Carboxypeptidase B2 gene polymorphisms and the risk of venous thromboembolism.
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Adult↗
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The gene for human carboxypeptidase U (CPU)--a proposed novel regulator of plasminogen activation--maps to 13q14.11.
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Amino Acid Sequence↗
Combined segregation-linkage analysis of plasma thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels with TAFI gene polymorphisms.
By decreasing plasminogen binding to fibrin surface, the thrombin activatable fibrinolysis inhibitor (TAFI) has been hypothesized to constitute an early marker for atherothrombotic diseases. Previous studies have shown that plasma TAFI levels exhibit a high interindividual variability that is only poorly explained by lifestyle factors. Several polymorphisms of the TAFI gene have been described, and a combination of a C+1542G substitution in the 3' untranslated region and an Ala147Thr amino acid change has been shown to explain 60% of TAFI variability in a sample of unrelated individuals. A segregation-linkage analysis was performed to determine whether these polymorphisms are directly involved in the genetic regulation of TAFI levels, or whether they are only markers in linkage disequilibrium (LD) with unmeasured TAFI-linked quantitative trait loci (QTLs). The sample consisted of 97 healthy nuclear families from the Stanislas Cohort. The C+1542G and Ala147Thr polymorphisms were in complete negative LD, with minor allele frequencies of 0.27 and 0.28, respectively. Results of the segregation-linkage analysis provided evidence of two TAFI-linked QTLs in LD with the two measured polymorphisms, which would explain 78% of the TAFI variance, as compared with 55% explained by the C+1542G and the Ala147Thr polymorphisms combined. The two putative QTLs would have minor allele frequencies of 0.45 and 0.32, respectively. The hypothesis that one of the measured polymorphisms is one of the QTLs was rejected. The putative QTLs also did not seem compatible with the other TAFI gene polymorphisms that we have previously described. More extensive sequencing of the TAFI gene is necessary to identify the functional variants.
Adolescent↗
Bioassays of smooth muscle contracting agents in cotton mill dust and bract extracts: arachidonic acid metabolites as possible mediators of the acute byssinotic reaction.
Byssinosis is an occupational respiratory disease contracted by cotton mill workers who inhale cotton mill dust. The acute byssinotic reaction is characterized by a drop in the 1-sec forced expiratory volume (FEV1.0) on Monday following a weekend's absence from work. This physiological reaction is the result of the contraction of bronchial smooth muscle, which causes narrowing of small airways. An isolated tissue bath technique was used to assay smooth muscle contractions induced by cotton dust extract (CDE) and cotton bract extract (CBE). CBE-induced contractions of rat stomach smooth muscle were blocked (82%) by 100 ng/ml methysergide (blocks 5-hydroxytryptamine (5HT) 100%); CDE was not significantly blocked by methysergide (13%). CDE-induced contractions were blocked (100%) by 25 micrograms/ml indomethacin and 100 micrograms/ml salicylic acid (blocks prostaglandin F2 alpha (PGF2 alpha) 100%). The portion of CBE contracting ability not blocked by methysergide was blocked totally by indomethacin. Blocking agents ineffective against CDE or CBE include atropine (acetylcholine blocker), pyrilamine maleate and diphenhydramine (histamine blockers), imidazole and 7-(1-imidazolyl)heptanoic acid (7IHA) (thromboxane blockers), and carboxypeptidase B2 (kinin blocker). The data suggest that cotton bract contains 5HT, which is responsible for a majority of the CBE-induced contraction and a minority of the CDE-induced contraction. The data also suggest that cotton dust and bract contain a substance which causes the release of PGF2 alpha which in turn causes the contraction of smooth muscle. This substance is responsible for a majority of the CDE-induced and a minority of the CBE-induced contractions. Radioimmunoassay (RIA) confirmed PGF2 alpha release from rat fundal smooth muscle when exposed to CDE and CBE. Increased synthesis and release of arachidonic acid metabolites might be a major mechanism in the bronchoconstriction observed in the acute byssinotic reaction.
Acute Disease↗
Electrochemiluminescence assay for basic carboxypeptidases: inhibition of basic carboxypeptidases and activation of thrombin-activatable fibrinolysis inhibitor.
Carboxypeptidases catalyze the removal of the C-terminal amino acid residues in peptides and proteins and exert important biological functions. Assays for carboxypeptidase activity that rely on change of absorbance generally suffer from low sensitivity and are difficult to adapt to high-throughput screening. We have developed a sensitive, robust assay for basic carboxypeptidase activity that makes use of electrochemiluminescent (ECL) detection of reaction product. In this assay, a peptide substrate contains the epitope for antibody (G2-10) binding which is masked by a C-terminal arginine. Carboxypeptidase activity exposes the epitope, allowing the binding of ruthenylated G2-10 which is then detected using ECL. High sensitivity allowed detection limits of 1-2 pM enzyme for carboxypeptidase B and activated thrombin-activatable fibrinolysis inhibitor (TAFIa). The inhibition of several basic carboxypeptidases by commercially available inhibitors was studied. This antibody-based method can be extended to other sensitive detection techniques such as amplified luminescent proximity homogeneous assay. The high sensitivity of the assay allowed the determination of the activatable levels of TAFI in human and other animal plasma in the presence of epsilon -aminocaproic acid, an active-site inhibitor that stabilizes TAFIa. A method to isolate in situ activated TAFIa from human serum in the presence of epsilon -aminocaproic acid was also developed.
Animals↗
Proteolytic activation of purified human procarboxypeptidase U.
Carboxypeptidase U (CPU, EC 3.4.17.20) is a recently described basic carboxypeptidase which circulates in plasma as an enzymatically inactive precursor procarboxypeptidase U (proCPU), also known as plasma carboxypeptidase B precursor or thrombin activatable fibrinolysis inhibitor (TAFI). The activation of the zymogen proceeds through a proteolytic cleavage at Arg-92. The active form - CPU - is able to retard the initial phase of fibrinolysis by cleaving C-terminal lysine residues exposed on fibrin partially degraded by the action of plasmin. These C-terminal lysine residues are essential for the high affinity binding of plasminogen to fibrin and the subsequent activation to plasmin. In this report, the activation of purified human proCPU was studied using trypsin and some key proteases of the coagulation and fibrinolytic cascade, i.e., kallikrein, plasmin and thrombin. The most efficient activation is obtained in the presence of thrombin in complex with thrombomodulin. After in vitro activation, CPU is unstable at 37 degrees C (T(1/2)=15 min). Its stability can be improved dramatically using lower temperatures.
Blood Coagulation↗
Human procarboxypeptidase B: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (TAFI).
Besides their classical role in alimentary protein degradation, zinc-dependant carboxypeptidases also participate in more selective regulatory processes like prohormone and neuropeptide processing or fibrinolysis inhibition in blood plasma. Human pancreatic procarboxypeptidase B (PCPB) is the prototype for those human exopeptidases that cleave off basic C-terminal residues and are secreted as inactive zymogens. One such protein is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma PCPB, which circulates in human plasma as a zymogen bound to plasminogen. The structure of human pancreatic PCPB displays a 95-residue pro-segment consisting of a globular region with an open-sandwich antiparallel-alpha antiparallel-beta topology and a C-terminal alpha-helix, which connects to the enzyme moiety. The latter is a 309-amino acid residue catalytic domain with alpha/beta hydrolase topology and a preformed active site, which is shielded by the globular domain of the pro-segment. The fold of the proenzyme is similar to previously reported procarboxypeptidase structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site of human procarboxypeptidase B has two alternate conformations in one of the zinc-binding residues, which account for subtle differences in some of the key residues for substrate binding. The reported crystal structure, refined with data to 1.6A resolution, permits in the absence of an experimental structure, accurate homology modelling of TAFI, which may help to explain its properties.
Amino Acid Sequence↗
An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombolysis model.
When activated in vitro, thrombin-activatable fibrinolysis inhibitor (TAFI) slows clot lysis by cleaving the C-terminal lysine and arginine residues from partially degraded fibrin. An inhibitor of carboxypeptidase isolated from potato (CPI) reverses prolongation of clot lysis by inhibiting activated TAFI. We investigated in vivo effect of TAFI inhibition on tissue-type plasminogen activator (t-PA)-induced clot lysis using CPI in a rabbit jugular vein thrombolysis model. It was found necessary to further purify the CPI preparations from commercial sources by HPLC chromatography to remove endotoxin and anti-plasmin activity that would affect the endogenous fibrinolytic system. The effect of intravenous administration of the purified CPI with t-PA was determined by measuring thrombus weight at the end of 90 minutes in six groups of animals. In the control group receiving saline, the median thrombus weight was 116 mg. In the group that received CPI only (0.5 mg/kg bolus injection followed by 0.3 mg/kg/h infusion), the median thrombus weight was 121 mg. In the group that received t-PA at a dose of 10 microg/kg bolus followed by 67 microg/kg/h infusion, the median thrombus weight decreased to 86 mg. When CPI was coadministered with the same regimen of t-PA, the median value further decreased to 58 mg. When animals were given three times higher the dose of t-PA (30 microg/kg bolus followed by 200 microg/kg/h infusion) in the absence or presence of CPI, median thrombus weights were 56 mg and 0 mg, respectively. Our results demonstrate that systemic coadministration of the purified CPI improves clot lysis induced by t-PA.
Animals↗
Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U).
Recently, a new inhibitor of fibrinolysis was described. This inhibitor downregulated fibrinolysis after it was activated by thrombin, and was therefore named TAFI (thrombin-activatable fibrinolysis inhibitor; EC 3.4.17.20). TAFI turned out to be identical to previously described proteins, procarboxypeptidase U, procarboxypeptidase R, and plasma procarboxypeptidase B. In this overview, the protein will be referred to as TAFI. TAFI is a procarboxypeptidase and a member of the family of metallocarboxypeptidases. These enzymes are circulating in plasma and are present in several tissues such as pancreas. In this review, we will describe the properties of basic carboxypeptidases with the emphasis on the role of TAFI in coagulation and fibrinolysis. It cannot be ruled out, however, that TAFI has other, yet undefined, functions in biology.
Animals↗
Thrombin-activatable fibrinolysis inhibitor antigen and TAFI activity in patients with APC resistance caused by factor V Leiden mutation.
Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as procarboxypeptidase U or plasma procarboxypeptidase B, is a relatively recently described plasma glycoprotein synthesised in the liver. It can be activated into active enzyme TAFIa (carboxypeptidase U or plasma carboxypeptidase B) by a complex of thrombin/thrombomodulin. TAFIa can potentially inhibit fibrinolysis by removing carboxyterminal lysine residues from partially degraded fibrin, decreasing plasminogen binding on the surface of fibrin, which thereby results in a decrease of the fibrinolytic activity. Since TAFI represents a connection between coagulation and fibrinolysis, it can be expected that TAFI levels are altered in different thrombotic and hemorrhagic diseases. Thrombin generation is increased in patients with activated protein C (APC) resistance, while it has been shown that APC has profibrinolytic effect. Therefore, changes in TAFI level should be found in patients with APC resistance due to factor V Leiden (FV Leiden) mutation. TAFI antigen (including TAFI, TAFIa and the inactive form TAFIai) and TAFI activity were determined in 17 female patients heterozygous for FV Leiden mutation while 13 healthy volunteers were controls. No statistically significant difference in levels of TAFI antigen was observed. TAFI activity was significantly reduced in APC resistance patients compared to control (P=.018). The nondifference in TAFI antigen, together with the decrease of TAFI activity level, can be explained by activation of TAFI to TAFIa and shifting of equilibrium towards an increase of the latter. This can be an indirect proof that TAFIa is increased in patients with APC resistance due to FV Leiden mutation, indicating that downregulation of fibrinolysis can be an additional risk factor for thrombosis in these patients.
Activated Protein C Resistance↗
No grip, no growth: the conceptual basis of excessive proteolysis in the treatment of cancer.
The formation of new bloodvessels, called angiogenesis, is critical for a tumour to grow beyond a few mm(3) in size. A provisional matrix promotes endothelial cell adhesion, migration, proliferation and survival. Synthesis and degradation of this matrix closely resemble processes that occur during coagulation and fibrinolysis. Degradation of the matrix and fibrinolysis are tightly controlled and balanced by stimulators and inhibitors of the plasminogen activation system. Here we give an overview of these processes during tumour progression. We postulate a novel way to inhibit angiogenesis by removal of the matrix through specific and localised overstimulation of the plasminogen activation system.
Angiogenesis Inhibitors↗
Dissociation of thrombin's substrate interactions using site-directed mutagenesis.
Thrombin is an allosteric enzyme that interacts with multiple procoagulant substrates such as specific clotting factors and cell surface thrombin receptors, as well as the anticoagulant substrate protein C. Functional mapping of thrombin's interactions with its various substrates has been carried out using a collection of thrombin mutants generated by systematic alanine scanning mutagenesis. A thrombin mutant, E229K, has been identified that has essentially lost all of its procoagulant properties while retaining its ability to activate protein C, thus functioning as an anticoagulant in vitro and in vivo. It is also found that specific and distinct domains are involved in thrombin's interaction with thrombomodulin (TM) and the subsequent activation by the thrombin/TM complex of protein C and the thrombin-activatable fibrinolysis inhibitor (TAFI).
Animals↗
Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B).
Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described human plasma zymogen that is related to pancreatic carboxypeptidase B. The active form of TAFI (TAFIa), which is formed by thrombin cleavage of the zymogen, likely inhibits fibrinolysis by removal from partially degraded fibrin of the carboxyl-terminal lysine residues which act to stimulate plasminogen activation. We have isolated and characterized genomic clones which encompass the entire human TAFI gene from lambda phage and bacterial artificial chromosome genomic libraries. The complete TAFI gene contains 11 exons and spans approximately 48 kb of genomic DNA. The positions of intron/exon boundaries are conserved between the TAFI gene and the rat pancreatic carboxypeptidase A1, A2, and B and the human mast cell carboxypeptidase A genes, indicating that these carboxypeptidases arose from a common ancestral gene. However, the intron lengths diverge significantly among all of these genes. The TAFI promoter lacks a consensus TATA sequence, and transcription is initiated from multiple sites. Transient transfection of reporter plasmids containing portions of the TAFI 5'-flanking region into mammalian cells allowed localization of the promoter and identified a approximately 70 bp region crucial for liver-specific transcription. Sequence analysis of cDNA clones obtained from human liver RNA indicated that the TAFI transcript is polyadenylated at three different sites. Our findings will facilitate the assessment of the regulation of TAFI expression by transcriptional and/or posttranscriptional mechanisms. Furthermore, knowledge of the genomic structure of the TAFI gene will aid in the identification of mutations that may be associated with the tendency to either bleed or thrombose.
3' Untranslated Regions↗
Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex.
The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.
Amino Acid Sequence↗
Reduced activity of TAFI (thrombin-activatable fibrinolysis inhibitor) in acute promyelocytic leukaemia.
Acute promyelocytic leukaemia (APL) is a disease that is distinguished from other leukaemias by the high potential for early haemorrhagic death. Several processes are involved, such as disseminated intravascular coagulation and hyperfibrinolysis. Recently, TAFI (thrombin-activatable fibrinolysis inhibitor) was identified as a link between coagulation and fibrinolysis. TAFI can be activated by thrombin, and in its activated form potently attenuates fibrinolysis by removing C-terminal lysine and arginine residues that are important for the binding and activation of plasminogen. Activation of TAFI by the coagulation system results in a down-regulation of fibrinolytic activity and, thereby, prevents a rapid dissolution of the fibrin clot. To establish whether TAFI was involved in the severity of the bleeding complications in APL, the TAFI antigen and activity levels were determined in a group of 15 patients. The TAFI antigen concentration was normal, but the activity of TAFI was severely reduced in APL by approximately 60%. The reduction of TAFI activity was most probably caused by the action of plasmin on TAFI because in vitro experiments revealed that plasmin slightly reduced antigen levels but severely reduced TAFI activity. The acquired functional TAFI deficiency in APL may contribute to the severity of the haemorrhagic diathesis because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis.
Adult↗
Activation of coagulation factor XI, without detectable contact activation in dengue haemorrhagic fever.
A prospective cohort study was performed in 50 patients with dengue haemorrhagic fever (DHF) to determine the potential role of the contact activation system and factor XI activation (intrinsic pathway) in the coagulation disorders in DHF. To establish whether TAFI (thrombin-activatable fibrinolysis inhibitor) was involved in the severity of the coagulation disorders, the TAFI antigen and activity levels were also determined. Markers of contact activation (kallikrein--C1-inhibitor complexes), the intrinsic pathway of coagulation (factor XIa--C1-inhibitor complexes) and TAFI were measured and correlated to thrombin generation markers (thrombin--anti-thrombin complexes (TAT), prothrombin fragment 1+2 (F1+2)) and a marker for fibrinolysis [plasmin--alpha 2--anti-plasmin complexes (PAP)]. Activation of the intrinsic pathway of coagulation was clearly demonstrated by elevated levels of factor XIa--C1-inhibitor complexes, without evidence of contact activation, reflected by undetectable kallikrein--C1-inhibitor complexes. Both TAFI antigen and activity levels were decreased in all patients, which may contribute to the severity of bleeding complications in DHF because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis. These findings in a human viral infection model are in accordance with earlier findings in bacterial sepsis.
Autoantigens↗
Very high TAFI antigen levels are associated with a lower risk of hard coronary events: the PRIME Study.
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Antigens↗
Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor.
Plasmin and other components of the plasminogen activation system play an important role in tissue repair by regulating extracellular matrix remodeling, including fibrin degradation. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, after activation, can attenuate plasmin-mediated fibrin degradation by removing the C-terminal lysine residues from fibrin, which play a role in the binding and activation of plasminogen. To test the hypothesis that TAFI is an important determinant in the control of tissue repair, we investigated the effect of TAFI deficiency on the healing of cutaneous wounds and colonic anastomoses. Histological examination revealed inappropriate organization of skin wound closure in the TAFI knockout mice, including an altered pattern of epithelial migration. The time required to completely heal the cutaneous wounds was slightly delayed in TAFI-deficient mice. Healing of colonic anastomoses was also impaired, as reflected by decreased strength of the tissue at the site of the suture, and by bleeding complications in 3 of 14 animals. Together, these abnormalities resulted in increased mortality in TAFI-deficient mice after colonic anastomoses. Although our study shows that tissue repair, including re-epithelialization and scar formation, occurs in TAFI-deficient mice, TAFI appears to be important for appropriate organization of the healing process.
Anastomosis, Surgical↗