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

Tissue kallikrein and factor Xa were found to activate tissue plasminogen activator (t-PA) at a rate comparable with that of plasmin. During the activation reaction, the single-chain molecule was converted into a two-chain form. A slight t-PA activating activity was also found in plasma kallikrein. Other activated coagulation factors, factor XIIa, factor XIa, factor IXa, factor VIIa, thrombin and activated protein C had no effect on t-PA activation. t-PA was also activated by a tissue kallikrein-like enzyme that was isolated from the culture medium of melanoma cells. These results indicate that tissue kallikrein and factor Xa may participate in the extrinsic pathway of human fibrinolysis.
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PMID:Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes. 656 16

DX-9065a is an orally active newly synthesized and specific inhibitor for factor Xa. We have examined the property of DX-9065a in vitro and ex vivo. DX-9065a prolonged human plasma recalcification time, APTT and PT. Its doubling concentrations for clotting times of each coagulation assay were 0.49, 0.97 and 0.52 microM, respectively. Kinetic study revealed that DX-9065a inhibited competitively human factor Xa (Ki value: 41 nM). Ki values (microM) for other human serine proteases were as follows; thrombin > 2000, trypsin 0.62, chymotrypsin > 2000, plasmin 23, t-PA 21, plasma kallikrein 2.3 and tissue kallikrein 1000. DX-9065a up to 100 microM had no effects on human platelet aggregation. After intravenous or oral administration, DX-9065a significantly prolonged APTT and PT with a dose dependent manner. These effects were well correlated with anti-Xa activity in plasma. These results suggest that DX-9065a may become an anticoagulant by means of the specific inhibition of factor Xa.
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PMID:DX-9065a, a new synthetic, potent anticoagulant and selective inhibitor for factor Xa. 802 95

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine-proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin-type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation-exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (M(r) 5866) revealed that hirustasin is the only antistasin-type protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight-binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactive-site peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin-type proteinase inhibitors.
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PMID:Isolation and characterization of hirustasin, an antistasin-type serine-proteinase inhibitor from the medical leech Hirudo medicinalis. 811 45

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.
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PMID:Interactions of mast cell tryptase with thrombin receptors and PAR-2. 902 Jan 12

We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202-12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin(1-170)) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C18 reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin(1-170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin(1-170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (Ki = 0.1 nM), human tissue kallikrein (Ki = 0.1 nM), human plasma kallikrein (Ki = 0.3 nM) and human factor XIa (Ki = 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (Ki = 1.6 microM), factor IXa (Ki = 206 nM), factor Xa (Ki = 364 nM), and factor XIIa (Ki = 430 nM). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitro specificity profile, recombinant placental bikunin(1-170) prolonged the clotting time in an activated partial thromboplastin time assay.
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PMID:Characterization of placental bikunin, a novel human serine protease inhibitor. 911 95

We have reported earlier the isolation and amino acid composition of bdellin A from medical leech, and characterised it as an inhibitor of trypsin, plasmin and acrosin [Fritz, H., Gebhardt, M., Meister, R. & Fink, E. (1971) in Proceedings of the international research conference on proteinase inhibitors (Fritz, H. & Tschesche, H., eds) pp. 271-280, Walter de Gruyter, Berlin]. In the present study, one of several chromatographic forms of this inhibitor was isolated from a semi-pure preparation. Elucidation of its amino acid sequence revealed that bdellin A is a member of the antistasin family. Therefore, it was renamed bdellastasin to avoid confusion with bdellin B, which is another trypsin-plasmin inhibitor from the medical leech, but of the Kazal type. Furthermore, a synthetic gene of bdellastasin was constructed, and the protein expressed in Saccharomyces cerevisiae with yields of 29 mg/l. The recombinant bdellastasin was purified by hydrophobic interaction and anion-exchange chromatography. Comparison by mass spectroscopy, far-ultraviolet circular dichroism studies, sequence determination, and inhibition characteristics demonstrated the identity of recombinant and native bdellastasin. The Ki values of bdellastasin for inhibition of bovine trypsin and human plasmin are in the nanomolar range; no inhibition was detected for factor Xa, thrombin, tissue kallikrein, plasma kallikrein and chymotrypsin. Circular dichroism analyses indicated that bdellastasin is devoid of secondary-structural elements.
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PMID:Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech (Hirudo medicinalis)--primary structure, expression in yeast, and characterisation of native and recombinant inhibitor. 957 79

The clearance of exogenous plasma kallikrein, its uptake by liver, spleen, kidneys, lungs and its extravasation in the paws were determined in normal Wistar rats, normal and kininogen-deficient Brown Norway rats. Kallikrein was purified from rat plasma and labelled with 125I. After intravenous injection of 125I-kallikrein, the disappearance of acid-precipitable kallikrein from the blood fits a biexponential curve similar in the three groups of rats: a rapid initial clearance (T1/2 around 3 min) followed by a phase of slower elimination (T1/2 around 50 min). Removal of kallikrein from the blood was associated with a large uptake of radioactivity by the liver: 67% of the 125I-kallikrein cleared from the blood at 10 min. The kidneys and the spleen accumulated small amounts of the radioactivity. The uptake of kallikrein by the spleen was slightly reduced in kininogen-deficient rats. The kininogen deficiency in Brown Norway rats from the strain BN/May Pfd was confirmed by the low levels of kinins released by tissue kallikrein and by a prolongation of activated thromboplastin times in the plasma of these animals. We concluded that plasma kallikrein is rapidly cleared from the circulation of the rat. The liver is the main clearing organ of plasma kallikrein. The disappearance of kallikrein from the circulation is not affected by the lack of high molecular weight kininogen, except in the case of the uptake of the enzyme performed by the cells of the spleen, which is reduced.
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PMID:The fate of plasma kallikrein in normal and kininogen-deficient rats. 978 57

A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.
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PMID:Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema. 1070 49

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.
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PMID:Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides. 1137 81

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.
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PMID:Proteinase inhibitors from the medicinal leech Hirudo medicinalis. 1156 48


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