Severe bleeding diatheses in an elderly patient with combined type autoantibody against factor XIII A subunit; novel approach to the diagnosis and classification of anti-factor XIII antibodies.

INTRODUCTION: Acquired factor XIII (FXIII) deficiency due to autoantibody is a rare, severe bleeding diathesis. Its laboratory diagnosis and classification represents a difficult task. AIM: Introduction of novel approaches into the diagnosis and characterization of anti-FXIII autoantibody and demonstration of their use in the diagnosis of a patient with autoimmune FXIII deficiency. METHODS: Factor XIII activity, FXIII antigen levels and the titre of anti-FXIII-A antibody were monitored throughout the course of the disease. FXIII activity was measured by ammonia release assay; FXIII-A2 B2 complex, total and free FXIII-B concentrations were determined by ELISAs. The binding constant for the interaction of the autoantibody with recombinant FXIII-A2 (rFXIII-A2 ) and FXIII-A2 B2 was determined by surface plasmon resonance (SPR). The inhibitory capacity of IgG was expressed as the concentration exerting 50% inhibition of FXIII activation/activity (IC50). The truncation of FXIII-A by thrombin was monitored by western blotting. The inhibition of Ca2+ -induced FXIII activation and active FXIII (FXIIIa) were assessed by FXIII activity assay. RESULTS: The antibody bound to rFXIII-A2 and FXIII-A2 B2 with high affinity and accelerated the decay of supplemented FXIII concentrate. An IC50 value of 170.1 µg IgG·mL-1 indicated effective FXIII neutralization. The main neutralizing effect of the autoantibody was the inhibition of FXIIIa. After 2 months, due to combined therapeutic modalities, the autoantibody disappeared and FXIII activity significantly elevated. CONCLUSION: The anti-FXIII-A autoantibody exerted a combined effect including inhibition of FXIIIa and acceleration of FXIII decay in the plasma. IC50 and binding constant determinations added important information to the characterization of the autoantibody.

Alloantibody developed in a factor XIII A subunit deficient patient during substitution therapy; characterization of the antibody.

INTRODUCTION: In factor XIII A subunit (FXIIIA) deficiency, the development of alloantibodies is extremely rare. Only four reports have been published and the antibodies were not characterized. AIM: The aim of this study was to describe the clinical course and the laboratory diagnosis of a FXIII-A deficient patient who developed alloantibodies. METHODS: FXIII activity was assessed with an ammonia release assay. FXIII-A, FXIII B subunit (FXIII-B) and the complex plasma FXIII (FXIII-A2 B2 ) antigens were determined by ELISA. The causative mutation was detected by fluorescent DNA sequencing. The binding of alloantibody to FXIII-A2 and FXIII-A2 B2 was studied by surface plasmon resonance. The cleavage of FXIII-A by thrombin and Ca2+ -induced activation of thrombin-cleaved FXIII were followed by western blotting and activity measurement, respectively. RESULTS: FXIII activity, FXIII-A2 B2 and FXIII-A antigens were below the limit of detection in the patient's plasma. The severe FXIII-A deficiency was due to a novel homozygous mutation resulting in early stop codon (c.127C>T, p.Gln42STOP). The alloantibody bound to FXIII-A2 and FXIII-A2 B2 with equally high affinity (Kd ~10-8 ). It accelerated the elimination of administered FXIII concentrate from the circulation, interfered with thrombin and Ca2+ -induced activation and inhibited FXIII activity. Attempts to eliminate the alloantibody resulted only in transient improvement. Patient developed intracerebral haemorrhage after a minor trauma and died in spite of aggressive replacement therapy with FXIII concentrate. CONCLUSION: The anti-FXIII-A alloantibody caused an unmanageable bleeding complication. The antibody was of combined subtype which accelerated the elimination of FXIII and exerted a multiple inhibitory effect on FXIII activation/activity.

Factor XIII deficiency: complete phenotypic characterization of two cases with novel causative mutations.

Coagulation factor XIII (FXIII) exists as heterotetramer (FXIII-A2B2) in the plasma and as dimer (FXIII-A2) in cells. Activated FXIII mechanically stabilizes fibrin and protects it from fibrinolysis by cross-linking fibrin chains and α2-plasmin inhibitor to fibrin. FXIII is essential to maintaining haemostasis, and its deficiency causes severe bleeding diathesis. Due to improper laboratory practices, FXIII deficiency is considered the most under-diagnosed bleeding disorder. The aim of this study was to demonstrate in two cases how FXIII deficiency is properly diagnosed and classified, and to compare results of laboratory analysis and clinical symptoms. FXIII activity from plasma and platelets was measured by a modified ammonia release assay, while FXIII-A2B2, FXIII-A and FXIII-B antigens were determined by ELISA. The exon-intron boundaries and the promoter region of F13A1 gene were amplified by PCR and the amplified products were analysed by direct fluorescent sequencing. FXIII-A mRNA in platelets was determined by RT-qPCR. Two children with severe bleeding symptoms were investigated. In both cases FXIII activity and FXIII-A antigen were undetectable in the plasma and platelet lysate. In the plasma no FXIII-A2B2 antigen was found, while FXIII-B antigen was >30% in both cases. Proband1 was a compound heterozygote possessing a known missense mutation (c.980G>A, p.Arg326Gln) and a novel splice-site mutation (c.1112+2T>C). Proband2 was homozygote for a novel single nucleotide deletion (c.212delA) leading to early stop codon. The discovered mutations explain the severity of clinical symptoms and the laboratory data. Methods precise in the low activity/antigen range are required to draw valid conclusion on phenotype-genotype relationship.

Impaired cerebral vasoreactivity in white coat hypertensive adolescents.

BACKGROUND AND PURPOSE: Although its incidence is not high, adolescent hypertension may predict hypertension and increased cardiovascular risk in adulthood. Therefore, the aim of the present study was to assess whether cerebrovascular reactivity is altered in adolescent white coat and sustained hypertensive patients compared to healthy teenagers. METHODS: Fifty-nine normotensive, 47 white coat hypertensive (WCH), and 73 sustained hypertensive (SH) adolescents were studied. WCH and SH were differentiated by ambulatory blood pressure monitoring. Cerebrovascular reactivity was assessed by transcranial Doppler breath-holding test and was expressed in percent (%) change to the resting cerebral blood flow velocity value. RESULTS: The percent increase in middle cerebral artery mean blood flow velocity after 30 s of breath holding was lower in both WCH (5.3 ± 3.1%) and SH (9.5 ± 2.6%) groups indicating lower vasodilatory reactivity compared to healthy adolescents (12.1 ± 2.2%). Additionally, serum nitric oxide (NOx) concentrations were lower in both WCH (30.6 ± 11 µM) and SH (30.7 ± 22.4 µM) groups compared to controls (38.8 ± 7.6 µM). CONCLUSIONS: Both white coat and sustained hypertension result in decreased vasodilatory reaction to CO(2) in adolescents, suggesting involvement of the cerebral arterioles. The present study underlines the importance of early recognition and proper treatment of adolescent hypertension in order to prevent long-term cardiovascular complications.

Plasma fibrin clots of pulmonary embolism patients present increased amounts of factor XIII and alpha2-antiplasmin at 3 months' anticoagulation since the acute phase.

Fibrin cross-linking by coagulation factor (F)XIII leads to clot stabilization. Reduced plasma FXIII levels have been reported in acute pulmonary embolism (PE) patients. We investigated the impact of anticoagulant therapy on clot-bound amounts of FXIII and α2-antiplasmin and their associations with fibrin clot properties in patients with PE. Clots generated from plasma of 18 acute symptomatic patients on admission and after a 3-month treatment with rivaroxaban were assessed off anticoagulation using mass spectrometry. Plasma FXIII and α2-antiplasmin activity were determined at the 2 time points along with thrombin generation markers, plasma fibrin clot permeability (Ks), and clot lysis time (CLT). Following anticoagulant therapy, clot-bound FXIII increased from 2.97 (interquartile range, 1.98 - 4.08) to 4.66 (3.5 - 6.9) mg/g protein and α2-antiplasmin from 9.4 (7.2 - 10.6) to 11 (9.5 - 14) mg/g protein (both p < 0.0001). The two parameters showed positive correlation at baseline only (r = 0.63, p = 0.0056). Similarly to clot-bound amounts, plasma FXIII (+25.8%) and α2-antiplasmin activity (+12%) increased at 3 months. Plasma FXIII activity on admission, but not after 3 months since the index PE, was associated with amounts of clot-bound FXIII (r = 0.35, p = 0.043) and α2-antiplasmin (r = 0.47, p = 0.048). At baseline, clot-bound FXIII correlated with plasma F1+2 prothrombin fragments levels (r = 0.51, p = 0.03), while clot-bound α2-antiplasmin correlated with CLT (r = 0.43, p = 0.036). At 3 months associations of clot-bound FXIII and α2-antiplasmin were abolished. This study assessed for the first time changes in the fibrin clot composition following acute PE, suggesting an increase of clot-bound and plasma FXIII and α2-antiplasmin levels after 3 months.

alpha1-Antitrypsin: the presence of excess mannose in the Z variant isolated from liver.

The Z variant of alpha1-antitrypsin was isolated by a new technique from the liver of a patient homozygous for the Z allele of the protease inhibitor locus. The material was homogenous and antigenically competent but had no protease inhibiting capacity. An interesting correlation was found between the subcellular localization and the carbohydrate composition of the Z variant from liver. Carbohydate analysis of this glycoprotein showed an absence of galactose and sialic acid, an appreciable decrease in N-acetylglucosamine, and an almost twofold increase in mannose residues. These data indicate a considerable slowdown in the processing of the oligosaccharides of liver Z variant. In spite of the absence of sialyl residues, the liver Z varant was microheterogeneous by analytical isoelectric focusing. The isoproteins of liver Z variant coincided with those of asialo M variant in the focusing field.

Auto- and alloantibodies against factor XIII: laboratory diagnosis and clinical consequences.

Acquired FXIII deficiencies caused by autoantibodies against FXIII subunits represent rare but very severe bleeding diatheses. Alloantibodies in FXIII-deficient patients also cause life-threatening bleeding complications, but they develop extremely rarely. In this review we provide an overview of the diagnosis and classification of anti-FXIII antibodies and analyze 48 patients with autoimmune FXIII deficiency and four additional FXIII-deficient patients who developed anti-FXIII alloantibody. The patients were collected from peer-reviewed publications from which relevant data could be extracted. With the exception of two cases the antibodies were directed against FXIII-A. The difficulties in the diagnosis of FXIII deficiency in the presence of anti-FXIII antibodies are discussed and a scheme for the functional classification of the anti-FXIII antibodies is recommended. The three main categories are neutralizing and non-neutralizing antibodies and antibodies with combined effect. The methods being used for detecting and quantifying the inhibitory effect on FXIII activation and on the transglutaminase activity of activated FXIII are summarized and techniques for the classification of neutralizing anti-FXIII antibodies are outlined. The importance of clearance studies in these cases is emphasized. Binding assays, useful for the identification of non-neutralizing and combined type antibodies, were collected from the literature and their informative power is demonstrated by examples. The most frequently occurring bleeding symptoms in patients with anti-FXIII antibodies were soft tissue bleeding; intracranial bleedings also occurred, but less frequently than in inherited FXIII deficiency. Treatment of such patients is extremely challenging; the main aim should be eradication of the antibody.

Minimal factor XIII activity level to prevent major spontaneous bleeds.

Essentials A strong association between bleeding severity and FXIII activity level (FXIII:C) was shown. The range 5-30 IU dL-1 of FXIII:C was associated with a high variability of bleeding severity. The PROspective study confirmed the association between FXIII:C activity and bleeding severity. A FXIII: C of 15 IU dL-1 is a proposed target to start prophylaxis for prevention of major bleeding. SUMMARY: Background Congenital factor XIII (FXIII) deficiency is a rare bleeding disorder associated with significant bleeding manifestations. The European Network of Rare Bleeding Disorders (EN-RBD) study, performed from 2007 to 2010, showed a strong association between bleeding severity and FXIII activity in plasma of patients with FXIII deficiency. Among these patients, variable levels of FXIII activity, from undetectable to 30%, were associated with a wide range of bleeding severity. Objectives and patients The present cross-sectional study, in the frame of the PRO-RBDD project, a prospective cohort study, analyzed data of 64 patients with FXIII deficiency and different types of clinical and laboratory severity. Results The results of this analysis confirmed that FXIII coagulant activity in plasma is well associated with clinical severity of patients. In addition, 15 IU dL-1 of FXIII activity was identified to be the level under which the probability of spontaneous major bleeding sharply increases (from 50% for levels of 15 IU dL-1 to more than 90% for levels of 5 IU dL-1 or lower). Conclusion The PRO-RBDD study suggests a FXIII coagulant activity level of 15 IU dL-1 as a target to start prophylaxis in order to prevent major bleedings, such as central nervous system or gastrointestinal tract hemorrhages.

Neutralizing autoantibody against factor XIII A subunit resulted in severe bleeding diathesis with a fatal outcome - characterization of the antibody.

UNLABELLED: Essentials Autoantibody against factor XIII (FXIII) is a rare but severe acquired hemorrhagic diathesis. In an elderly patient, anti-FXIII-A antibody led to severe bleedings with fatal outcome. The neutralizing autoantibody bound to FXIII with high affinity (Ka≈10(9) m(-1) ). The dominant effect of the autoantibody was the inhibition of activated FXIII. SUMMARY: Autoantibodies may develop against the catalytic A subunit of factor XIII (FXIII-A) or the carrier B subunit (FXIII-B). Autoimmune FXIII-A deficiency was diagnosed in an elderly (75 years) patient with severe bleeding symptoms. The patient had 3% FXIII activity, and unmeasurable FXIII-A2 B2 and FXIII-A antigens in the plasma, whereas, in the platelet lysate, activity and FXIII-A antigen values were normal. As revealed by western blotting, FXIII antigen was present in the plasma, but the autoantibody interfered with the immunoassays. A mixing study indicated the presence of inhibitor with a titer of 63.2 Bethesda units (BU). The patient's IgG bound to FXIII-A2 B2 and to FXIII-A2 with equally high affinity (Ka in the range of 10(9) m(-1) ). It exerted a multiple inhibitory effect on FXIII activation/activity (IC50: 50 µg mL(-1) ). Immunosupressive therapy gradually decreased the autoantibody titer to 8.0 BU, but FXIII activity remained very low, and, owing to recurrent bleeding, the patient died.

The temperature dependence of the surface tension of aqueous solutions of plasma proteins.

The surface behavior of aqueous solutions of fibrinogen, transferrin, gamma-globulin and albumin at the liquid-gas interface has been investigated by a modified Wilhelmy technique. The temperature dependence of the surface tension was studied over a temperature range of 20--80 degrees C and a pH range of 2--12. Most pronounced conformational changes of fibrinogen with this technique were found in physiological conditions: 35--45 degrees C and pH 7--8. A conformational change was found for gamma-globulin and transferrin solutions, but at a higher temperature and less pronounced than fibrinogen. Albumin did not undergo conformational transitions to a significant extent.

Kinetics of nerve impulse blocking by protein cross-linking aldehydes. Apparent critical thermal points.

The effect of formaldehyde, crotonaldehyde, butyraldehyde, glutaraldehyde and cinnamaldehyde on the compound action potential of frog sciatic nerve was studied in the temperature domain 20-35 degrees C at various aldehyde concentrations. All these reagents gradually decrease the amplitude of nerve action potential, up to the complete block, the order of effectiveness being: crotonaldehyde greater than cinnamaldehyde greater than butyraldehyde greater than formaldehyde greater than glutaraldehyde. The effect of cinnamaldehyde is almost completely reversible, while all others have irreversible action. The dependence of the blocking time on temperature and concentration is well expressed in all cases by the same empirical equation. This dependence points to the existence of critical temperatures, specific for each aldehyde, at which impulse blocking would be instantaneous, regardless of concentration. These temperatures (obtained by extrapolation) lie between 43 degrees C (for crotonaldehyde) and 57.5 degrees C (for butyraldehyde). The existence of free amino groups within ionic channels, as main sites of aldehyde attack, is inferred.

Sequential sodium-proton exchange in thrombin-induced human platelets.

Thrombin stimulation of human platelets initiates a membrane depolarization attributable to a Na+ influx into, and an alkalinization of, the cytoplasm, both of which follow a similar rapid time scale and thrombin-dose dependence. These responses precede secretion of the contents of the dense granules (serotonin) and, after 1 minute, of lysosomes (beta-glucuronidase). We have evaluated these parameters in the presence of 2H2O in order to determine if the Na+ influx and H+ efflux are sequential or simultaneous. NMR evidence indicates that 2H2O equilibration in rapid, and virtually complete within the 3 min prestimulation platelet equilibration period. In response to an 0.05 U/ml addition of thrombin, the rate of depolarization is 70-80% slower in 2H2O than in H2O. The time to reach maximal depolarization is 5 to 10 seconds longer in 2H2O, the extent of depolarization 60% inhibited, and the pH change 85% inhibited. The serotonin secretion is unaltered, while the beta-glucuronidase secretion is 130-180% enhanced. Dimethylamiloride inhibits the Na+ influx and the pH change completely. These results suggest that the Na+ and H+ fluxes across the plasma membrane are interdependent but neither simultaneous nor electroneutral. Furthermore, granule secretion, previously shown by us to be independent of the existent Na+ gradient, depends on the cytoplasmic K+ and H+ concentrations.

Factor XIII in bronchoalveolar lavage fluid from children with chronic bronchoalveolar inflammation.

BACKGROUND: Extravascular activation of the coagulation system and consequent fibrin deposition is involved in the pathomechanism of chronic bronchoalveolar inflammatory diseases. The turnover of extravascular fibrin is attenuated by its cross-linking with activated factor XIII (FXIII). OBJECTIVES: Determination of cellular and plasmatic forms of FXIII and their correlation with D-dimer level in the bronchoalveolar lavage fluid (BALF) from healthy children and from children with bronchoalveolar inflammation. PATIENTS AND METHODS: Highly sensitive immunoassays were used for the quantitation of cellular and plasma FXIII and D-dimer in the BALF of children with recurrent wheezy bronchitis and fibrosing alveolitis. BALF was investigated for FXIII-containing cells by flow cytometry. RESULTS AND CONCLUSIONS: In the BALF of controls a low amount of the cellular form of FXIII (FXIII A2) and D-dimer were measured, while plasma FXIII (FXIII A2B2) was absent. Alveolar macrophages represented the single cell population in BALF that contained FXIII. In the BALF of both patients' groups the concentration and the total amount of FXIII A2 was significantly elevated, and plasma FXIII also appeared in the BALF of most patients. The D-dimer concentration was also elevated in the patients' groups and it correlated both with plasma FXIII and neutrophil count. These findings suggest that FXIII A2 is released from activated or injured alveolar macrophages into the bronchoalveolar lining fluid and in bronchoalveolar inflammatory diseases, FXIII A2B2 also leaks out from the capillaries. By cross-linking fibrin and inhibitors of fibrinolysis to fibrin, FXIII might be a key regulator of fibrin turnover in the extravascular compartment.

Enzyme-linked immunosorbent assay for the determination of blood coagulation factor XIII A-subunit in plasma and in cell lysates.

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.

A simple, quick one-step ELISA assay for the determination of complex plasma factor XIII (A2B2).

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.

Fibronectin in bronchoalveolar lavage fluid and plasma of dogs with acute inflammation of the lungs.

Bronchoalveolar inflammation, which was generated in dogs by Broncho-Vaxom instilled into the right lower lobe, was characterized first of all by an increased influx of macrophages. In this non-purulent acute-subacute inflammatory reaction, the lavage fibronectin decreased rapidly three hours after the incubation and then a marked gradual elevation was observed, which persisted throughout the whole two-week process, while plasma fibronectin concentrations were not altered significantly. Changes in the levels of lavage fibronectin may be an important sign for the control of the inflammatory reaction activity in the lungs.

Development of ELISA and enzyme-linked immunofiltration assay (ELIFA) methods for monitoring cyclodextrin glycosyltransferase (CGTase) production and bacterial growth in Bacillus macerans batch cultures.

Immunochemical methods were developed for monitoring cyclodextrin (CD) glycosyltransferase (CGTase) production and growth of an industrial CD-producing Bacillus macerans strain. Extracellular concentrations of CGTase released into a non-transparent culture medium during a 44 h long fermentation were detected by an indirect antigen inhibition enzyme-linked immunosorbent assay (ELISA). The ELISA was sensitive (minimal detection level 6 ng ml-1) and highly reproducible (coefficients of variation < or = 1.2 and 5.9%, within-runs and between-runs, respectively) compared to assays of CGTase activity (coefficients of variation < or = 4.2 and 7.0%, respectively). The ELISA, in combination with enzyme activity measurements, was useful to detect the decrease in the specific CGTase activities after 36 h of incubation, which was clearly indicative of the proteolytic degradation of CGTase. B. macerans cell numbers were estimated using an enzyme-linked immunofilter assay (ELIFA). The assay took less than 1 h and the coefficients of variation within and between-runs (2.9-6.4%) were considerably less than for viable counting (10.6-15.4%). In the exponential phase of growth, ELIFA results correlated more closely with the cell counting based on total protein than with viable counts. Nevertheless, in the phase of cell lysis, the bacterial cell number was systematically underestimated by ELIFA in comparison to both viable cell number and total protein determinations. Thus cell antigens detected with immunological procedures might be lost during the transition from vegetative cells to spores. On the other hand, the ELIFA procedure was specific for B. macerans cells and was a better indicator of the onset of the different growth phases than the cell numbers calculated from the protein assay.

Anesthesia and the golgi apparatus: enzyme activity changes in a golgi-rich fraction of rat liver after a short ether anesthesia.

Activity changes of enzymes in isolated rat liver Golgi preparations at different times (1-48 h) after a short ether anesthesia are reported. Activity of galactosyl-transferase showed a slight gradual increase but thiamine pyrophosphatase decreased sharply, and after 24 h increased to above control level. Arylsulphatase-A remained largely unchanged, and B was significantly decreased. Acid phosphatase activity did remain at the control level, but alkali phosphatase showed a gradual and highly significant increase. Five other enzymes representing probable contaminations from other subcellular organelles, have also been assayed. Correlation is sought between the enzyme activity changes and some other metabolic effects of anesthesia.

[Vestibular side effects of contraceptives]. / Az anticoncipiensek vestibuláris mellékhatásairól.

PIP: The authors describe the results of otoscopic examination of 18 patients who complained of dizziness and in whom, after other etiological factors were excluded, an effect of the oral contraceptive (OC) could be suspected. In every case the cochlear function was undamaged, but the central vestibular areas were affected. These symptoms gradually disappeared when the women were taken off the OC and symptomatic treatment was applied. They call attention to the possibility of vestibular side effects from contraceptives, i.e., the need for rigorous medical supervision of the patient.^ieng