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Antiphospholipid syndrome (APS) is a systemic autoimmune disorder caused by the presence of aPLs (antiphospholipid antibodies, i.e., anti-β2-glycoprotein I and anti-cardiolipin). Everyday practice in terms of laboratory diagnostics of APS includes determination of aPLs and well-known functional assays assessing for lupus anticoagulant (LA), in turn using various tests. According to recent guidelines, the recommended method for LA identification or exclusion is based on the Russell Viper Venom test and a sensitive activated partial thromboplastin time assay. Despite the fact that LA can be quantified in laboratory practice in this way, LA is still used as a binary parameter that is just one of the risk factors of thrombosis in APS. As of today, there are no other functional assays to routinely assess the risk of thrombosis in APS. It is well-known that APS patients display a wide range of clinical outcomes although they may express very similar laboratory findings. One way to solve this dilemma, could be if antibodies could be further delineated using more advanced functional tests. Therefore, we review the diagnostic approaches to test the function of aPLs. We further discuss how thrombin generation assays, and rotational thromboelastometry tests can be influenced by LA, and how experimental methods, such as flow cytometric platelet activation, surface plasmon resonance, or nano differential scanning fluorimetry can bring us closer to the puzzling interaction of aPLs with platelets as well as with their soluble protein ligand. These novel approaches may eventually enable better characterization of aPL, and also provide a better linkage to APS pathophysiology.

Nucleoside and nucleic acid analogues are known to possess a considerable therapeutic potential. In this work, by coupling cysteine to nucleosides, we successfully synthesized compounds that may not only have interesting biological properties in their monomeric form, but can be used beyond that, for oligomerization, in order to produce new types of synthetic nucleic acids. We elaborated different strategies for the synthesis of cysteinyl nucleosides as monomers of cysteinyl nucleic acids using nucleophilic substitution or thiol–ene coupling as a synthetic tool, and utilised on two complementary nucleosides, uridine and adenosine. Dipeptidyl dinucleosides and pentameric cysteinyl uridine were prepared from the monomeric building blocks, which are the first members of a new class of peptide nucleic acids containing the entire ribofuranosyl nucleoside units bound to the peptide backbone.

Hemostasis disorder in patients with end-stage renal disease (ESRD) is frequently associated with bleeding diathesis but it may also manifest in thrombotic complications. Analysis of individual coagulation and fibrinolytic factors may shed light on the background of this paradox situation. Here we explored components essential for fibrin formation/stabilization in ESRD patients being on maintenance hemodiafiltration (HDF) or hemodialysis (HD). Pre-dialysis fibrinogen, factor XIII (FXIII) antigen concentrations and FXIII activity were elevated, while &alpha, 2-plasmin inhibitor (&alpha, 2PI) activity decreased. The inflammatory status, as characterized by C-reactive protein (CRP) was a key determinant of fibrinogen concentration, but not of FXIII and &alpha, 2PI levels. During a 4-h course of HDF or HD, fibrinogen concentration and FXIII levels gradually elevated. When compensated for the change in plasma water, i.e., normalized for plasma albumin concentration, only FXIII elevation remained significant. There was no difference between HDF and HD treatments. Individual HDF treatment did not influence &alpha, 2PI activity, however after normalization it decreased significantly. HD treatment had a different effect, &alpha, 2PI activities became elevated but the elevation disappeared after normalization. Elevated fibrinogen and FXIII levels in ESRD patients might contribute to the increased thrombosis risk, while decreased &alpha, 2PI activity might be associated with elevated fibrinolytic potential.

One of the most abundant coagulation proteins is β

One of the most abundant coagulation proteins is β2-glycoprotein I (β2GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) β2GPI and converted it into an open conformation. The effectiveness of these procedures was assessed by Western blot and negative-staining electron microscopy. We found that in coagulation assays the open form of β2GPI had a significant prolonging effect on fibrin formation in a dilute prothrombin time test (p

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.

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.

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-A2B2) antigens were determined by ELISA. The causative mutation was detected by fluorescent DNA sequencing. The binding of alloantibody to FXIII-A2 and FXIII-A2B2 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-A2B2 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-A2B2 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.

Coagulation factor XIII (FXIII) is a heterotetramer consisting of 2 catalytic A subunits (FXIII-A2) and 2 protective/inhibitory B subunits (FXIII-B2). FXIII-B, a mosaic protein consisting of 10 sushi domains, significantly prolongs the lifespan of catalytic subunits in the circulation and prevents their slow progressive activation in plasmatic conditions. In this study, the biochemistry of the interaction between the 2 FXIII subunits was investigated. Using a surface plasmon resonance technique and an enzyme-linked immunosorbent assay-type binding assay, the equilibrium dissociation constant (Kd) for the interaction was established in the range of 10(-10) M. Based on the measured Kd, it was calculated that in plasma approximately 1% of FXIII-A2 should be in free form. This value was confirmed experimentally by measuring FXIII-A2 in plasma samples immunodepleted of FXIII-A2B2. Free plasma FXIII-A2 is functionally active, and when activated by thrombin and Ca(2+), it can cross-link fibrin. In cerebrospinal fluid and tears with much lower FXIII subunit concentrations, >80% of FXIII-A2 existed in free form. A monoclonal anti-FXIII-B antibody that prevented the interaction between the 2 subunits reacted with the recombinant combined first and second sushi domains of FXIII-B, and its epitope was localized to the peptide spanning positions 96 to 103 in the second sushi domain.

Coagulation factor XIII (FXIII) is converted by thrombin and Ca2+ into an active transglutaminase (FXIIIa) in the final phase of coagulation cascade. Its main function is the mechanical stabilization of fibrin clot and its protection from fibrinolysis by cross-linking of fibrin chains and α2-plasmin inhibitor to fibrin. In non-substituted patients FXIII deficiency is a severe hemorrhagic diathesis, not infrequently with fatal consequences. The main reason for using FXIII assays is the diagnosis of FXIII deficiency. The aim of this review is to provide a comprehensive critical evaluation of the methods reported for the determination of FXIII activity in the plasma. Such methods are based on two principles: 1) measurement of labeled amines incorporated by FXIIIa into a glutamine residue of a substrate protein, 2) monitoring ammonia released from a peptide bound glutamine residue by FXIIIa using NAD(P)H dependent glutamate dehydrogenase indicator reaction. The incorporation assays are sensitive, but cumbersome and time-consuming, they are difficult to standardize and cannot be automated. The ammonia release assays are less sensitive, but quick, well standardized, and can be automated; this type of assay is recommended for the screening of FXIII deficiency. The traditional clot solubility assay should not be used for this purpose.

Summary. 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 α2-plasmin inhibitor (α2PI) molecules become truncated by a plasma protease, and the truncated isoform (N1-α2PI) is an important substrate of FXIII-A2*. The crosslinking of N1-α2PI 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-α2PI(1–12), the sequence of which corresponds to the N-terminal sequence of N1-α2PI. Kinetic parameters for N1-α2PI(1–12) and for its truncated or synthetic mutants were determined by a spectrophotometric assay. The interaction of N1-α2PI(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-α2PI(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-α2PI(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.

Background Von Hippel-Lindau disease is an autosomal dominantly inherited highly penetrant tumor syndrome predisposing to retinal and central nervous system hemangioblastomas, renal cell carcinoma and phaeochromocytoma among other less frequent complications. Methods Molecular genetic testing of the VHL gene was performed in five unrelated families affetced with type I VHL disease, including seven patients and their available family members. Results Molecular genetic investigations detected three novel (c.163 G > T, c.232A > T and c.555C > A causing p.Glu55X, p.Asn78Tyr and p.Tyr185X protein changes, respectively) and two previously described (c.340 + 1 G > A and c.583C > T, resulting in p.Gly114AspfsX6 and p.195GlnX protein changes, respectively) germline point mutations in the VHL gene. Molecular modeling of the VHL-ElonginC-HIF-1alpha complex predicted that the p.Asn78Tyr amino acid exchange remarkably alters the 77-83 loop structure of VHL protein and destabilizes the VHL-HIF-1alpha complex suggesting that the mutation causes type I phenotype and has high risk to associate to renal cell carcinoma. The novel p.55X nonsense mutation associated to bilateral RCC and retinal angioma in a 15-year-old male patient. Conclusion We describe the earliest onset renal cell carcinoma in VHL disease reported so far in a 15-year-old boy with a nonsense VHL mutation. Individual tailoring of screening schedule based on molecular genetic status should be considered in order to diagnose serious complications as early as possible. Our observations add to the understanding of genotype-phenotype correlation in VHL disease and can be useful for genetic counseling and follow-up of VHL patients.