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)

Addition of protease inhibitors to the culture medium has been shown to enhance neurite outgrowth by cultured mouse dorsal root ganglia (DRG). Those results are now extended to show that a diffusible source of soybean trypsin inhibitor (STI) or zones of immobilized STI can orient the direction of outgrowth towards the region of STI. However, a high concentration of diffusible STI promotes outgrowth in the opposite direction from the STI source. Immobilized leupeptin, L-lysine, or D-Phe-Pro-Arg-chloromethyl ketone can also direct outgrowth towards their immobilized areas, as do zones of laminin or fibronectin. However, derivatized zones containing urokinase or thrombin preferentially direct outgrowth away from those zones. These data support the hypothesis that a balance between extracellular protease and inhibitor is important in mediating interactions between neurite growth cone and extracellular matrix.
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PMID:Protease inhibitors influence the direction of neurite outgrowth. 271 79

A method based on active-site affinity chromatography on soybean trypsin inhibitor (SBTI)-Sepharose was developed for isolation of human factor Xa in primarily the undergraded alpha-form. The chromatography procedure separated factor Xa from factor X, the Russel's viper venom proteinase used to activate factor X, and traces of contaminating thrombin. alpha-Factor Xa was unstable at pH 7.6 and 25 degrees C, undergoing slow proteolytic degradation to functionally heterogeneous products as evidenced by the greater loss of coagulation assay activity compared to activity measured with a chromogenic substrate. The results of monitoring factor Xa degradation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with proteolysis of the light chain as a major component reaction occurring in parallel with slower proteolysis of the heavy chain. The decreased rates of these reactions at pH 6.0 enabled isolation and storage of factor Xa in greater than or equal to 88% alpha-form and minimized the heterogeneity due to proteolytic degradation. Characterization of the reaction of fluorescein mono-p-guanidinobenzoate (FMGB) with human and bovine factor Xa isolated by SBTI-Sepharose chromatography demonstrated its utility as a sensitive reagent for continuous fluorometric active-site titration. Analysis of the reaction kinetics as a function of FMGB and human factor Xa concentrations in G/2 0.3, pH 7.4, buffer at 25 degrees C indicated that the ratio of acylation to deacylation rate constants was greater than 200 and that the Km for FMGB was 0.06-0.11 microM, predicting pre-steady-state burst amplitudes of greater than or equal to 96-98% of the active-site concentration at FMGB concentrations greater than or equal to 5 microM. Human factor Xa active-site concentrations were consistent with 82-99% active preparations when compared with the protein concentrations determined from the 280-nm absorbance. Concentrations of human alpha-factor Xa as low as 20 nM could be measured with FMGB, indicating a sensitivity approximately 50 times greater than that measured by spectrophotometric active-site titration with p-nitophenyl p'-guanidinobenzoate.
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PMID:Isolation of human blood coagulation alpha-factor Xa by soybean trypsin inhibitor-sepharose chromatography and its active-site titration with fluorescein mono-p-guanidinobenzoate. 277 57

The Egyptian Sand Viper (Cerastes cerastes) crude venom and subfractions were, for the first time, shown to induce platelet aggregation with agonist activities present in two subfractions. The combined activities of the crude venom components behaved in a unique fashion as compared to the platelet agonists, ADP, collagen and thrombin. The action of the venom was inhibited by conditions that increased cAMP, partially required the formation of thromboxane A2 and was inhibited by the serine protease inhibitor PMSF while being only partially sensitive to leupeptin or soybean trypsin inhibitor. One of the fractionated venom agonists strongly induced serotonin release while the other venom agonist essentially did not. Further characterization of the Cerastes cerastes venom components should broaden our knowledge of the pathology of snake venoms, platelet aggregation and their potential therapeutic value.
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PMID:Cerastes cerastes (Egyptian sand viper) venom induced platelet aggregation as compared to other agonists. 282 99

Plasma from insulin-dependent diabetics shows an increased ability to specifically activate the (Na-K)ATPase from different sources. Several protease inhibitors like phenyl methyl sulfonyl fluoride, trypsin inhibitor, antithrombin III and aprotinin, produced a significant dose-dependent inhibition of the stimulatory effect produced by a 1/100 final dilution of plasma on the beef heart (Na-K)ATPase activity. Serine proteases employed at scalar concentrations in the ATPase medium gave a dose-dependent stimulation of the enzyme activity as did diabetic plasma. The maximum percent stimulation of the (Na-K)ATPase activity (about 60%) was reached by 0.56 microgram/ml of thrombin, 0.50 microgram/ml of kallikrein and 0.55 microgram/ml of trypsin. The protease-induced ATPase stimulation was significantly reduced by antithrombin III, trypsin inhibitor and by aprotinin. A partial purification of the activating plasma factor was obtained by eluting plasma on a heparin-Sepharose column. Two (Na-K)ATPase stimulating fractions were found, which eluted with 1.0 and 3.0 mol/l NaCl, respectively. Half-maximal stimulation of the enzyme occurred with 3.4 micrograms/ml proteins of fraction 1.0 mol/l and with 45 ng/ml proteins of fraction 3.0 mol/l, this last representing the most purified plasma fraction (about 8890-fold purification). The proteolytic activity of both plasma and purified plasma fractions was tested on Tos-Arg-OMe substrate which was hydrolyzed to a much higher degree by the most purified plasma fraction. Like the (Na-K)ATPase stimulation, the esterolytic activity was inhibited by protease inhibitors, the most effective to this regard being antithrombin.
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PMID:Identification and partial purification of a (Na-K)ATPase stimulating serine protease from plasma of insulin-dependent diabetics. 283 59

The influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein.
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PMID:Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets. 283 22

Plasmin was recently reported to inhibit platelet aggregation. We report here on the interaction of plasmin with the adenylate cyclase system of human platelets. Human plasmin caused a dose- and time-dependent increase in adenylate cyclase activity when added to a crude platelet membrane preparation. Both basal and prostaglandin E1-stimulated adenylate cyclase activity doubled in presence of plasmin. This stimulatory activity was shared by papain and alpha-chymotrypsin, but not by thrombin which displayed a slightly inhibitory effect. Plasmin not only stimulated platelet adenylate cyclase activity, but also suppressed the GTP-dependent alpha 2-adrenergic inhibition, thereby producing a five- to six-fold increased activity measured in the presence of adrenaline and GTP. These effects of plasmin on the adenylate cyclase system were suppressed by the addition of the protease inhibitor leupeptin, and of soybean trypsin inhibitor, indicating that proteolysis mediated these effects. We also examined the adenylate cyclase activity in membranes prepared from intact platelets incubated with increasing doses of plasmin. Incubation of platelets with plasmin concentrations as low as 0.25 mg/ml resulted in an irreversible increase in membrane adenylate cyclase activity and suppression of the adrenaline-mediated inhibition of enzyme activity. These results suggest that the proteolytic stimulating effect of plasmin on the platelet adenylate cyclase system may account for the inhibition of platelet aggregation.
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PMID:Plasmin: a possible physiological modulator of the human platelet adenylate cyclase system. 295 Oct 52

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme-linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.
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PMID:High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s). 300 Apr 74

Strong activity of acid-stable trypsin inhibitor (ASTI) was confirmed in some clinical thrombin preparations. Thrombin preparations of human plasma origin had no detectable ASTI activity, whereas some preparations of bovine plasma origin revealed more than 5,000 U/vial (5,000 thrombin units), indicating a higher content of ASTI than of thrombin in terms of protein concentration. Contamination by other biologically active substances was also suggested by variations in amidolytic activity with several synthetic substrates (S-2238, S-2251, S-2444, S-2266 and Bz-L-Arg-pNA). On isoelectric focussing, the ASTI activities migrated in acidic positions with pI values of 3.9, 4.5, 5.0, 5.9 and 6.5, respectively. They were almost parallel to the thrombin Bz-L-Arg-pNA hydrolytic activity, and differed from that of the purified thrombin preparation (pI = 7.0). By gel filtration on Sephadex G-100, the molecular weights of the inhibitors as calculated using standard proteins were 140,000 (main), 70,000 and less than 10,000 (minor), respectively. An immunological difference between the main inhibitor (pI = 3.9, mol wt 140,000) and previously reported plasma ASTI was also confirmed with goat anti-UTI serum by the double immunodiffusion and ELISA methods. The inhibitor exerted a strong inhibitory effect not only on trypsin and chymotrypsin, but also on non-plasmic fibrinolysis with human leukocyte elastase, and to a lesser extent on the blood coagulation system (lengthening of APTT and PT). Clearly, when using thrombin preparations and analyzing the data obtained after their administration, the effects of this and other contaminant biologically active substances must be taken into account.
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PMID:Strong activity of acid-stable trypsin inhibitor in bovine thrombin for clinical use. 314 Oct 90

Kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human alpha-, beta- and gamma-thrombin have been determined, between 5 and 45 degrees C, at pH 7.5. BPTI-binding properties to human thrombins have been analyzed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates, with particular reference to the bovine beta-trypsin/BPTI system. The observed binding behaviour of BPTI to human alpha-, beta- and gamma-thrombin has been related to the inferred stereochemistry of the enzyme/inhibitor contact region(s).
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PMID:Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human alpha-, beta- and gamma-thrombin; a kinetic and thermodynamic study. 316 67

A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly.
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PMID:Kunitz-type protease inhibitor found in rat mast cells. Purification, properties, and amino acid sequence. 326 66


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