific inhibitor of thrombin, inhibits the cellular internalization of AT-T complexes, indicating a possible role for thrombin activity in the internalization process of complexes. Monoclonal antibodies (mAb) against AT, whose epitopes on the AT molecule have been located, affect the binding of AT-T to cultured cells. Two mAb, A10 whose epitope is found outside of the cellular binding site, and B108 whose epitope may overlap this sequence, totally inhibit the binding of the complex to cells. Inhibition of binding results either from steric hindrance or induced changes in the binding site.
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PMID:Characterization of the cellular binding domain and the effects of monoclonal antibodies and thrombin inhibitors on the binding and internalization of the antithrombin-III--thrombin complex by cultured cells. 199 98

Using strips of human basilar arteries mounted in organ chambers to record isometric tension, we investigated vascular reactivity to thrombin and bradykinin. Both agents produced endothelium-dependent relaxation of basilar artery strips precontracted with phenylephrine but had no effect on resting tension in strips with or without endothelium. The relaxations caused by thrombin were abolished by antithrombin III/heparin, hirudin, and MD805. Thrombin but not bradykinin caused complete tachyphylaxis toward a second exposure. Indomethacin did not inhibit the relaxations induced by thrombin or bradykinin, whereas bromophenacyl bromide and methylene blue did. Aging decreased the relaxation induced by thrombin but did not affect the concentration needed to reach 50% maximal relaxation, nor did it affect the maximal relaxation in response to bradykinin, calcium ionophore A23187, and sodium nitroprusside. Our results suggest that thrombin and bradykinin produce endothelium-dependent relaxations mediated by an endothelium-derived relaxing substance and that the relaxation caused by thrombin is mediated by a proteolytic action on endothelial cells. The decrease in relaxations in response to thrombin with increasing age might be due to a decrease in the number or sensitivity of thrombin receptors on endothelial cells.
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PMID:Effect of aging on endothelium-dependent vascular relaxation of isolated human basilar artery to thrombin and bradykinin. 211 73


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