RESUMEN
BACKGROUND: Activated factor XIII (FXIII), a dimer of truncated A-subunits (FXIII-A2*), is a transglutaminase that crosslinks primary amines to peptide-bound glutamine residues. Because in the few natural substrates of FXIII-A2* no consensus sequence could be identified around the reactive glutamine, studying the interaction between individual substrates and FXIII-A2* is of primary importance. Most of the alpha2-plasmin inhibitor (alpha2PI) molecules become truncated by a plasma protease, and the truncated isoform (N1-alpha2PI) is an important substrate of FXIII-A2*. The crosslinking of N1-alpha2PI to fibrin plays a major role in protecting fibrin from fibrinolysis. METHODS: We studied the interaction of FXIII-A2* with its dodecapeptide glutamine donor substrate, N1-alpha2PI(1-12), the sequence of which corresponds to the N-terminal sequence of N1-alpha2PI. Kinetic parameters for N1-alpha2PI(1-12) and for its truncated or synthetic mutants were determined by a spectrophotometric assay. The interaction of N1-alpha2PI(1-12) with FXIII-A2* was investigated by proton nuclear magnetic resonance (NMR) and saturating transfer difference (STD) NMR. RESULTS AND CONCLUSIONS: Kinetic experiments with peptides in which the Asn1 residue was either truncated or replaced by alanine and proton NMR analysis of the FXIII-A2*-N1-alpha2PI(1-12) complex demonstrated that Asn1 is essential for effective enzyme-substrate interaction. Experiments with C-terminally truncated peptides proved that amino acids 7-12 are essential for the interaction of N1-alpha2PI(1-12) with the enzyme, and suggested the existence of a secondary binding site on FXIII-A2*. Hydrophobic residues, particularly Leu10 and the C-terminal Lys12, seemed to be especially important in this respect, and direct interaction between hydrophobic C-terminal residues and FXIII-A2* was demonstrated by STD NMR.
Asunto(s)
Factor XIIIa/metabolismo , Glutamina/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Asparagina , Sitios de Unión , Bovinos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Espectroscopía de Resonancia Magnética , Mutación , Unión Proteica , Especificidad por SustratoRESUMEN
Development of autoantibody against coagulation factor V (FV) is a rare clinical condition with hemorrhagic complications of varying severity. The aim of this study was to establish the pathomechanism of an acquired FV deficiency and characterize the FV inhibitor responsible for the clinical symptoms. A 78-year-old female was admitted to hospital with severe gastrointestinal bleeding. General clotting tests and determination of clotting factors were performed by standard methods. FV antigen and FV containing immune complexes were measured by ELISA. The FV molecule was investigated by Western blotting and by sequencing the f5 gene. The binding of patient's IgG to FV and activated FV (FVa) was demonstrated in an ELISA system and its effect on the procoagulant activity of FVa was tested in clotting tests and in a chromogenic prothrombinase assay. Localization of the epitope for the antibody was performed by blocking ELISA. FV activity was severely suppressed both in plasma and platelets. FV antigen levels were normal by ELISA using polyclonal anti-FV antibody or monoclonal antibody against the connecting region of FV, but depressed when HV1 monoclonal antibody against the C2 domain in the FV light-chain was used as capture antibody. The FV molecule was found intact. An IgG reacting with both FV and FVa was present in the patient's plasma and its binding to FV was inhibited by HV1 antibody. FV-containing immune complexes were detected in the patient's plasma and platelet lysate. The patient's IgG inhibited the procoagulant function of FVa. An anti-FV IgG was present in the patient's plasma and platelets. The autoantibody reacted with an epitope in the C2 domain of FV light chain and neutralized the procoagulant function of FVa.
Asunto(s)
Autoanticuerpos/sangre , Plaquetas/inmunología , Deficiencia del Factor V/complicaciones , Factor V/inmunología , Hemorragia Gastrointestinal/inmunología , Anciano , Pruebas de Coagulación Sanguínea , Ensayo de Inmunoadsorción Enzimática , Epítopos , Deficiencia del Factor V/diagnóstico , Deficiencia del Factor V/inmunología , Femenino , Hemorragia Gastrointestinal/etiología , Humanos , Inmunoglobulina G/inmunologíaRESUMEN
Coagulation factor XIII (FXIII) is a protransglutaminase involved in the last step of the coagulation cascade by stabilising the fibrin clot. Recently, a common variation (FXIII Val34Leu) has been associated with a decreased risk of myocardial infarction and deep venous thrombosis. Val34Leu is critically located near the thrombin activation site of FXIII-A. In this study we investigated its effects on the activation of FXIII. Both recombinant and platelet-derived FXIII Val34Leu variants were shown to be more susceptible to thrombin cleavage than the wild type FXIII. The rate of enzymatic activation of FXIII Val34Leu was found increased, however, the specific activity of fully activated wild type FXIII and the Val34Leu mutant did not differ. During the course of thrombin-induced activation of FXIII fibrin gamma-chain dimerisation and alpha-chain polymerisation developed more rapidly with the Val34Leu mutant. The increased rate of fibrin stabilisation brought about by the Val34Leu FXIII seems to be paradoxically associated with a protective effect against pathological thrombosis.
Asunto(s)
Polimorfismo Genético , Transglutaminasas/genética , Animales , Coagulación Sanguínea , Trastornos de la Coagulación Sanguínea/etiología , Trastornos de la Coagulación Sanguínea/genética , Células COS , Humanos , Mutación , Factores de Riesgo , Transglutaminasas/metabolismoRESUMEN
Val34Leu polymorphism of the A subunit of coagulation factor XIII (FXIII-A) is located in the activation peptide (AP) just 3 amino acids away from the thrombin cleavage site. This mutation has been associated with a protective effect against occlusive arterial diseases and venous thrombosis; however, its biochemical consequences have not been explored. In the current study it was demonstrated that the intracellular stability and the plasma concentration of FXIII of different Val34Leu genotypes are identical, which suggests that there is no difference in the rate of synthesis and externalization of wild-type and mutant FXIII-A. In contrast, the release of AP by thrombin from the Leu34 allele proceeded significantly faster than from its wild-type Val34 counterpart. By molecular modeling larger interaction energy was calculated between the Leu34 variant and the respective domains of thrombin than between the Val34 variant and thrombin. In agreement with these findings, the activation of mutant plasma FXIII by thrombin was faster and required less thrombin than that of the wild-type variant. Full thrombin activation of purified plasma FXIII of different genotypes, however, resulted in identical specific transglutaminase activities. Similarly, the mean specific FXIII activity in the plasma was the same in the groups with wild-type, heterozygous, and homozygous variants. Faster activation of the Leu34 allele hardly could be associated with its presumed protective effect against venous thrombosis. No such protective effect was observed in a large group of patients with familial thrombophilia.
Asunto(s)
Factor XIII/genética , Leucina , Polimorfismo Genético , Trombofilia/epidemiología , Trombofilia/genética , Valina , Adulto , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión , Factor XIII/química , Factor XIII/metabolismo , Femenino , Expresión Génica , Genotipo , Humanos , Masculino , Modelos Moleculares , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Recombinantes , Trombina/metabolismo , Trombina/farmacología , Transglutaminasas/metabolismoRESUMEN
A new highly sensitive sandwich ELISA assay was developed for the determination of plasma factor XIII (FXIII). Plasma FXIII is a tetrameric complex of two types of subunits (A2B2). A biotinylated monoclonal capture-antibody against the B-subunit and a peroxidase-labelled monoclonal tag-antibody against the A-subunit were added to the plasma dilution and the amount of the complex attached to streptavidin-coated microplate was quantitated by measuring peroxidase activity. Only the tetrameric plasma FXIII reacted in the assay, non-complexed A or B subunits showed no reaction. The assay is linear up-to 40 microg/L of FXIII in the assay mixture. It is a quick one-step assay which can be performed within two hours. At normal and low FXIII concentration within batch reproducibility was 2.0% and 3.3%, day to day variation was 5.5% and 8.7%, respectively. Its high sensitivity allows reliable measurement at FXIII concentrations below 1% of normal average. Plasma samples can be stored for the assay at -20 degrees C for at least one month. Plasma levels of healthy individuals were normally distributed and no gender difference was observed. A reference interval of 14-28 mg/L (67-133%) was established.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Factor XIII/análisis , Anticuerpos Monoclonales , Biotinilación , Reacciones Cruzadas , Factor XIII/química , Factor XIII/inmunología , Factor XIII/farmacocinética , Deficiencia del Factor XIII/sangre , Deficiencia del Factor XIII/tratamiento farmacológico , Fibrinógeno/metabolismo , Peroxidasa de Rábano Silvestre , Humanos , Estructura Cuaternaria de Proteína , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrofotometría , Transglutaminasas/inmunologíaRESUMEN
The goals of this investigation were, first, to develop a chemical strategy to identify and quantitate the mass of fatty acid which is covalently bound to proteins by thioester linkage in unactivated platelets, and, second, to determine whether exogeneously added fatty acids can alter the fatty acid composition of thioester bound fatty acids. Studies with radiolabeled fatty acids cannot identify and quantitate the actual fatty acids bound to proteins because they permit analysis of only the radiolabeled fatty acids added and their metabolites. Therefore, in the absence of metabolic labeling by radiolabeled fatty acids, we isolated the thioester-linked fatty acids from platelet proteins using hydroxylamine at neutral pH to form fatty acid hydroxamates. The hydroxamates were subsequently converted to fatty acid methyl esters by acid methanolysis for quantitation by gas chromatography-mass spectrometry. Using platelet specimens from 14 subjects, 74% of the fatty acid recovered from the unactivated platelet proteins as thioester linked was palmitate. Importantly, however, 22% was stearic acid, and oleate was 4% of the total thioester bound fatty acid. There was minimal variability (2.6-fold at maximum) between the subjects in the amount of the thioester-linked palmitate and thioester-linked stearate. However, there was substantial variability (>100-fold at maximum) between subjects in the amount of thioester-linked oleate. We also demonstrated that incubation of platelets with exogenous fatty acids can alter the profile of fatty acids bound to platelet proteins by thioester linkages. Incubation of platelets with 100 microM palmitate for 3 h increased the amount of thioester-linked palmitate by up to 26%, and incubation of platelets with 100 microM stearate increased the amount of thioester-linked stearate up to 30%. In support of the observation that radiolabeled fatty acids other than palmitate were shown to be capable of binding to platelet proteins by thioester linkage, our results indicate that the fatty acids actually bound to unactivated platelet proteins include a significant amount of stearate, and variable amounts of oleate, as well as palmitate. In addition, the data show that palmitate and stearate can be increased, as a percentage of total protein-bound fatty acid, by incubation with exogenous palmitate and stearate, respectively.
Asunto(s)
Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Ácidos Grasos no Esterificados/sangre , Albúmina Sérica/metabolismo , Ésteres , Humanos , Técnicas In Vitro , Ácido Palmítico/sangre , Ácidos Esteáricos/sangreRESUMEN
Factor XIII deficiency is an autosomal recessive bleeding disorder that is largely caused by various mutations in FXIII A-subunit gene. Characteristically, the patients lack both A-subunit activity and antigen in the circulation. Here we have analysed the consequences of four missense mutations (Met242-->Thr, Arg252-->Ile, Arg326-->Gln, Leu498 to Pro) and one stop mutation (Arg661-->Stop) in the FXIII A-subunit gene by expression in COS-cells. After transient transfection each mutant cDNA expressed mRNA at an equal level to the wild type FXIII. However, the mutant polypeptides accumulated in the cells in significantly reduced quantities and demonstrated only very low enzymatic activity. Analysis of immunoprecipitated metabolically labelled polypeptides demonstrated remarkable instability and intracellular degradation of all mutant FXIII proteins. These results verify the deleterious nature of the individual amino acid changes and confirm that protein instability and susceptibility to proteolysis are consequences of the mutations, as predicted from the three-dimensional model of crystallised FXIII A-subunit.
Asunto(s)
Deficiencia del Factor XIII/genética , Factor XIII/genética , Mutación , Animales , Células COS , ADN Complementario/genética , Factor XIII/metabolismo , Deficiencia del Factor XIII/metabolismo , Humanos , TransfecciónRESUMEN
Congenital factor XIII (FXIII) deficiency is potentially a severe bleeding disorder, but in some cases, the symptoms may be fairly mild. In this study, we have characterized the molecular mechanism of a mild phenotype of FXIII A-subunit deficiency in a Finnish family with two affected sisters, one of whom has even had two successful pregnancies without regular substitution therapy. In the screening tests for FXIII deficiency, no A-subunit could be detected, but by using more sensitive assays, a minute amount of functional A-subunit was seen. 3H-putrescine incorporation assay showed distinct FXIII activity at the level of 0.35% of controls, and also the fibrin cross-linking pattern in the patients clotted plasma showed partial gamma-gamma dimerization. In Western blot analysis, a faint band of full-length FXIII A-subunit was detected in the patients' platelets. The patients have previously been identified as heterozygotes for the Arg661 --> Stop mutation. Here we report a T --> C transition at position +6 of intron C in their other allele. The transition affected splicing of FXIII mRNA resulting in low steady state levels of several variant mRNA transcripts. One transcript contained sequences of intron C, whereas two transcripts resulted from skipping of one or two exons. Additionally, correctly spliced mRNA lacking the Arg661 --> Stop mutation of the maternal allele could be detected. These results demonstrate that a mutation in splice donor site of intron C can result in several variant mRNA transcripts and even permit partial correct splicing of FXIII mRNA. Further, even the minute amount of correctly processed mRNA is sufficient for producing protein capable of gamma-gamma dimerization of fibrin. This is a rare example of an inherited functional human disorder in which a mutation affecting splicing still permits some correct splicing to occur and this has a beneficial effect to the phenotype of the patients.
Asunto(s)
Deficiencia del Factor XIII/genética , Factor XIII/genética , Mutación Puntual , Empalme del ARN , ARN Mensajero/metabolismo , Análisis Mutacional de ADN , Dimerización , Factor XIII/química , Factor XIII/inmunología , Deficiencia del Factor XIII/sangre , Femenino , Heterocigoto , Humanos , Isoanticuerpos/inmunología , Linaje , Polimorfismo Conformacional Retorcido-Simple , Embarazo , Complicaciones del Embarazo , ARN Mensajero/genéticaRESUMEN
Factor XIII (FXIII) is of high importance in the regulation of fibrinolysis. It crosslinks alpha 2-antiplasmin (alpha 2AP) and fibrin and by this way protects fibrin from the prompt elimination by plasmin. Although FXIII of platelets has been implicated in this protective mechanism, the role of platelets and platelet FXIII in the crosslinking process is far from being elucidated. As demonstrated by SDS PAGE and by immunoblotting for alpha 2AP, intact normal platelets resuspended in FXIII-free plasma or FXIII-free fibrinogen solution catalyzed the crosslinking of fibrin chains and also the crosslinking of alpha 2AP to fibrin alpha-chains. With FXIII-deficient platelets no crosslinking reaction could be observed indicating that the crosslinking with normal platelets was, indeed, due to platelet FXIII and not to another, putative platelet transglutaminase. However, the crosslinking of alpha 2AP to fibrin induced by the FXIII of intact platelets resuspended in FXIII-free plasma was considerably less extensive than the crosslinking carried out by the FXIII of normal plasma in the presence of FXIII-free platelets. Furthermore, the replacement of FXIII-free platelets by normal platelets in normal FXIII-containing plasma resulted in little, if any, difference in the crosslinking process. When crosslinking was induced by highly purified plasma FXIII the presence of intact FXIII-free platelets significantly accelerated the formation of alpha-chain polymers as well as the incorporation of alpha 2AP-fibrin alpha-chain hetero-dimer into these polymers. The results indicate that, in physiological conditions, platelet FXIII plays only a minor role in the crosslinking of alpha 2AP and fibrin; however, platelets, independently of their FXIII content, promote the crosslinking reaction by providing a catalytic surface on which the formation of highly crosslinked fibrin polymers is accelerated.
Asunto(s)
Plaquetas/química , Factor XIII/fisiología , Fibrina/química , Fibrinólisis/fisiología , alfa 2-Antiplasmina/química , Deficiencia del Factor XIII/sangre , Humanos , Immunoblotting , Sustancias MacromolecularesRESUMEN
The cellular form of blood coagulation factor XIII (FXIII) is present in platelets, monocytes and macrophages. During long-term stimulation of platelets by thrombin cellular FXIII becomes activated and cross-links proteins, however, the mechanism of its activation has not been elucidated. It was shown that, contrary to plasma FXIII, the intracellular activation of platelet FXIII does not involve proteolysis. FXIII remained intact in thrombin-activated platelets, i.e., the activation peptide was not removed from the molecule. Part of the zymogen FXIII molecules, however, assumed an active configuration as was demonstrated both by the measurement of transglutaminase activity and by active-site-SH titration. These findings clearly indicate that during platelet activation, when intracellular Ca2+ concentration is raised, a slow non-proteolytic transformation of FXIII zymogen into an active transglutaminase occurs.