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)

The authors report the effects of four proteases (trypsin, plasmin, chymotrypsin and thrombin) on human heart adenylate cyclase (HHAC) activity. Trypsin and plasmin inhibited HHAC at concentrations higher than 0.3 and 1 microgram/ml, respectively. Chymotrypsin had a biphasic effect, with a stimulation (from 0.5 to 10 micrograms/ml) followed by an inhibition at higher concentrations. Maximal stimulation was obtained at 3 micrograms/ml and averaged 67.2 +/- 5.4%. Thrombin had no significant effect at concentrations up to 1 mg/ml. These data indicate that proteases might regulate HHAC and therefore influence cardiac function.
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PMID:The effects of trypsin, plasmin, chymotrypsin and thrombin on human heart adenylate cyclase activity. 295 13

Thrombomodulin is an endothelial cell surface protein which complexes with thrombin to accelerate protein C activation. To gain insight into the mechanisms of thrombomodulin-membrane association, limited proteolytic digestions of thrombomodulin with trypsin and elastase were performed. Trypsin digestion resulted in two major fragments (Mr = 54,000 and 27,000), both of which bound to phosphatidylcholine/phosphatidylserine vesicles. Elastase digestion also yielded two major fragments (Mr = 50,000 and 25,000), but only the smaller fragment bound to the phospholipid vesicles. The larger fragment obtained from both enzymatic digestions retained the ability to accelerate protein C activation. The Mr = 54,000 fragment from the trypsin digest retained a high affinity for thrombin (Kd less than or equal to 0.5 nM), a Km for protein C of approximately equal to 8 microM, and a half-maximal Ca2+ dependence of 0.3 mM. The Mr = 50,000 fragment from elastase digestion had a lower affinity for thrombin (Kd approximately equal to 6 nM) than intact thrombomodulin, and the Km for protein C was decreased to 0.3 microM in the presence of 0.3 mM Ca2+. The Ca2+ dependence of protein C activation with the Mr = 50,000 fragment was distinctly different from that of thrombomodulin or the active tryptic fragment. The active elastase fragment exhibited a Ca2+ optimum at 0.3 mM and activity rapidly decreased with further increases in Ca2+. At the Ca2+ optimum, the Km for protein C was similar to that observed on endothelial cell surfaces or with thrombomodulin reconstituted into liposomes. Our data demonstrate that thrombomodulin has one or more membrane-binding domains and that an active soluble form with catalytic activity can be generated by limited proteolytic digestion. Digestion with elastase appears to expose a site on thrombomodulin capable of recognizing the gamma-carboxyglutamic acid domain of protein C (residues 1-44 of the light chain). Whether this is the same site which is exposed on thrombomodulin upon incorporation into phospholipid vesicles (see accompanying manuscript) remains to be determined.
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PMID:Proteolytic formation and properties of functional domains of thrombomodulin. 302 70

Standard isolated smooth muscle tissue preparations were used to screen the musculotropic actions of serine proteases and other substances that are generated during hemostatic activation. Sera obtained from human, rabbit, and guinea pigs produced a dose-dependent contraction of these isolated tissue preparations. These sera also augmented the actions of epinephrine and other agonists on different tissue preparations. Both contractile and relaxant actions of proteases were observed with various serine proteases and their complexes. Thrombins (alpha, beta, and gamma) produced varying degrees of contractile actions of rabbit aortic strips; however, Factor Xa produced a marked relaxation of this preparation. Trypsin and kallikrein did not produce any action on the aortic strip and thrombin, however, produced contraction of the guinea pig ileum. Protease complexes produced varying musculotropic actions on the isolated tissue preparations, which may be related to their composition. The musculotropic actions of serum and thrombin were not blocked by conventional pharmacologic antagonists. However, protease inhibitors, such as hirudin, D-Phe-Pro-Arginal, and other derivatives, produced varying effects of the musculotropic actions of serum and thrombins. These results indicate that thrombin and related proteases generated during the activation of the hemostatic system are capable of producing direct musculotropic actions of various smooth muscles. Various serine proteases and serine protease complexes were found to produce strong hemodynamic effects in a rabbit model for the study of blood plasma. The smooth muscle modulant actions of serine proteases should be taken into account to clarify the vascular pathologic changes in relation to spasmogenic and spasmolytic syndromes observed during hemostasis disorders.
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PMID:Modulation of smooth muscle responses by serine proteases and related enzymes. 309 26

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.
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PMID:Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants. 316 17

We investigated the binding of C4 and C3 to red cell surfaces by non-complement enzymes. Cell bound C components were quantitated by a radioimmunoassay, the chain structure of bound components was analyzed by Western blotting and the hemolytic activity of bound components was determined. Trypsin, chymotrypsin, plasmin, elastase, thrombin, kallikrein and enzymes from Bacillus subtilis, Staphylococcus aureus and Streptomyces griseus all were found capable of binding C4b and C3b to sheep red cells. C4b bound by any of these enzymes was hemolytically active; both classical and alternate pathway activity of C3 could be demonstrated for most enzymes except plasmin and thrombin. In addition, trypsin and the bacterial enzymes were also able to generate the classical pathway C3-convertase from C4b + C2. The hemolytic efficiency of enzyme bound C4b and C3b was about the same as for these molecules bound by complement enzymes. In contrast, the process of binding by the non-complement enzymes was several hundred-fold less efficient than by cell bound complement enzymes. The results demonstrate that several enzymes can replace the C1 and C42 enzymes in the classical pathway and are able to initiate the alternative pathway by activating C3 and binding C3b to the cell surface.
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PMID:Binding and activation of C4 and C3 on the red cell surface by non-complement enzymes. 341 32

Permeabilization of human platelets with saponin (15-25 micrograms/ml) allows the determination of the ADP-ribosylation of a 41-kDa protein by pertussis toxin. The ADP-ribosylated protein is present in the particulate fraction. ADP-ribosylation of the 41-kDa protein increases for 20 min; it is not affected by indomethacin, prostacyclin, and 1,2-diacylglycerols but is inhibited by 1 mM Ca2+ and phorbol esters. Treatment of platelets with trypsin, thrombin, or collagen before saponin addition precludes subsequent pertussis toxin-induced ADP-ribosylation of the 41-kDa protein. The effect of trypsin or thrombin is blocked by soybean trypsin inhibitor and leupeptin. Trypsin proteolytically cleaves the ADP-ribosylated 41-kDa protein to an ADP-ribosylated fragment slightly smaller than 20 kDa. The results suggest that a modification of a guanine nucleotide-binding regulatory protein is associated with the actions of trypsin, thrombin, and collagen on platelet activation.
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PMID:Treatment of human platelets with trypsin, thrombin, or collagen inhibits the pertussis toxin-induced ADP-ribosylation of a 41-kDa protein. 346 64

This study examined whether the neurointermediate lobe (NIL) of the rat pituitary contains latent kallikrein- and thrombin-like proteases activated by trypsin. Partial characterization of such proteases was attempted. Also examined were the distribution of proteolytic activity within the NIL and levels in both male and female lobes. NIL homogenates were assayed for proteolytic activity at pH 8.0 before and after incubation with trypsin (10 micrograms/ml). Trypsin caused a 10-fold activation of kallikrein-like activity and a 40-fold activation of thrombin-like activity in NIL homogenates. The kallikrein-like activity was separated into two components using diethylaminoethyl-Sephadex. The predominant kallikrein-like protease was a potent kininogenase closely related or identical to glandular kallikrein and was almost exclusively localized to the intermediate lobe. The second kallikrein-like protease (kallikrein A) was a weak kininogenase sensitive to inhibition by both soybean trypsin inhibitor and aprotinin and was similarly concentrated in both the neural lobe and the intermediate lobe. The thrombin-like protease was sensitive to inhibition by hirudin (a specific thrombin inhibitor), clotted fibrinogen, and was slightly more concentrated in the neural lobe than in the intermediate lobe. NILs from female rats contained approximately 40% less kallikrein activity than NILs from male rats but did not differ in their content of thrombin-like activity.
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PMID:Trypsin activation, partial characterization, and distribution of kallikrein-like and thrombin-like proteases in the neurointermediate lobe of the rat pituitary. 351 68

We have used both proteolysis and reconstitution experiments to characterize the determinants for LDL receptor binding of HTG-VLDL. In these studies, we showed that the removal of approximately one mole of apo E per mole of HTG-VLDL (Sf 100-400) and HTG-VLDL (Sf 60-100) by thrombin-specific cleavage results in loss of receptor binding and concomitant loss of suppression of HMG-CoA reductase. This is in direct contrast to the lack of effect thrombin cleavage has on the receptor-mediated uptake of LDL, an apo B-mediated process. We were able to reconstitute receptor binding in thrombin-treated HTG-VLDL (Sf 100-400) by the specific reincorporation of one mole of apo E into the VLDL. The incorporation of one mole of apo E into normal non-suppressive VLDL (Sf 60-400) also enables this lipoprotein to bind to the receptor as effectively as LDL. Trypsin, which destroys apo E-mediated, but not apo B-mediated binding to the LDL receptor, abolishes binding of HTG-VLDL (Sf 100-400) and HTG-VLDL (Sf 60-100), but not that of HTG-VLDL (Sf 20-60), IDL, or LDL to the LDL receptor. Therefore, we conclude that apo E of the appropriate conformation is required for receptor-mediated uptake by the LDL receptor of large TG-rich lipoproteins (Sf greater than 60). This conformation of apo E is probably related to the surface on which it is found (i.e., size of the particle) and the mode of incorporation into the phospholipid surface (i.e., transferred from plasma HDL). In large TG-rich particles, it appears that the intact apo E is necessary for the proper orientation of the molecule on the surface, with the carboxy-terminal one-third needed to anchor the apoprotein to the phospholipid surface. We believe that the binding of apo E to the LDL receptor is a redundant system and is used as a backup system in abnormal pathological states such as hypertriglyceridemia, abetalipoproteinemia, and hypobetalipoproteinemia. In the case of hypertriglyceridemia, where the lipolysis mechanism is overloaded, the abnormal binding of HTG-VLDL (Sf greater than 60) provides an alternate catabolic route for their removal from plasma. In the cases of a beta- and hypobetalipoproteinemia, where the normal particles for cholesterol delivery are either absent or at low levels, apo E-containing particles can serve to deliver cholesterol to cells as has been recently observed in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Expression of LDL receptor binding determinants in very low density lipoproteins. 390 64

Large triglyceride-rich very low density lipoproteins (VLDL) Sf 60-400 from hypertriglyceridemic (HTG) patients, but not VLDL from normal subjects, bind to the LDL receptor of human skin fibroblasts because they contain apolipoprotein E (apoE) of the correct conformation, accessible both to the LDL receptor and to specific proteolysis by alpha-thrombin. Trypsin treatment of HTG-VLDL Sf 60-400 causes extensive apoB hydrolysis (fragments less than 100,000 mol wt), total degradation of apoE, and thus complete loss of LDL receptor binding. The reincorporation of apoE (1 mol/mol VLDL) into trypsin-treated HTG-VLDL completely restored the ability of HTG-VLDL to interact with the LDL receptor, suggesting that apoE probably does not induce a conformational change in apoB which results in receptor recognition, nor is intact apoB necessary to maintain the appropriate conformation of apoE for LDL receptor binding. As a model of large triglyceride-rich VLDL Sf greater than 60, we fractionated Intralipid by the Lindgren method of cumulative flotation and prepared apoE-Intralipid complexes. Competitive binding studies demonstrated that apoE-Intralipid is at least as effective as LDL for uptake and degradation of 125I-labeled LDL. Control Intralipid complexes containing apoA-I instead of apoE do not compete with iodinated LDL. Since these TG-rich complexes contain no apoB, apoB is, therefore, not only not sufficient for receptor-mediated uptake of large particles, it is not necessary. ApoE of the correct conformation is not only necessary but is sufficient to mediate receptor binding of large triglyceride-rich particles to the LDL receptor.
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PMID:ApoE is necessary and sufficient for the binding of large triglyceride-rich lipoproteins to the LDL receptor; apoB is unnecessary. 395 13

The platelet aggregating component from murine 15091A mammary adenocarcinoma cells was purified by solubilization of activity with CHAPS (3-[(3-cholidamidopropyl)-dimethylammonio]-1-propane sulfonate), fractionation with ammonium sulfate, ion exchange chromatography on DEAE cellulose, and hydrophobic interaction chromatography on dodecyl agarose. A purification of 90-100 fold over the initial cell homogenate was achieved. SDS-PAGE of the purified material resulted in a single major band with a molecular weight of 51,000 +/- 2,000. Procoagulant activity was found to copurify with platelet aggregating activity. Reconstitution with phospholipids was necessary to obtain platelet aggregating activity and procoagulant activity. Trypsin abolished both platelet aggregating and procoagulant activities. The irreversible proteinase inhibitors phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethyl ketone, iodoacetamide or phenanthroline had no effect on platelet aggregating or procoagulant activities. Platelet aggregation induced by this material was inhibited by low concentrations of the specific irreversible thrombin inhibitors, dansylarginine N-(3-ethyl-1, 5-pentanediyl) amide and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone. This is the first report of copurification of tumor cell platelet aggregating and coagulating activities.
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PMID:Purification and characterization of platelet aggregating activity from tumor cells: copurification with procoagulant activity. 397 74


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