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)

Factor VIII is a large glycoprotein which can be separated into a high molecular weight component which retains factor VIII-related antigens and supports ristocetin-induced platelet agglutination, and a low molecular weight component which has procoagulant activity. It has been suggested that this separation, observed in high ionic strength buffers, might be the consequence of proteolysis by plasma proteins. To consider this possibility, we have examined the interaction of the proteolytic enzyme thrombin with factor VIII. This study, carried out with highly purified materials, has used thrombin-mediated factor VIII proagulant activation as an indicator of this interaction. Several proteolytic inhibitors have been studied to determine their ability to inhibit factor VIII activation by thrombin. Under the current experimental conditions, diisopropylfluorophosphate (DFP) and hirudin inhibited the reaction, while heparin was an effective inhibitor only when plasma proteins were added. Benzamidine inhibited factor VIII activation when used at high concentrations, and phenylmethyl sulphonylfluoride and soybean trypsin inhibitor were found to be ineffective. These results show that DFP and hirudin prevent thrombin alteration of factor VIII and will be useful in purification and characterization studies of the factor VIII molecule.
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PMID:Thrombin activation of factor VIII: the effect of inhibitors. 88 21

Single-chain human recombinant factor VII produced by transfected baby hamster kidney cells was purified to homogeneity in the presence of benzamidine. The amidolytic activity of single-chain recombinant factor VII with a peptidylnitroanilide substrate, methoxycarbonyl-D-cyclohexanylglycyl-L-arginine-p-nitroanilide, was less than 1% of that obtained with factor VIIa. Purified single-chain recombinant factor VII spontaneously activated in the absence of inhibitor. The activation reaction was enhanced by at least 2 orders of magnitude in the presence of a positively charged surface, provided either as an anion-exchange matrix or as poly(D-lysine). The progress curve for factor VIIa generation was sigmoidal. Benzamidine inhibits recombinant factor VIIa activity and factor VII activation with identical inhibition constants (Ki) of 11 mM. In contrast, benzamidine inhibition of bovine factor Xa and bovine factor IIa was observed at Ki values equal to 0.3 and 0.5 mM, respectively. Bovine factors Xa and IIa are known activators of factor VII and the most likely contaminants of our recombinant factor VII preparations. Single-chain recombinant factor VII purified from cells cultured in the absence of bovine serum activated at the same rate as factor VII from cells cultured in the presence of bovine serum. This also excluded the possibility that the activation reaction was caused by contaminating bovine proteases. On the basis of these observations, we propose that factor VII is autoactivated in vitro in the presence of a positively charged surface.
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PMID:Autoactivation of human recombinant coagulation factor VII. 261 Dec 33

1. v([I]) data were obtained for the hydrolysis of the chromogenic substrate H-D-Phe-L-Pip-L-Arg-pNA (S-2238) by native human thrombin in the presence of the synthetic inhibitors Benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581). v([S]) data were also obtained in the absence and presence of fixed concentrations of each of the inhibitors. 2. Analysis of the kinetic data was based on the numerical fitting to rate equations of the polynomial quotient type of degree n:m using nonlinear regression methods. The discrimination between rate equations with different degrees was performed by application of the statistical F test. 3. Of eight v([I]) curves fitted, in six cases it was found that degree 1:2 was significantly better than degree 1:1 at a confidence level of 95% or higher; in no case was a significant improvement found with rate equations with a higher number of parameters. For the v([S]) data, of eleven curves fitted it was found that in nine cases degree 2:2 significantly improved degree 1:1 at confidence levels 99% and in one case at a level of 95%; no significant improvement was found with rate equations of higher degree for these data either. 4. Our findings allow us to propose that inhibition of the amidolytic activity of native human thrombin by benzamidine and I-2581 may be accounted for by mechanisms whose v([I]) rate equation will be a minimum of degree 1:2, thus implying a pure inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic study of the inhibition of the amidolytic activity of thrombin by benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581). 342 80

Benzamidine derivatives which are competitive inhibitors of trypsin-like serine proteinases also inhibited the enzymatic activity of batroxobin, a thrombin-like snake venom proteinase. Structure-activity relationships showed that primary amides of 4-amidinophenyl-alpha-aminobutyric acid have pronounced, relatively selective antibatroxobin activity. Identical effects were found on batroxobin isolated from the venoms of Bothrops atrox or Bothrops moojeni. Esters containing a benzamidine moiety acylated the active centre serine hydroxyl of either batroxobin, however, the inhibition was temporary. Such compounds, especially 4-amidinophenyl esters of substituted benzoic acids, are a particularly useful tool for designing acyl-batroxobin intermediates with different deacylation rates. With 4-nitrophenyl 4'-guanidinobenzoate, the acyl enzyme was formed so rapidly that titration of the active site of batroxobin was possible. Irreversible inhibition of batroxobin was caused only by the selective thrombin inhibitor D-Phe-Pro-ArgCH2Cl.
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PMID:Inhibition of batroxobin, a serine proteinase from Bothrops snake venom, by derivatives of benzamidine. 352 3

An anticoagulant activity was identified and isolated from the leaves of a West African plant, Aspilia africana by gel filtration on Sephadex G-100. The anticoagulant factor had an apparent molecular weight of approximately 60,000 d. Upon incubation with plasma, it prolonged the partial thromboplastin time, prothrombin time, thrombin and reptilase time. The factor decreased the fibrinogen content of plasma as well as the activity of coagulation factors V, VIII and IX but not factor VII, X or XI activities. After incubation with fibrinogen, the thrombin clotting time was prolonged and the quantity of clottable fibrinogen reduced. The action on fibrinogen was characterized by sequential lytic breakdown of the A-alpha-chain and B-beta-chain, the gamma-chain being lysed last, after prolonged incubation. Benzamidine, Epsilon aminocaproic acid or soybean trypsin inhibitor did not impede lysis.
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PMID:Studies on the anticoagulant action of Aspilia africana. 366 Mar 50

Serine proteinases are involved in several physiological processes and elicit profound cellular effects in a variety of tissues. Besides the thrombin receptor a second receptor, activated by trypsin, the proteinase-activated receptor 2 (PAR-2), was cloned and characterized. Both enzymes generate a new extracellular N-terminus by limited proteolytic cleavage which functions as tethered ligand to activate the receptor. Synthetic peptides corresponding to the sequences of the newly generated N-terminus are able to mimic the effects of the enzymes. In porcine pulmonary arteries trypsin and the receptor-derived peptide SLIGRL elicited an endothelium-dependent transient relaxation of PGF2alpha-precontracted vessels. The EC50 values for trypsin and SLIGRL amounted to 1.1 +/- 0.2 nM and 5.4 +/- 0.6 microM, respectively. Trypsin and SLIGRL caused a homologous desensitization but thrombin and the thrombin receptor-activating peptide SFLLRN were still able to elicit pronounced relaxant effects. The trypsin- and SLIGRL-induced relaxant responses were markedly diminished after blockade of the nitric oxide synthesis by N(G)-nitro-L-arginine methyl ester (200 microM) and were absent in endothelium-denuded vessels. Indomethacin and hirudin did not influence the relaxant effects. The effect of trypsin but not that of SLIGRL was blocked by the proteinase inhibitor aprotinin suggesting that only proteolytically active trypsin activates the receptor. Benzamidine derivatives of the 3-amidinophenylalanine type with different affinity for trypsin and thrombin inhibited the vascular effects of trypsin (IC50 0.007-0.7 microM) correlating with its antitrypsin activity. The data suggest that the vascular effects of trypsin and SLIGRL are mediated through activation of PAR-2 which differs from the thrombin receptor.
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PMID:Trypsin- and SLIGRL-induced vascular relaxation and the inhibition by benzamidine derivatives. 940 26

A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bb chain of fibrinogen while the Aa chain and g chain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bb chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mm x 0.2 mm x 0.2 mm) and used for X-ray diffraction experiments.
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PMID:Purification and characterization of jararassin-I, A thrombin-like enzyme from Bothrops jararaca snake venom. 1559 46

Duvernoy's gland secretion of Philodryas patagoniensis exhibits high hemorrhagic activity, containing enzymes that are able to degrade the vascular wall. In this work we aim to determine if the secretion can also affect the hemostatic system by causing changes in blood coagulation. Procoagulant and coagulant activities were evaluated on plasma and fibrinogen, respectively. The delay in the thrombin clotting time of fibrinogen previously incubated with the secretion was also determined. Specific hydrolysis of fibrinogen and fibrin incubated with the secretion at different time intervals was shown by electrophoresis on polyacrylamide gel. To determine the structural characteristics of the enzymes degrading fibrinogen and fibrin, secretion were incubated in the presence of 45 mM Na(2)EDTA, 40 mM Benzamidine, and/or 2 mM PMSF before the incubation with fibrinogen or fibrin, respectively. The effect in vivo was investigated in adult male rats injected with different dose of secretion, aliquots of blood were withdrawn at different time intervals, and the fibrinogen concentration was determined. Duvernoy's gland secretion of P. patagoniensis did not clot plasma or fibrinogen. It exhibited a potent fibrinogenolytic activity degrading the Aalpha-chain faster than the Bbeta-chain, whereas gamma-chain was resistant. This latter corresponded with a strong delay in the thrombin clotting time of fibrinogen (4 mg/ml) pre-incubated with the secretion, being 9.53 microg the amount of protein from Duvernoy's gland secretion that increased the thrombin clotting time from 20 to 60 s. In vivo, the loss of rat plasma fibrinogen was proportional to the amount of secretion injected. The secretion also hydrolyzed fibrin degrading the alpha-monomer. Inhibition studies with Na(2)EDTA, Benzamidine, and/or PMSF showed that metalloproteinases and serinoproteinases are the main enzymes responsible for the hydrolyzing activity on fibrinogen and fibrin. All these results demonstrate that Duvernoy's gland secretion of P. patagoniensis possesses enzymes able to hydrolyze plasma components playing a relevant role in the blood coagulation. These hydrolyzing activities and those acting on the wall of blood vessels let the secretion exhibit a high hemorrhagic activity, which may result in permanent sequelae or even cause the death of the victims bitten by this colubrid snake.
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PMID:Duvernoy's gland secretion of Philodryas patagoniensis from the northeast of Argentina: its effects on blood coagulation. 1573 75

The synthesis and evaluation of inhibitors of activated protein C (aPC) are reported. This serine protease is partly responsible for the degradation of factor VIIIa, involved in the regulation of bleeding in hemophilia A. Benzamidine-containing derivatives were found to be potent aPC inhibitors, some of them showing selectivity against the procoagulant protease thrombin. Moreover, compound 1 significantly restored the generation of thrombin in hemophiliac plasma.
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PMID:Low molecular weight activated protein C inhibitors as a potential treatment for hemophilic disorders. 1691 94

The venom of Bothrops insularis snake, known in Brazil as jararaca ilhoa, contains a variety of proteolytic enzymes such as a thrombin-like substance that is responsible for various pharmacological effects. B. insularis venom chromatography profile showed an elution of seven main fractions. The thrombin-like activity was detected in fractions I and III, the latter being subjected to two other chromatographic procedures, so to say DEAE and Hi Trap Benzamidine. The purity degree of this fraction was confirmed by analytical reverse phase HPLC, which displayed only one main fraction confirmed by SDS-PAGE constituting fraction III. About 5 microg of fraction III protein potentiated the secretion of insulin induced by 2.8 mM of glucose in rats isolated pancreatic beta-cells treated; the increase being around 3-fold higher than its respective control. B. insularis lectin (BiLec; 10 microg/mL) was also studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. BiLec increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, the thrombin-like substance isolated from B. insularis venom induced an increase in insulin secretion, in vitro, and transiently altered vascular, glomerular and tubular parameters in the isolated rat kidney.
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PMID:Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom. 1716 57


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