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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site.
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PMID:Amide H/2H exchange reveals a mechanism of thrombin activation. 1678 23

Ecotin is a Escherichia coli-derived protein that has been characterized as a potent inhibitor of serine-proteases. This protein is highly effective against several mammalian enzymes, which includes pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. In this work we showed that ecotin binds to human alpha-thrombin via its secondary binding site, and modulates thrombin catalytic activity. Formation of wild type ecotin-alpha-thrombin complex was observed by native PAGE and remarkably, gel filtration chromatography showed an unusual 2:1 ecotin:enzyme stoichiometry. Analysis of the protease inhibitor effects on thrombin biological activities showed that (i) it decreases the inhibition of thrombin by heparin/antithrombin complex (IC50=3.2 microM); (ii) it produces a two-fold increase in the thrombin-induced fibrinogen clotting; and (iii) it inhibits thrombin-induced platelet aggregation (IC50=4.5 microM). Allosteric changes on thrombin structure were then evaluated. Complex formation with ecotin caused a three-fold increase in the rate of thrombin inhibition by BPTI, suggesting a displacement of the enzyme's 60-loop. In addition, ecotin modulated the enzyme's catalytic site, as demonstrated by changes in the fluorescence emission of fluorescein-FPRCK-alpha-thrombin (EC50=3.5 microM). Finally, solid phase competition assays demonstrated that heparin and prothrombin fragment 2 prevents thrombin interaction with ecotin. Altogether, these observations strongly support an ecotin interaction with thrombin anion-binding exosite-2, resulting in modulation of its biological activities. At this point, ecotin might be useful as a new tool for studying thrombin allosteric modulation.
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PMID:Ecotin modulates thrombin activity through exosite-2 interactions. 1684

Prothrombin is a vitamin K-dependent serine protease and plays pivotal roles in both procoagulant and anticoagulant pathway of hemostasis. In this study, we cloned the full-length cDNA of porcine prothrombin by cDNA library screening and SMART RACE technique. The full-length cDNA is 2027 bp, with a 1869 bp Open Reading Frame (ORF) coding 623 amino acids. The deduced protein of porcine prothrombin contains signal peptide, propeptide, Gla domain, two kringle domains and trypsin domain. Porcine prothrombin shares 86.15% nucleotide similarity and 83% amino acid similarity with human prothrombin. The trypsin domain is highly conserved between the two species with 92.1% amino acid identity. Macromolecular interaction sites comparison between porcine and human prothrombin suggests that the Gla domain in porcine prothrombin contains an additional potential gamma-carboxyglutamic acid site. However, a thrombin cleavage site (Arg284-Thr285) in its light chain is lost. When thrombin heavy chain is concerned, the most important functional sites such as catalytic triad DHS, RGD site, Na+ binding site and anion-binding exosite-I and II are highly conserved. However, great differences have been observed between residues 145 and 158 of heavy chain which is associated with thrombomodulin binding. Two important limited proteolysis sites at Ala150 and Lys154 were lost in porcine sequence, which would affect epsilon-thrombin and gammaT-thrombin generation. Comparison on 3-D protein models demonstrates that these proteins are obviously different in autolysis loop (Lys145 to Gly155). Compared with that of human prothrombin, variation at critical recognition sites would likely alter its binding affinity and reaction velocity, which would contribute to coagulation disorder when porcine liver is transplanted into human body.
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PMID:Full-length cDNA cloning and protein three-dimensional structure modeling of porcine prothrombin. 1718 33

In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in various ways. At present, to devitalize tumor-bearing osteochondral segments, extracorporeal irradiation or autoclaving is mainly used, although both methods have substantial disadvantages, leading to a significant loss of biomechanical and biological integrity of the bone. As an alternative approach, a new technology to achieve bone sterilization, the high hydrostatic pressure (HHP) treatment of bone, has been suggested, which is currently being preclinically tested. This novel technique leads to the inactivation of tumor cells without impairing biomechanical properties of the bone, cartilage, or tendons. HHP may not only exert an effect on tumor and normal cells present in the bone but also on tumor-associated proteases released by these cells, which are conductive to tumor bone turnover. In order to investigate this, proteolytic key enzymes, e.g. MMP-9, uPA, t-PA, plasmin, trypsin, and thrombin were subjected to HHP <or=600 MPa. Thereafter, compared to the non-pressurized enzymes, the proteolytic activity of the pressurized enzymes was determined. The proteases studied showed varying degrees of susceptibility to HHP, depending on the pressure level applied. The latent activity of the inactive zymogens prothrombin, plasminogen, and pro-uPA, in addition to the proteolytically active forms of plasmin, thrombin, HMW-uPA, and trypsin were minimally affected by HHP (10 min, 20 degrees C, 600 MPa) with a reduction of activity up to 13% only, whereas t-PA was significantly impaired by a reduction of activity of 30%. In contrast, for pressurized pro-MMP-9 (10 min, 5 degrees C, 400 MPa) a 3-fold increase in enzymatic activity was observed after activation compared to non-pressurized pro-MMP-9. No activation of pro-MMP-9 due to HHP was observed. These data encourage further exploration of the potential of HHP to sterilize tumor-affected bone segments prior to reimplantation. During this treatment tumor cells are irreversibly impaired, while HHP treatment of proteases may not exert any significant autolytic effect on bone tissue.
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PMID:Quantitative analysis of the impact of short-time high hydrostatic pressure on bone tumor-associated proteases. 1733 43

Serum mast cell tryptase levels are used as a diagnostic criterion and surrogate marker of disease severity in mastocytosis. Approximately 29% of the healthy population lacks alpha tryptase genes; however, it is not known whether lack of alpha tryptase genes leads to variability in tryptase levels or impacts on disease severity in mastocytosis. We have thus analyzed tryptase haplotype in patients with mastocytosis, computing correlations between haplotype and plasma total and mature tryptase levels; and disease category. We found: (1) the distribution of tryptase haplotype in patients with mastocytosis appeared consistent with Hardy-Weinberg equilibrium and the distribution in the general population; (2) the disease severity and plasma tryptase levels were not affected by the number of alpha or beta tryptase alleles in this study; and (3) information about the tryptase haplotype did not provide any prognostic value about the severity of disease. Total and mature tryptase levels positively correlated with disease severity, as well as prothrombin time and partial thromboplastin time, and negatively correlated with the hemoglobin concentration.
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PMID:Tryptase haplotype in mastocytosis: relationship to disease variant and diagnostic utility of total tryptase levels. 1744 30

Fractionated Plasma Separation, Adsorption and Dialysis (Prometheus) has a well-documented capacity to remove protein-bound organic toxins in patients with liver failure. However, the compositions of adsorbed proteins remain unknown. Elution of both adsorbers constituting Prometheus system was performed following a 6-h session in a patient with acute on chronic liver failure. Sodium dodecylsulphate was employed to elute proteins from the neutral adsorber (P1), while acetic acid was applied to the cationic one (P2). Eluted proteins were resolved by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS). Totally, 4113 and 8280 mg of proteins were obtained from P1 and P2 eluates, 2-DE yielded 148 and 163 protein fractions in P1 and P2, respectively. MS identified 18 unique proteins in P1, and 30 unique proteins in P2 sample. Proteins with the highest selective adsorption (as determined by eluate to plasma ratio) included transthyretin (37), trypsin-2 (29), prothrombin (23), hyaluronan-binding protein 2 (13) and plasma retinol-binding protein (8.7), all of which adsorbed to P2. We identified a large number of proteins removed by extracorporeal liver support system. A selective adsorption was demonstrated in a subset of proteins depending on the type of adsorber and proteins' characteristics.
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PMID:Proteomic analysis of proteins bound to adsorption units of extracorporeal liver support system under clinical conditions. 1926 3

AZD0837 is in development as a new oral anticoagulant for use in thromboembolic disorders. In vivo, AZD0837 is converted to AR-H067637, a selective and reversible direct thrombin inhibitor. Established biochemical methods were used to assess and measure the biochemical and pharmacological properties of AR-H067637. Both direct Biacore binding studies of AR-H067637 with immobilised alpha-thrombin and inhibition studies using pre-steady state kinetics with thrombin in the fluid phase confirmed that AR-H067637 is a rapid-binding, reversible and potent (inhibition constant K(i) = 2-4 nM), competitive inhibitor of thrombin, as well as of thrombin bound to fibrin (clot-bound thrombin) or to thrombomodulin. The total amount of free thrombin generated in platelet-poor clotting plasma was inhibited concentration-dependently by AR-H067637, with a concentration giving half maximal inhibition (IC(50)) of 0.6 microM. Moreover, AR-H067637 is, with the exception of trypsin, a selective inhibitor for thrombin without inhibiting other serine proteases involved in haemostasis. Furthermore, no anticoagulant effect of the prodrug was found. AR-H067637 prolonged the clotting time concentration-dependently in a range of plasma coagulation assays including activated partial thromboplastin time, prothrombin time, prothrombinase-induced clotting time, thrombin time and ecarin clotting time. The two latter assays were found to be most sensitive for assessing the anticoagulant effect of AR-H067637 (plasma IC(50) 93 and 220 nM, respectively). AR-H067637 also inhibited thrombin-induced platelet activation (by glycoprotein IIb/IIIa exposure, IC(50) 8.4 nM) and aggregation (IC(50) 0.9 nM). In conclusion, AR-H067637 is a selective, reversible, competitive inhibitor of alpha-thrombin, with a predictable anticoagulant effect demonstrated in plasma coagulation assays.
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PMID:Biochemical and pharmacological effects of the direct thrombin inhibitor AR-H067637. 1949 47

Nine of the 17 venoms here tested were found capable of coagulating citrated blood or plasma. As has been believed by most workers in the field, 7 of these 9 coagulant venoms convert fibrinogen to an insoluble modification resembling fibrin (Bothrops atrox, Bothrops jararaca, Bothrops nummifera, Crotalus adamanteus, Crotalus horridus, Crotalus terrificus basiliscus, Crotalus terrificus terrificus). The optimum pH for this coagulation was determined for 3 of these, and was found in each case to be approximately pH 6.5, the same as that for the action of thrombin on fibrinogen. Unlike thrombin, however, the fibrinogen-coagulating activity of the venoms was unaffected by the antithrombin elaborated in the course of anaphylactic shock. In addition to coagulating fibrinogen directly, 3 of these venoms (Bothrops atrox, Bothrops jararaca, and to a less extent, Crotalus terrificus basiliscus) acted on prothrombin to convert it to thrombin, without the necessary intervention of either calcium or platelets. Finally, 2 venoms (Notechis scutatus, and to a slight extent, a mixed Micrurus venom), which had no demonstrable effect on purified fibrinogen, nevertheless converted prothrombin to thrombin. Unlike the reaction between the venoms and fibrinogen, this activation of prothrombin has no definite pH optimum, but takes place over a wide zone (pH 5.6-8.3). In the case of Bothrops atrox, there was some indication that the initial velocity of the reaction increased with increasing alkalinity, but that the amount of thrombin ultimately formed decreased. Extraordinarily minute quantities of some of these venoms sufficed to produce a demonstrable activation of prothrombin. Thus, the fer de lance (Bothrops atrox) venom was active in a 1:25,000,000 dilution, and that of the Australian tiger snake (Notechis scutatus) was active in a 1:4,000,000 dilution. The thrombin formed was indistinguishable from that produced by the action of calcium + platelets on prothrombin. Like the latter type of thrombin, and unlike venoms which act directly on fibrinogen, thrombin formed from prothrombin by venom was inhibited by antithrombin. Every one of the 9 non-coagulant venoms in this series destroyed prothrombin; and 5 of these destroyed fibrinogen as well. As is discussed in the text, there is reason to believe that these several properties of the venoms (coagulation and destruction of fibrinogen; activation and destruction of prothrombin) depend on the proteolytic enzymes which they were found to contain. These observations lend further support to the thesis that, in the course of physiological coagulation, (a) calcium plus platelets (or tissue derivative) constitute an enzyme system which reacts with prothrombin to form thrombin, and which is thus analogous to trypsin and to several of the proteolytic venoms here discussed, and (b) the thrombin so formed is itself a proteolytic enzyme which, like papain and the majority of the coagulant and proteolytic snake venoms here studied, reacts with fibrinogen to form a fibrillar gel, fibrin.
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PMID:THE COAGULATION OF BLOOD BY SNAKE VENOMS AND ITS PHYSIOLOGIC SIGNIFICANCE. 1987 Jun 22

Certain specimens of human semen shorten the coagulation time of whole blood because of the presence of active thromboplastic agents, while other samples prolong its coagulation time. Human prostatic fluid in large amounts always delays or abolishes blood coagulation. The delay or absence of clotting is counteracted by adding calcium ions and is due to the large concentration of citrate in prostatic fluid and in some semens. While most specimens of dog semen shorten the coagulation time of blood because of their thromboplastic activity, certain specimens render blood incoagulable or delay coagulation; in contrast to human semen, this adverse effect on coagulation is not overcome with calcium ions and is due to a different mechanism, the lysis of fibrinogen. The citrate content of dog prostatic fluid is small. Human semen which has become liquefied does not contain thrombin or prothrombin, but fibrinogen and thromboplastic substances are present. Beef fibrinogen added to semen is destroyed by incubation for 18 hours, but added prothrombin and thromboplastic substances are still present after this treatment. Dog semen, in some instances, contains small amounts of thrombin. The semens of man and dog contain a fibrinolysin for human blood which seems not to differ greatly from the fibrinolysin associated with hemolytic streptococci. The blood of the donor of prostatic fluid is susceptible to fibrinolysis by this fluid. However, the blood of persons with some diseases, is absolutely resistant to the action of seminal fibrinolysin. In how many diseases this happens has not yet been determined. The semens of man and dog both contain an agent capable of inactivating fibrinogen, but in different amounts. This activity may be called fibrinogenase. Human semen is rich in fibrinolysin, poor in fibrinogenase; dog semen is rich in fibrinogenase, poor in fibrinolysin. These species differences, together with the fact that it is easy by appropriate dilution to retain the stronger proteolytic agent and eliminate the weaker one, imply that fibrinolysin and fibrinogenase are different entities. Dog semen, and less constantly human semen, contain very small amounts of trypsin. All of these proteolytic agents derive from the prostate gland; their secretion in prostatic fluid constitutes a hitherto undescribed function for the prostate gland.
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PMID:COAGULATION AND LIQUEFACTION OF SEMEN : PROTEOLYTIC ENZYMES AND CITRATE IN PROSTATIC FLUID. 1987 Dec 56

Crude or crystalline trypsin in proper concentration causes the blood or plasma of human beings, dogs, rabbits, guinea pigs, and horses to coagulate. It does not clot the fibrinogen directly, but reacts with prothrombin to form thrombin. Since trypsin thus has the same effect as the physiological system Ca plus platelets (or Ca plus tissue extracts), it is suggested as a tentative working hypothesis that the latter system contains a proteolytic enzyme with a specific affinity for prothrombin. Other implications of this trypsin effect with respect to the mechanism of physiological coagulation are discussed in the text (pages 557-558). The proteolytic enzyme papain also coagulates blood. In this case the enzyme does not activate prothrombin, but acts directly on fibrinogen to form a fibrillar gel resembling fibrin. If one admits this clot to be fibrin, this constitutes strong evidence that thrombin, the physiological coagulant, is also a proteolytic enzyme with a specific action on fibrinogen.
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PMID:STUDIES IN BLOOD COAGULATION : V. THE COAGULATION OF BLOOD BY PROTEOLYTIC ENZYMES (TRYPSIN, PAPAIN). 1987 9


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