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 precursor of plasma carboxypeptidase B (pCPB) also known as thrombin-activable fibrinolysis inhibitor can be converted by thrombin to an active enzyme capable of eliminating C-terminal Lys- and Arg-residues from proteins. The activation is about 1000-fold more efficient in the presence of thrombomodulin (TM). We investigated the antifibrinolytic potency of maximally activated pCPB in plasma and explored the antifibrinolytic mechanism of pCPB. During clotting of plasma in the presence of 3.3 NIH units/ml thrombin and 1 microg/ml soluble TM, more than 80% pro-pCPB was converted into the active form causing an increase of plasma carboxypeptidase activity from 100 units/liter (constitutive activity ascribed to plasma carboxypeptidase N) to 430 units/liter as measured with furoylacroleyl-alanyl-arginine substrate. Under these conditions, lysis of a plasma clot induced by a range of tissue-type plasminogen activator (t-PA) concentrations (0.2-2 microg/ml) was retarded more than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration comparable with physiological concentrations of soluble TM in human plasma. The presence of Ca2+ appeared to be a critical requirement for effective activation of pro-pCPB by thrombin-TM in plasma. Plasminogen-binding sites (C-terminal lysines) on the surface of a plasmin-treated fibrin clot were eliminated within 1-3 min by plasma with maximally activated pCPB, as studied in a recently described model involving fluorescence microscopy. Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers during t-PA-induced lysis of a plasma clot. In the presence of TM (and a concomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers. In conclusion, generation of active pCPB during clotting of plasma in the presence of Ca2+ and TM leads to a retardation of plasma clot lysis in a wide range of t-PA concentrations, from low to therapeutic, and to a fast elimination of plasminogen-binding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.
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PMID:On the mechanism of the antifibrinolytic activity of plasma carboxypeptidase B. 916 90

Carboxypeptidase R (EC 3.4.17.20) (CPR) and carboxypeptidase N (EC 3.4.17.3) (CPN) cleave carboxy-terminal arginine or lysine residues from biologically active peptides such as kinins or anaphylatoxins in the circulation thereby regulating their activities. Although CPN is present in a stable active form in plasma, CPR is generated from proCPR, a plasma zymogen, by proteolytic enzymes such as thrombin, thrombin-thrombomodulin complex and plasmin. We have isolated rat proCPR and CPN cDNA clones which can induce enzymatic activities in culture supernatants of the transfected cells. mRNA of proCPR was detected only in rat liver by Northern hybridization and showed hepatocyte-specific expression. Expression of proCPR mRNA was enhanced following LPS injection, indicating that proCPR production is increased under inflammatory conditions.
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PMID:Molecular cloning and partial characterization of rat procarboxypeptidase R and carboxypeptidase N. 1102 4

Pro-carboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.
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PMID:Inactivation of C3a and C5a octapeptides by carboxypeptidase R and carboxypeptidase N. 1193 78

The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation.
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PMID:The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase. 1193 91

Carboxypeptidase R (EC 3.4.17.20; CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is a stable enzyme. CPR in fresh serum is generated from its zymogen (proCPR) during coagulation by trypsin-like enzymes such as thrombin and thrombin/thrombomodulin complexes. Since removal of the C-terminal arginine abrogates the anaphylatoxin activity of C3a and C5a, CPR and CPN are regarded as anaphylatoxin inactivators. We report here that the culture supernatant of activated human neutrophils converts proCPR to CPR. Addition of an elastase specific inhibitor, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MSAAPVCK) to the supernatant of stimulated neutrophils completely inhibited activation of proCPR. On the other hand, a thrombin specific inhibitor, p-Nitrophenyl-p'-amidinophenyl-methanesulfonate hydrochloride (pNP-pAPMS) inhibited only 16% of proCPR activation by the neutrophil supernatant. Furthermore, purified elastase converted proCPR to CPR. Therefore, elastase can activate proCPR directly, or indirectly through activation of some proteases, which have been contaminating in reagents. Release of CPR generating enzymes from neutrophils should play an important role in regulation of excess inflammation.
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PMID:Elastase from activated human neutrophils activates procarboxypeptidase R. 1200 33

We designed complementary peptides (C-peptides) using a novel computer program (MIMETIC), which generates a series of peptides designed to interact with a target peptide sequence. Carboxypeptidase R (CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is stable. CPR is generated from its precursor form (proCPR) by trypsin-like enzymes, and its activation is mediated by thrombin generated in the coagulation cascade. The efficiency of activation is enhanced approximately 1,200-fold when thrombin (T) is bound to thrombomodulin (TM). We attempted to generate C-peptides which recognize the T-binding site within TM assuming that some of these might interfere with the generation of T and TM complexes (T-TM). Among three peptides designed, two inhibited the enhancement in activation of proCPR by T in the presence of TM. One of the peptides at 16 microM reduced the activation of proCPR to the level obtained by T alone.
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PMID:Modulation of procarboxypeptidase R (ProCPR) activation by complementary peptides to thrombomodulin. 1272 95

Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity.
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PMID:Mutations in the substrate binding site of thrombin-activatable fibrinolysis inhibitor (TAFI) alter its substrate specificity. 1279 75

The latent plasma carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. In this study, we examined the effect of activated TAFI (TAFIa) in modulating the proinflammatory functions of bradykinin, complement C5a, and thrombin-cleaved osteopontin. Hydrolysis of bradykinin and C5a and thrombin-cleaved osteopontin peptides by TAFIa was as efficient as that of plasmin-cleaved fibrin peptides, indicating that these are also good substrates for TAFIa. Plasma carboxypeptidase N, generally regarded as the physiological regulator of kinins, was much less efficient than TAFIa. TAFIa abrogated C5a-induced neutrophil activation in vitro. Jurkat cell adhesion to osteopontin was markedly enhanced by thrombin cleavage of osteopontin. This was abolished by TAFIa treatment due to the removal of the C-terminal Arg168 by TAFIa from the exposed SVVYGLR alpha 4 beta 1 integrin-binding site in thrombin-cleaved osteopontin. Thus, thrombin cleavage of osteopontin followed by TAFIa treatment may sequentially up- and down-modulate the pro-inflammatory properties of osteopontin. An engineered anticoagulant thrombin, E229K, was able to activate endogenous plasma TAFI in mice, and E229K thrombin infusion effectively blocked bradykinin-induced hypotension in wild-type, but not in TAFI-deficient, mice in vivo. Our data suggest that TAFIa may have a broad anti-inflammatory role, and its function is not restricted to fibrinolysis.
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PMID:Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation. 1452 95

Carboxypeptidase R (CPR) is a heat-labile enzyme found in serum in addition to stable carboxypeptidase N. CPR cleaves the C-terminal basic amino acids, arginine and lysine, from inflammatory peptides such as complement C3a and C5a, bradykinin, and enkephalin. This enzyme is generated from procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor, following cleavage by proteolytic enzymes such as thrombin, plasmin, and trypsin. We generated proCPR-deficient mice by knocking out exons 4 and 5 of the proCPR gene, which are regarded as essential for CPR function. At LPS challenge, there was virtually no difference in lethality among proCPR(+/+), proCPR(+/-), and proCPR(-/-) mice. However, challenge with cobra venom factor, which can activate and deplete almost all complement in vivo, induced a lethal effect on proCPR(-/-) mice following LPS sensitization which up-regulates C5a receptor expression. In contrast, proCPR(+/+) and proCPR(+/-) mice were able to tolerate the cobra venom factor challenge with the limited dose (30 U). Although carboxypeptidase N plays a role in inactivation of inflammatory peptides in vivo, CPR may also be important in the regulation of hyperinflammation.
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PMID:Absence of procarboxypeptidase R induces complement-mediated lethal inflammation in lipopolysaccharide-primed mice. 1538 2

A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (-)2a displays TAFIa IC50 = 3 nM and 600-fold selectivity against CPN. Inhibition of TAFIa with (rac)2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was efficacious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.
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PMID:3-Mercaptopropionic acids as efficacious inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa). 1718 88


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