RESUMEN
BACKGROUND: Portal vein embolization (PVE) is used to increase future remnant liver size in patients requiring major hepatic resection. PVE using permanent embolization, however, predisposes to complications and excludes the use of PVE in living donor liver transplantation. In the present study, an absorbable embolization material containing fibrin glue and different concentrations of the fibrinolysis inhibitor aprotinin was used in an experimental animal model. METHODS: PVE of the cranial liver lobes was performed in 30 New Zealand White rabbits, which were divided into five groups, fibrin glue + 1000, 700, 500, 300 or 150 kunits/ml aprotinin, and were compared with a previous series of permanent embolization using the same experimental set-up. Caudal liver lobe hypertrophy was determined by CT volumetry, and portal recanalization was identified on contrast-enhanced CT images. Animals were killed after 7 or 42 days, and the results were compared with those of permanent embolization. RESULTS: PVE using fibrin glue with aprotinin as embolic material was effective, with 500 kunits/ml providing the optimal hypertrophic response. Lower concentrations of aprotinin (150 and 300 kunits/ml) led to reduced hypertrophy owing to early recanalization of the embolized segments. The regeneration rate over the first 3 days was higher in the group with 500 kunits/ml aprotinin than in the groups with 300 or 150 kunits/ml or permanent embolization. In the 500-kunits/ml group, four of five animals showed recanalization 42 days after embolization, with minimal histological changes in the cranial lobes following recanalization. CONCLUSION: Fibrin glue combined with 500 kunits/ml aprotinin resulted in reversible PVE in 80 per cent of animals, with a hypertrophy response comparable to that achieved with permanent embolization material. Surgical relevance Portal vein embolization (PVE) is used to increase future remnant liver volume in patients scheduled for major liver resection who have insufficient future remnant liver size to perform a safe resection. The current standard is PVE with permanent embolization materials, which renders patients found to have unresectable disease prone to complications owing to the permanently deportalized liver segments. Absorbable embolization might prevent the PVE-associated morbidity and lower the threshold for its application. In this study, PVE using fibrin glue and aprotinin resulted in an adequate hypertrophy response with 80 per cent recanalization after 42 days. Considering the minor histological changes following recanalization of embolized segments and potentially preserved function, reversible PVE might also be applied in living donor liver transplantation.
Asunto(s)
Aprotinina , Embolización Terapéutica/métodos , Adhesivo de Tejido de Fibrina , Regeneración Hepática , Hígado/crecimiento & desarrollo , Vena Porta , Animales , Femenino , Hígado/irrigación sanguínea , Hígado/diagnóstico por imagen , Conejos , Tomografía Computarizada por Rayos XRESUMEN
Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator in the balance of coagulation and fibrinolysis. TAFI is a metallocarboxypeptidase that circulates in plasma as zymogen. Activated TAFI (TAFIa) cleaves C-terminal lysine or arginine residues from peptide substrates. The removal of C-terminal lysine residues from partially degraded fibrin leads to reduced plasmin formation and thus attenuation of fibrinolysis. TAFI also plays a role in inflammatory processes via the removal of C-terminal arginine or lysine residues from bradykinin, thrombin-cleaved osteopontin, C3a, C5a and chemerin. TAFI has been studied extensively over the past three decades and recent publications provide a wealth of information, including crystal structures, mutants and structural data obtained with antibodies and peptides. In this review, we combined and compared available data on structure/function relationships of TAFI.
Asunto(s)
Carboxipeptidasa B2/metabolismo , Animales , Anticuerpos/química , Arginina/química , Coagulación Sanguínea , Bradiquinina/química , Catálisis , Bovinos , Quimiocinas/química , Complemento C3a/química , Complemento C5a/química , Cristalografía por Rayos X , Precursores Enzimáticos/química , Fibrinólisis , Humanos , Inflamación , Péptidos y Proteínas de Señalización Intercelular/química , Lisina/química , Ratones , Mutación , Osteopontina/química , Péptidos/química , Dominios Proteicos , Relación Estructura-Actividad , Especificidad por Sustrato , Trombina/químicaRESUMEN
BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for coronary heart disease. TAFI is proteolytically activated by thrombin, the thrombin-thrombomodulin complex and plasmin. Once active, it dampens fibrinolysis and inflammation. The aim of this study was to generate TAFI-derived peptides that specifically modulate TAFI activation and activity. METHODS: Thirty-four overlapping TAFI peptides, and modifications thereof, were synthesized. The effects of these peptides on TAFI activation and TAFIa activity were determined. In addition, the binding of the peptides to thrombin were determined. RESULTS: Four peptides (peptides 2, 18, 19 and 34) inhibited TAFI activation and two peptides (peptides 14 and 24) inhibited TAFIa activity directly. Peptide 2 (Arg12-Glu28) and peptide 34 (Cys383-Val401) inhibited TAFI activation by the thrombin-thrombomodulin complex with IC50 values of 7.3 ± 1.8 and 6.1 ± 0.9 µm, respectively. However, no inhibition was observed in the absence of thrombomodulin. This suggests that the regions Arg12-Glu28 and Cys383-Val401 in TAFI are involved in thrombomodulin-mediated TAFI activation. Peptide 18 (Gly205-Ser221) and peptide 19 (Arg214-Asp232) inhibited TAFI activation by thrombin and the thrombin-thrombomodulin complex. Furthermore, these peptides bound to thrombin (KD : 1.5 ± 0.4 and 0.52 ± 0.07 µm for peptides 18 and 19, respectively), suggesting that Gly205-Asp232 of TAFI is involved in binding to thrombin. Peptide 14 (His159-His175) inhibited TAFIa activity. The inhibition was TAFIa specific, because no effect on the homologous enzyme carboxypeptidase B was observed. CONCLUSIONS: Thrombin-activatable fibrinolysis inhibitor-derived peptides show promise as new tools to modulate TAFI activation and TAFIa activity. Furthermore, these peptides revealed potential binding sites on TAFI for thrombin and the thrombin-thrombomodulin complex.
Asunto(s)
Carboxipeptidasa B2/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Trombina/farmacología , Secuencia de Aminoácidos , Carboxipeptidasa B2/química , Activación Enzimática/efectos de los fármacos , Semivida , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Trombina/metabolismo , Trombomodulina/metabolismoRESUMEN
BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a proenzyme that links coagulation and fibrinolysis. TAFI can be activated by thrombin, the thrombin-thrombomodulin complex and plasmin through cleavage of the first 92 amino acids from the enzyme. In silico analysis of the TAFI sequence revealed a potential thrombin cleavage site at Arg12. The aim of this study was to determine whether TAFI can be cleaved at Arg12 and whether this cleavage plays a role in TAFI activation. METHODS: A peptide based on the first 18 amino acids of TAFI was used to determine whether thrombin was able to cleave at Arg12. Mass spectrometry was performed to determine whether the Arg12-cleaved peptide was released from full-length TAFI. Furthermore, a TAFI mutant in which Arg12 was replaced by a glutamine (TAFI-R12Q) was constructed and characterized with respect to its activation kinetics. RESULTS: The peptide and mass spectrometry data showed that thrombin was able to cleave TAFI at Arg12, but with low efficiency in full-length TAFI. Characterization of TAFI-R12Q showed no difference in thrombin-mediated activation from wild-type TAFI. However, there was an approximately 60-fold impairment in activation of TAFI-R12Q by the thrombin-thrombomodulin complex. CONCLUSIONS: Arg12 of TAFI plays an important role in thrombomodulin-mediated TAFI activation by thrombin. Thrombin is able to cleave TAFI at Arg12, but it remains to be determined whether Arg12 is part of an exosite for thrombomodulin or whether cleavage at Arg12 accelerates thrombomodulin-mediated TAFI activation.
Asunto(s)
Arginina/química , Carboxipeptidasa B2/química , Espectrometría de Masas , Trombina/química , Trombomodulina/química , Aminoácidos/química , Coagulación Sanguínea , Clonación Molecular , Activación Enzimática , Fibrinolisina/química , Fibrinólisis , Glutamina/química , Humanos , Péptidos/químicaRESUMEN
BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 56-kDa procarboxypeptidase. Proteolytic enzymes activate TAFI into TAFIa, an inhibitor of fibrinolysis, by cleaving off the N-terminal activation peptide (amino acids 1-92), from the enzyme moiety. Activated TAFI is unstable, with a half-life of approximately 10 min at 37 degrees C. So far, it is unknown whether the activation peptide is released or remains attached to the catalytic domain, and whether it influences TAFIa's properties. The current study was performed to clarify these issues. METHODS: TAFI was activated, and the activity and half-life of the enzyme were determined in the presence and absence of the activation peptide. RESULTS: TAFIa was active both before and after removal of the activation peptide, and the half-life of TAFIa was identical in the two preparations. Furthermore, we observed that intrinsically inactivated TAFIa (TAFIai) aggregated into large, insoluble complexes that could be removed by centrifugation. CONCLUSIONS: The data presented in this article show that the activation peptide of TAFI is not required for TAFIa activity and that the activation peptide has no effect on the stability of the enzyme. These results are in favour of a model in which the activation peptide solely stabilizes the structure of the proenzyme. After activation of TAFI and subsequent breakage of interactions between the activation peptide and the catalytic domain, the activation peptide is no longer capable of performing this stabilizing task, and the integrity of the catalytic domain is lost rapidly. The resulting TAFIai is more prone to proteolysis and aggregation.