[Show abstract][Hide abstract] ABSTRACT: We investigated the effect of elevated levels of humoral 5HT and DA on the feeding latency of Helix pomatia, 1 day, 3 days and 10 days following satiation, by injecting monoamines into the haemocoel. HPLC assay of monoamines showed that both 5HT and DA are present in pmol/ml concentrations in the haemolymph of both starved and non-starved animals. Elevated levels of 5HT and DA were most effective at decreasing the feeding latency 10 days following satiation when DA decreased the feeding latency in a concentration dependent manner between 10(-7) and 10(-5) M whereas 5HT levels decreased the feeding latency only at 10(-6) M but increased it at 10(-5) M. Immunocytochemistry revealed that both 5HT3 and D1 receptor-like immuno-reactivity are present in cell bodies located in the same areas of the buccal ganglia. Our observations suggest that both humoral DA and 5HT mutually modulate the activity of the feeding CPG through neurons which have these receptors.
[Show abstract][Hide abstract] ABSTRACT: The effect of factor XIII A subunit (FXIII-A) Val34Leu polymorphism on the risk of coronary artery disease (CAD) has been extensively studied. In this study we investigated how FXIII-A Val34Leu genotypes influence plasma factor XIII levels in patients with coronary sclerosis (CS) and myocardial infarction (MI) and how fibrinogen level modulates this effect.
955 consecutive patients admitted for coronary angiography were categorized according to the presence or absence of significant CS and the history of MI. The frequency of FXIII-A Val34Leu polymorphism, fibrinogen, FXIII activity and antigen levels were determined.
CS or MI decreased FXIII levels in patients homozygous for FXIII-A Leu34 allele, but not in heterozygous or wild type patients. In the subgroup of patients with CS, but without the history of MI no significant effect was detected, which suggests that MI has a more prominent role. The specific activity of plasma FXIII was independent of FXIII-A Val34Leu genotype. FXIII and fibrinogen levels significantly correlated in CS+ and MI+ patients. In MI+ patients of Leu/Val or Leu/Leu genotypes and with fibrinogen levels in the lowest quartile, FXIII levels were lower than in the same patient groups, but with higher fibrinogen level. The low-scale continuous activation of blood coagulation in CAD patients could lead to parallel FXIII and fibrinogen consumption. As the same amount of thrombin activates more Leu34 FXIII than Val34 FXIII, increased FXIII consumption might be responsible for the decreased FXIII levels in Leu34 homozygous CAD patients.
Thrombosis Research 02/2008; 121(4):469-76. DOI:10.1016/j.thromres.2007.05.012 · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The results on the association of factor XIII (FXIII) A subunit (FXIII-A) Val34Leu polymorphism with the risk of myocardial infarction (MI) are rather inconclusive. The original paper and confirmatory reports demonstrated a protective effect of the mutation, but results demonstrating the lack of protection have also been published. Gene-gene and gene-environmental interactions have been proposed to be responsible for the opposing results. As the rate of change in fibrin clot permeability with increasing fibrinogen concentrations decreased stepwise with increasing number of Leu34 alleles it was proposed that the protection by Val34Leu polymorphism become effective only at higher fibrinogen concentrations. However, this hypothesis has not been tested on patients with coronary artery disease.
955 consecutive patients admitted for coronary angiography were categorized according to the presence or absence of significant coronary sclerosis (CS) and according to positive or negative history of MI. The frequency of FXIII-A Val34Leu polymorphism, and a number of risk factors, including fibrinogen were determined in the patients. FXIII-A Val34Leu polymorphism was also investigated in a population control group of 1146 subjects.
The presence of FXIII-A Leu34 allele or homozygous Leu34 genotype did not change the risk of CS or MI in the general Hungarian population. However, when patients with fibrinogen level in the upper quartile were separately investigated, the Leu34 allele provided a statistically significant protection against MI.
Fibrinogen concentration modulates the effect of Leu34 allele on the risk of MI; its protective effect emerges at increasing fibrinogen concentration.
Thrombosis Research 02/2007; 120(4):567-73. DOI:10.1016/j.thromres.2006.12.013 · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the transglutaminase enzyme family are involved in a broad range of biological phenomena, including haemostasis, apoptosis, semen coagulation, skin formation, and wound healing. A new and rapid method for measurement of transglutaminase activity is described in this article. The enzyme links tritium-labeled putrescine to biotinylated oligoglutamine, and the tritiated peptide is bound to a streptavidin-coated microtiter plate permanently covered by a thin layer of scintillant. Only the radioisotope incorporated into the peptide substrate is close enough to the scintillant molecules for photons to be produced. The signal generation depends on the transglutaminase activity, and it can be detected by appropriate light-measuring instrumentation without separation steps. The assay is sensitive, specific, linear at concentrations of tissue transglutaminase between 0.05 and 1.6m U/ml, and suitable for high-throughput measurements.
[Show abstract][Hide abstract] ABSTRACT: We combine electrophysiological and immunocytochemical analyses in the snail Lymnaea stagnalis of M-CCAP1 and M-CCAP2, two molluscan peptides with structure similar to crustacean cardioactive peptide CCAP, originally isolated from the snail Helix pomatia. Both M-CCAP peptides (M-CCAP1 and M-CCAP2, 1 microM) had an excitatory effect, depolarizing all the identified neurons of the buccal feeding network (including motoneurons: B1, B2, B4 and modulatory interneurons SO, OC: 62 neurons in 33 preparations). Additionally, in 67% of preparations, rhythmic activity (fictive feeding) was recorded with a mean rate of 7 cycles/min. No significant difference in the proportion of preparations showing fictive feeding or mean feeding rate was found between M-CCAP1 and M-CCAP2. The extrinsic feeding modulator, the serotonergic CGC neuron, responds by increase of the spontaneous activity after M-CCAP application (9 of 18 preparations). Crustacean CCAP (1 microM) evokes a slight membrane depolarization in 3 out of 8 preparations but never evokes fictive feeding. Immunostaining revealed no cell bodies in the buccal ganglia, but a dense network of CCAP immunopositive fibers arborizing in the buccal neuropil. Many of these fibers originate from a symmetrical pair of CCAP-immunoreactive cerebro-buccal interneurons, which are the most likely candidates for extrinsic modulatory interneurons in the buccal feeding network. Our data are the first results suggesting that M-CCAP-peptides exist as effective modulators in mollusc.
[Show abstract][Hide abstract] ABSTRACT: Factor XIII subunit A (FXIII A) is synthesized by megakaryocytes, and monocytes/macrophages. In addition, the liver has been reported as an extrahaematopoietic source of FXIII A. At present, the extent of contribution of either haematopoietic or extrahaematopoietic sources to the plasma FXIII A level is unknown. We studied the effect of bone marrow aplasia due to high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ASCT) on plasma FXIII A activity and concentration in 20 patients with haematological or solid tumour malignancies. A multiple linear regression model was used to assess the effect of gender, age, malignancy and treatment types, platelet and monocyte counts, abnormal liver function tests, prothrombin time, and number of platelet transfusions on FXIII activity measured in plasma before and following ASCT. Significant correlation between platelet counts and FXIII A activity in plasma was observed (r = 0.51, P = 0.0001), which remained after the adjustment for the aforementioned parameters (multiple R = 0.52, P = 0.0001). In contrast, no significant correlation between FXIII A levels and monocyte counts was observed (r = 0.19), and this lack of correlation persisted after the adjustment. These results suggest that in the ASCT model, following myeloablation, platelets but not monocytes are the haematopoietic cells that contribute significantly to plasma FXIII A levels. In addition, extra-haematopoietic sources of FXIII synthesis may also contribute to FXIII levels in plasma.
[Show abstract][Hide abstract] ABSTRACT: A new one-step ELISA was developed for the determination of the concentration of blood coagulation factor XIII subunit A (FXIII-A) in plasma and in cell lysates. Monoclonal antibodies directed against different epitopes on FXIII-A were used for the assay. The capture antibody was biotinylated on its carbohydrate moiety and the detection antibody was labelled with horseradish peroxidase. The antigen-antibody reaction was carried out in the well of a streptavidin-coated microplate. Complex formation with FXIII subunit B (FXIII-B) and association to fibrinogen did not influence the accessibility of the antibodies to FXIII-A. The method could be performed within 2 h and demonstrated good reproducibility, recovery and sensitivity. Plasma samples could be assayed after storage at -20 degrees C for at least 6 months. However, in the case of platelet lysates freezing and rethawing resulted in a significant loss of FXIII-A. FXIII-A concentrations measured in the plasma samples of healthy individuals and patients correlated well with the concentrations of complexed plasma FXIII (A2B2) and with the results of FXIII activity measurements. A reference range of 46-82 fg/platelet was established for platelet FXIII-A.
[Show abstract][Hide abstract] ABSTRACT: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca(2+). FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory.
In the assay, FXIII was activated by thrombin and Ca(2+). Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogenase and NADPH.
The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was <8% even at very low FXIII activities. A reference interval of 108-224 U/L (69-143%) was established. The results correlated well with results obtained by an immunoassay specific for plasma FXIII.
The optimized FXIII assay is a simple, rapid method for the diagnosis of inherited or acquired FXIII deficiencies and increased FXIII concentrations. It can be easily adapted to clinical chemistry analyzers.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Thrombosis and Haemostasis 11/2000; 84(4):595-600. · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Thrombosis and Haemostasis 03/2000; 83(2):268-73. · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Új megállapításokat tettünk a FXIII plazmában történő aktivációjára, a két alegység (FXIII-A és FXIII-B) egymáshoz kapcsolódásának strukturális elemeire és a FXIIIa-glutamin szubsztrát kapcsolatra vonatkozóan. Új módszereket dolgoztunk ki a FXIII aktivitás mérésére, a FXIII-A intracelluláris detektálására. Utóbbi bevezetésre került a leukémiák diagnosztikájában. Immunoassay-t dolgoztunk ki az a2 plazmin inhibitor két izoformájának mennyiségi meghatározására. 10 FXIII-A hiányos betegen derítettük fel a háttérben álló mutációkat és ezek következményeit a fehérje strukturájára-funkciójára. Kísérletesen bizonyítottuk, hogy a plazma FXIII hiánya sebgyógyulási zavart okoz. Kimutattuk, hogy myocardiális infarctuson (MI) átesett nőkben emelkedett a plasma FXIII szintje és az emelkedett FXIII szint 2,5-3,0 szorosra fokozza az MI rizikóját, ami kizárólag nőkön érvényesül. 16 cikk metaanalízisiével a FXIII-A L34 allél szignifikáns védőhatását lehetett kimutatni a coronaria betegség ellen, a polimorfizmus a nagy rizikóju magyar populációban azonban csak emelkedett fibrinogén szint esetén védő hatású. A L/L homozigóták FXIII szintje MI-ben szignifikánsan alacsonyabb a vad típusúakénál. Csontvelő abláció után csökken, magas thrombocyta számmal járó myeloproliferatív betegségben emelkedik a FXIII szintje. Bronchoalveoláris mosófolyadékban kimutatható az alveoláris macrophagokból származó FXIII-A, chronicus bronchitisben ennek szintje emelkedik, s esetenként megjelenik a plazma FXIII is. | New results were reported on the activation of factor XIII (FXIII) in plasma, on the structural elements involved in the association of FXIII subunits (FXIII-A and FXIII-B) and on the interaction of activated FXIII (FXIIIa) with its glutamine substrate. Methods were developed for the determination of FXIII activity and the intracellular detection of FXIII-A by flow cytometry. The latter was introduced in the diagnostics of leukemias. Immunoassay was developed for the determination of the two isoforms of a2 plasmin inhibitor. The mutations causing FXIII-A deficiency were identified in 10 patients and their consequences were explored at the protein level. The involvement of FXIII in would healing was proven. It was shown that in women with the history of myocardial infarction (MI) FXIII level was elevated and elevated FXIII level represented a 2.5-3.0-fold increased risk of MI in women, but not in men. A protective effect of the FXIII-A L34 allele against MI was demonstrated by metaanalysis of 16 articles. In the Hungarian population this protective effect prevailed only at high fibrinogen level. FXIII level was decreased in L/L homozygotes with the history of MI. Bone marrow ablation decreased plasma FXIII level, while myeloproliferative diseases increased it. In the bronchoalveolar lavage fluid FXIII-A derived from alveolar macrophages was detected, in inflammatory bronchoalveolar diseases FXIII-A level increased and occasionally plasma FXIII was also be present.
[Show abstract][Hide abstract] ABSTRACT: I. A XIII-as faktor (FXIII) struktúrája és funkciója Az aktivációs peptid nélkül a FXIII-A instabil. A FXIII aktivációt a plazmában a fibrin polimerizáció determinálja, a FXIII-A Val34Leu polimorfizmusnak csak moduláló hatása van. A FXIII A és B alegységének kapcsolódását gátló monoklonális antitestek előállítása. A celluláris FXIII szerepet játszik a monocyták/macrophagok phagocytosisában. Az alvadékban aktiválódó granulocytákból felszabadult proteázok lebontják a FXIIIa-t. II. Klinikai FXIII kutatások Új típusú módszer a FXIII (ill. más transzglutaminázok) mérésére, ill. a FXIII-A Val34Leu polimorfizmus kimutatására. Az intracelluláris FXIII-A áramlásos citometriás kimutatására új módszer, mely kiválóan alkalmazható az acut myleoid leukemiák diagnosztikájában. Csontvelő abláció során szignifikánsan csökken a plazma FXIII szintje, magas thrombocyta számmal járó myeloproliferatív megbetegedésekben viszont emelkedik. Nőkben az emelkediett FXIII szint több mint kétszeresére növeli a myocardiális infarctus rizikóját nőkben. Krónikus bronchoalveoláris megbetegedésekben jelentősen emelkedik a bronchoalveoláris mosófolyadékban a FXIII mennyisége. 4 új mutációt írtunk le FXIII hiányos betegekben, s analizáltuk ezek fehérje biokémiai következményeit. FXIII hiányíos transzgén egereken igazoltuk, hogy a FXIII szükséges a normális sebgyógyuláshoz. | I. Structure and function of factor XIII (FXIII) The absence of activation peptide makes FXIII-A instable. The activation of FXIII in the plasma is determined by fibrin polymerization; FXIII-A Val34Leu polymorphism only modulates activation. Production of monoclonal antibodies with inhibitory effect on the association of FXIII A and B subunits. Cellular FXIII plays a role in the phagocytosis by monocytes/macrophages. Proteases released from granulocytes in the clot break down activated FXIII. Clinical studies on FXIII New methods on the measurement of FXIII activity, and on the detection of FXIII-A Val34Leu polymorphism. Method on the detection of intracellular FXIII-A and its application to the diagnosis of acute myeloid leukemias. Bone marrow ablation results in the significant decrease of plasma FXIII level; in myeloproliferative diseases with high platelet count plasma FXIII is elevated. In women elevated FXIII level increases the risk for myocardial infarction by more than two-folds. In chronic bronchoalveolar inflammation the amount of FXIII in the lavage fluid is significantly increased. Description of four new mutations in FXIII deficient patients, and protein structural analysis of their consequences. It was demonstrated on FXIII deficient transgene mice that FXIII is required for normal wound healing.