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.

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.

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.

Molecular mechanism of the interaction between activated factor XIII and its glutamine donor peptide substrate.

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.

Investigation of cyclic depsipeptides by fast atom bombardment-mass spectrometry.

In recent years, the synthetic analogue elcatonin has been used successfully in the treatment of osteoporosis. During an investigation of synthetic strategies for the production of elcatonin, a number of peptide products were generated. These could be purified by liquid chromatography and were shown to have identical amino acid compositions. Investigation by fast atom bombardment-mass spectrometry revealed three of the peptides to have a common pseudomolecular ion but notably dissimilar fragmentation patterns. Further studies involving chemical modification, mass spectrometry and chromatographic separation showed these products to be novel depsipeptides, cyclized with an ester bond between either the serine or the threonine residue and the side chain of aminosuberic acid.