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 review of different mechanisms of non-physiological non-specific prothrombin activation is given as compared with the specific activation by the factor Xa. The use of snake venom enzymes or staphylocoagulase makes possible the thrombin generation from pathological forms of prothrombin lacking of gamma-carboxyglutamic acid and incapable of complete activation into thrombin by the specific activator, factor Xa. This fact stimulated the use of non-specific activators in medicine. Investigation of non-specific prothrombin activators made possible to reveal and to trace pathways of the formation of thrombin active site. It is demonstrated that prothrombin, like other serine proenzymes (trypsinogen, chymotrypsinogen), has already formed active site. This site can be revealed under changes of the conformation of the prothrombin molecule due to the chemical modification or the complex formation with staphylocoagulase.
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PMID:[Mechanisms of non-specific prothrombin activation]. 73 9

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

Factor VIII is present in plasma in a precursor or inactive form. When bovine factor VIII that has been purified approximately 10,000-fold is incubated with thrombin, an activated product is formed which participates in the conversion of factor X to factor Xa in the presence of factor IXa, calcium ions, and phospholipid. This activated product, which has been tentatively identified as activated factor XIII, was stable when formed in the presence of 0.25M CaCl2 but was rapidly inactivated in the absence of CaCl2. It was inhibited by diisopropyl phosphorofluoridate and antithrombin III, suggesting that it is a serine enzyme. The exact role of this serine enzyme in the intrinsic pathway of coagulation remains to be established.
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PMID:Formation of a serine enzyme in the presence of bovine factor VIII (antihemophilic factor) and thrombin. 87 60

The esterolytic activity of bovine alpha-thrombin on the synthetic substrate N-alpha-p-tosyl-L-arninine methyl ester (TosArgOMe) is stimulated when the prothrombin activation fragment, prothrombin fragment 2, is added as previously reported by this laboratory (Heldebrant, C. M., and Mann, K. G. (1973), J. Biol. Chem. 248, 3642). A similar stimulation of beta-thrombin is observed upon addition of prothrombin fragment 2. The binding constant of prothrombin fragment 2 to alpha-thrombin has been determined by the method of Gutfreund ((1972), Enzymes, Physical Principles, Wiley, New York, N.Y., pp 67-71). The dissociation constant is 7.7 X 10(-10)M, and there is one molecule of prothrombin fragment 2 bound per molecule of alpha-thrombin. Prethrombin-2 competes for prothrombin fragment 2, so the enhancement of the esterolytic activity of alpha-thrombin by prothrombin fragment 2 was used as a probe to determine the dissociation constant for the binding of prothrombin fragment 2 to prethrombin 2. The dissociation constant for this association is 1.3 X 10(-10)M. The kinetic parameters for the reaction of alpha-thrombin on TosArgOMe were determined in the absence and presence of prothrombin fragment 2 and are as follows: (a) in the absence of prothrombin fragment 2, Km(app) = 1.92 X 10(-4)M, and k3(app) = 35.8 mol of TosArgOMe/mol of alpha-thrombin s(-1); (b) in the presence of prothrombin fragment 2,Km(app = 1.76 X 10(-4)M, and k3(app) = 60.5 mol of TosArgOMe/mol of alpha-thrombin s(-1). Thus, the stimulatory effect of bovine prothrombin fragment 2 on bovine alpha-thrombin is reflected in k3(app) and not in Km(app). In contrast to the stimulatory effect of prothrombin fragment 2 on the thrombin-catalyzed hydrolysis of TosArgOMe, it inhibits the activity of alpha-thrombin toward N-alpha-benzoyl-L-arginine ethyl ester and N-alpha-benzoyl-L-arginine p-nitroanilide. The inhibition of activity toward these substrates by prothrombin fragment 2 is also reflected in k3(app). Activity toward the nonspecific substrate p-nitrophenyl butyrate was completely inhibited by the addition of prothrombin fragment 2. Prothrombin fragment 2 has no effect on the inhibition of alpha-thrombin activity by the active-site serine inhibitors diisopropyl phosphofluoridate, phenylmethanesulfonyl fluoride, or p-nitrophenyl guanidinobenzoate. Inhibition by the active-site-histidine-modifying inhibitor, N-alpha-p-tosyl-L-arginine chloromethyl ketone, was enhanced by the addition of prothrombin fragment 2. Soybean trypsin inhibitor reduces the stimulation by prothrombin fragment 2, but only at high molar ratios. Prothrombin fragment 2 has no effect on the clotting activity of alpha-thrombin, nor inhibition of this activity by heparin, hirudin, or diisopropyl phosphafluoridate. Bovine prothrombin fragment 2 enhances the esterolytic activity of both human and bovine alpha-thrombin, but human prothrombin fragment 2 does not enhance the esterolytic activity of either human or bovine alpha-thrombin.
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PMID:Characteristics of the association between prothrombin fragment 2 and alpha-thrombin. 94 91

Bovine prothrombin was activated, in both the absence and presence of dissopropyphosphofluoridate (DEP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecysulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28,000 and 57,000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong alpha-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35,000 of 27,000 daltons. The former was indentified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocaralytically yielded a fragment (inner fragment) of 14,000 daltons with N-terminal serine and the clotting enzyme alpha-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51,000 and 34,000 daltons, of which the former consisted of N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with antithrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with p-nitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activaton of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond liniking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates alpha-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator...
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PMID:The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates. 95 36

The effect of organophosphorus inhibitors of serine esterases (proteases) on secretion from washed rabbit platelets was examined. Five noncytotoxic stimuli were employed: collagen, thrombin, heterologous anti-platelet antibody (in the absence of complement), rabbit C3 bound to zymosan, and platelet activating factor derived from antigen-stimulated, IgE-sensitized rabbit basophils. Diisoprophyl phosphofluoridate, three series of p-nitrophenyl ethyl phosphonates, and a series of cyclohexyl phenylalkylphosphonofluridates were all found to be inhibitory to the platelet secretion. These are irreversible inhibitors of serine proteases but in this system were only inhibitory if added to the platelets concurrently with the stimuli. Pretreatment of either the platelets or the stimuli with the inhibitors followed by washing, was without effect on the subsequent reaction. This suggested the involvement of stimulus-activatable serine proteases in the secretory process. The concept was supported by finding that nonphosphorylating phosphonates or hydrolyzed phosphonates or phosphonofluoridates were without inhibitory action. The effect of a series of phosphonates or phosphonoflouridates in inhibiting each stimulus exhibited a unique activity-structure profile. The demonstration of such unique profiles with four series of inhibitors for each of the five stimuli was interpreted as demonstrating that a specific activatable serine protease was involved in the platelet secretory response to each stimulus.
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PMID:Activation of stimulus-specific serine esterases (proteases) in the initiation of platelet secretion. I. Demonstration with organophosphorus inhibitors. 100 7

A possibility of formation of the thrombin-phosphatidyl serine and prothrombin-phosphatidyl serine complex is discussed. Prothrombin incubation with 131J-labelled phosphatidyl serine and its subsequent activation results in a formation of a thrombin-phosphatidyl serine--131J-complex. The radioactive label is also detected in the protein precipitate after thermodenaturation of thrombin preincubated with 131J-labelled phosphatidyl serine, which suggests that thrombin is firmly bound to phosphatidyl serine. The formation of the thrombin-phosphatidyl serine and prothrombin-phosphatidyl serine complex is supported by data of gel-filtration on Sephadex G-200 and acrylexes P-150 and P-60, as well as by differential spectrophotometry.
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PMID:[Formation of the prothrombin-phosphatidyl serine and thrombin-phosphatidyl serine complex]. 102 77

A 38-residue fragment is isolated from carboxymethylated plasminogen. Residues 29-38 have the same sequence as the amino-terminal end of the light chain of plasmin. The sequence 1-28 is therefore the sequence of the carboxyl-terminal end of the heavy chain and contains the specific sequence at which urokinase (EC 3.4.99.26) and other plasminogen-activating serine proteases split. Two of the five carboxymethyl-cysteine residues in the isolated fragment are situated close to the cleavage site and the fragment is not itself a substrate for plasminogen-activators. Residues 1-11 show extensive sequence homology with residues 137-147 and 242-252 in prothrombin, which are located in corresponding regions of the two internally homologous 83-residue structures in the non-thrombin part of the molecule, indicating that such structures may be a common feature of the non-protease part of the larger serine protease zymogens.
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PMID:Amino-acid sequence of activation cleavage site in plasminogen: homology with "pro" part of prothrombin. 105 75

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

Both the clotting and esterase activities of thrombin are inhibited by alpha1-proteinase inhibitor (alpha1-antitrypsin). The inhibition is a time-and temperature-dependent reaction which is proportional to the molar ratio of thrombin to inhibitor. Both the active-site serine residue of thrombin and the reactive-site lysine residue of alpha1-proteinase inhibitor are involved. alpha1-Proteinase inhibitor forms a 1:1 complex with thrombin that is comparable with the complex formed with trypsin and other proteinases. Incubation of the inhibitor with excess of thrombin, however, results in inactivation of nearly all the enzyme, even though only as much complex is formed as alpha1-proteinase inhibitor present. A portion of the remaining thrombin apparently aggregates. These results suggest that the mechanism for inhibition of thrombin may not be exactly the same as for trypsin, which is inhibited only to the extent to which complex is formed.
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PMID:Inactivation of human thrombin in the presence of human alpha1-proteinase inhibitor. 108 57


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