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

---

Query: EC:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The second domain (R-020) of human urinary trypsin inhibitor (UTI) exerts similar inhibitory activities on trypsin, alpha-chymotrypsin, leukocyte elastase, and plasmin to those of UTI itself, and additionally inhibits coagulation factor Xa (FXa) and plasma kallikrein, on both of which UTI has no inhibitory effect. In the present study, we attempted to increase this FXa-inhibitory activity by modeling the structure of R-020-FXa complex and substituting one or two amino acids in R-020 using recombinant DNA technology. Molecular modeling of R-020 and FXa was performed with reference to X-ray analysis of the complex of bovine pancreatic trypsin inhibitor (BPTI) and bovine trypsin to determine the site of amino acid modification. The expression plasmids into which R-020 genes with base substitution were inserted were prepared and introduced into Escherichia coli to express R-020 variants. The resulting variants were purified and their enzyme inhibitory activities were measured. The FXa-inhibitory activity was increased in four variants with single amino acid substitution. With another four variants having two amino acid substitutions involving combinations of the above single amino acid substitutions, the FXa-inhibitory activity was further increased. Because the electrostatic interaction within R-020-FXa complex seemed stronger in these R-020 variants, this increase in FXa-inhibitory effect was speculated to be a consequence of more potent binding between the enzyme and the inhibitor.
...
PMID:Design of variants of the second domain of urinary trypsin inhibitor (R-020) with increased factor Xa inhibitory activity. 798 90

Protein C is a vitamin K-dependent serine protease zymogen that upon activation inhibits the coagulation cascade by inactivating factors Va and VIIIa. In an attempt to improve the anticoagulant activity of activated protein C (APC), we have prepared a mutant of protein C in mammalian cells in which Glu at position 192 (chymotrypsin numbering system) has been replaced with Gln (PC E192Q). Our strategy is based on the observation that the same substitution in thrombin improves the catalytic activity toward natural and synthetic substrates that contain Asp residues at P3 and P3'. Since factor Va also has an Asp at position P3 in the APC cleavage site of the factor Va heavy chain, we hypothesized that APC E192Q would inactivate factor Va more rapidly than wild type APC. The mutant inactivated factor Va approximately 2-3-fold faster than wild type. In plasma the mutant exhibited slightly less anticoagulant activity than wild type enzyme. Further characterization revealed that APC E192Q is inhibited 280 times faster than APC by alpha 1-antitrypsin (K2 = 2.8 x 10(3) M-1S-1 versus 10 M-1 S-1), and unlike APC, APC E192Q is inhibited by antithrombin III in the presence of heparin (K2 = 1.17 x 10(3) M-1 S-1) M-1 S-1) and absence of heparin (K2 = 57 M-1 S-1). Ca2+ increased K2 more than 4-fold with or without heparin. Unlike wild type APC, APC E192Q was effectively inhibited by pancreatic trypsin inhibitor (Ki = 10.6 +/- 0.26 nM) and tissue factor pathway inhibitor (58 +/- 5 nM). Like factor Xa, APC E192Q rapidly processed factor IX to factor IX alpha. These observations suggest that even though Glu at position 192 is not an optimal residue for catalyzing factor Va inactivation, it is an evolutionary adaptation to slow inhibition by plasma protease inhibitors.
...
PMID:Conversion of glutamic acid 192 to glutamine in activated protein C changes the substrate specificity and increases reactivity toward macromolecular inhibitors. 810 82

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.
...
PMID:Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. 819 13

An integral membrane protease was solubilized and purified to homogeneity from rat submaxillary mitochondria. The purified enzyme could coagulate rabbit plasma. The molecular mass of the enzyme is 22 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions and 24 kDa on gel filtration on a Sephadex G-100 column. Its isoelectric point is 4.2-4.25. Enzyme activity is strongly inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, benzamidine, aprotinin, and antipain, suggesting the enzyme as a serine protease. Its pH optimum for activity is 8.5. Zn2+ is strongly inhibitory; at 1 mM concentration it produced 72% inhibition. The enzyme is active toward different synthetic substrates (p-nitroanilide derivatives) containing Arg at the P1 position with blocked NH2 terminus. Kcat/Km was highest with the substrate N-Bz-Pro-Arg-pNa (where Bz is benzoyl and pNA is paranitroanilide). The purified enzyme coagulates rabbit plasma in a dose-dependent manner. Plasma coagulation by the enzyme is completely blocked in the presence of aprotinin or soybean trypsin inhibitor, suggesting that protease activity is required for this coagulation reaction. Antibody raised against the purified enzyme inhibits the plasma coagulation initiated by the enzyme. The enzyme can correct the prolonged clotting time of factor X-deficient human plasma but is unable to convert purified fibrinogen to fibrin clots, indicating factor Xa-like activity of the enzyme. The enzyme has the ability to activate prothrombin. Several properties of the enzyme distinguish it from other reported submaxillary proteases.
...
PMID:A new blood-coagulating protease in mitochondrial membranes of rat submaxillary glands. Purification and characterization of protease and its blood-coagulating activity. 820 26

Previous investigations have indicated that interference with the initial level of the blood coagulation may lead to effective antithrombotic therapy. Recently a series of potential coagulation inhibitors derived from bovine pancreatic trypsin inhibitor (BPTI, aprotinin) was described. We have determined their inhibition constants, effects on coagulation assays, effects in an in vitro human thrombosis model and pharmacological profiles in hamsters. The aprotinin-derived analogues (4C2, 7L22, 5L15, 6L15, 5L84) showed significantly increased inhibitory activity towards factor Xa, factor VIIa-tissue factor (TF) complex, factor XIa and plasma kallikrein or a combination of them, and a significantly decreased plasmin inhibition as compared to aprotinin. In the coagulation assays, 4C2 and 7L22 mainly inhibited factor Xa, 5L15 and 6L15 inhibited factor VIIa-TF complex and 5L84 inhibited factor Xa, factor VIIa-TF complex and the contact activation. In flow chamber experiments with human blood 7L22, 5L15, 6L15, 5L84 and rTAP significantly inhibited fibrin formation and platelet deposition on extracellular matrix from phorbol ester stimulated human endothelial cells both under high and low shear stress and in the presence of low molecular weight heparin. The pharmacological profiles of the aprotinin analogues and rTAP with a mean residence time of 64 to 140 min were not significantly different. Modification of an aprotinin analogue with PEG (5L15-PEG) resulted in a 10-fold decrease of the inhibition constant for the factor VIIa-TF complex and in a significant prolongation of the secondary half-life, while the initial half-life was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Characterisation of a novel series of aprotinin-derived anticoagulants. I. In vitro and pharmacological properties. 858 1

Upon vascular damage platelet activation and blood coagulation are initiated. Interference at the initial level of the activation of the coagulation cascade can result in effective inhibition of thrombus formation. The in vivo antithrombotic properties of a series of bovine pancreatic trypsin inhibitor mutants (BPTI, aprotinin) 4C2, 7L22, 5L15, 5L15-PEG, 6L15 and 5L84, as described in the accompanying paper, with a combined inhibitory activity on factor Xa, factor VIIa-tissue factor complex, factor XIa and plasma kallikrein were compared to rTAP, r-hirudin, heparin and enoxaparin in a platelet rich thrombosis model in hamsters. Platelet dependent thrombus deposition was quantified by dedicated image analysis after transillumination of the femoral vein to which a standardised vascular trauma was applied. After increasing intravenous bolus injections all tested agents, except for aprotinin, induced a dose dependent decrease of thrombus formation and a concomitant prolongation of the aPTT. From the linear correlation between these two parameters it was found that 5 out of the 6 tested aprotinin analogues, rTAP and r-hirudin completely inhibited thrombus formation at a therapeutical (2- to 3-fold) aPTT prolongation while 4C2, heparin and enoxaparin only inhibited thrombus formation for 40 to 50 percent at a 2-fold aPTT prolongation. Based on the calculated IC50 values for thrombus formation rTAP was found to be the most active compound in this model. It is concluded that acceptable interference at the initial level of the blood coagulation, e.g. within a therapeutical aPTT prolongation, can significantly inhibit platelet deposition at a site of vascular injury.
...
PMID:Characterisation of a novel series of aprotinin-derived anticoagulants. II. Comparative antithrombotic effects on primary thrombus formation in vivo. 858 2

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.
...
PMID:Isolation and properties of a blood coagulation factor X activator from the venom of king cobra (Ophiophagus hannah). 859 78

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.
...
PMID:An activator of blood coagulation factor X from the venom of Bungarus fasciatus. 859 79

The pathogenesis of intestinal damage caused by bolus intravenous injection of endotoxin (ETX; 3 mg/kg) was investigated. Administration of ETX to rats induced reddish discoloration suggestive of bleeding, increased hemoglobin amounts, and leakage of plasma protein in the intestine. However, light microscopic examination of the intestine demonstrated blood congestion of the microvessels. Plasma accumulation was partially inhibited by combined pretreatment with a histamine H1 antagonist and a serotonin (5-HT) antagonist. Neither a 5-lipoxygenase inhibitor, a soybean trypsin inhibitor, nor atropine was observed to inhibit plasma accumulation. Both the intestinal leakage of plasma and the accumulation of hemoglobin were completely inhibited by indomethacin, a selective thromboxane A synthetase inhibitor (OKY 1581), and a stable PGI2 analogue (beraprost). Intravital microscopic observation of the microvessels of the small intestinal villi demonstrated microthrombus formation within several minutes after the injection of ETX, and pretreatment with OKY 1581 attenuated the formation of microthrombus. Platelet counts decreased significantly 10 min after ETX administration, and the decrease was not inhibited by pretreatment with either OKY 1581 or beraprost. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were not prolonged. These observations thus suggest that microcirculatory disturbances by platelet thrombus, which are mediated by thromboxane A2 at least in part, play an important role in ETX-induced intestinal damage.
...
PMID:Induction mechanism of small intestinal lesions caused by intravenous injection of endotoxin in rats. 885 94

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds of Torresea acreana, one of the two known species of Torresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears as M(r) 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2, Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.
...
PMID:Sequence of a new Bowman-Birk inhibitor from Torresea acreana seeds and comparison with Torresea cearensis trypsin inhibitor (TcTI2). 889 2


<< Previous 1 2 3 4 5 6 7 Next >>

---