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BACKGROUND: Factor XIII (FXIII) promotes fibrin crosslinking and red blood cell (RBC) retention in clots. The FXIII-A polymorphism, Val34Leu, is associated with protection against venous thrombosis. This effect is hypothesized to result from fibrinogen concentration-dependent changes in fibrin structure. Effects of the FXIII-A Val34Leu polymorphism in whole blood clots have not been investigated. AIM: Characterize effects of FXIII-A Val34Leu polymorphism and fibrinogen on whole blood clots. METHODS: We isolated platelet-poor plasmas from human donors (FXIII(Val/Val), FXIII(Val/Leu), FXIII(Leu/Leu)), reconstituted plasmas with platelets and RBCs, and triggered clotting. We assessed contributions of gender, age, clotting times, thrombin generation, FXIII activity, FXIII-A Val34Leu polymorphism, and fibrinogen to clot mass. We also reconstituted FXIII-depleted plasma with platelets, RBCs, and purified FXIII(Val/Val) or FXIII(Leu/Leu), varied fibrinogen, and characterized effects on clot mass. RESULTS: Clot mass was associated with age, fibrinogen, prothrombin time, and thrombin generation. Clots reconstituted with plasmas from individuals with FXIII-A(Val/Val) and FXIII-A(Val/Leu) did not differ in mass from clots with FXIII-A(Leu/Leu). However, clots containing a 34Val allele demonstrated a fibrinogen concentration-dependent increase in mass, whereas clots with homozygous 34Leu did not. In plasmas with high fibrinogen, mass was higher for clots with 34Val alleles compared to clots with homozygous 34Leu. In clots reconstituted with purified FXIII, increasing fibrinogen enhanced clot mass in the presence of 34Val, but decreased mass in the presence of 34Leu. CONCLUSIONS: FXIII 34Leu mitigates the effect of elevated fibrinogen on whole blood clot mass. The Val34Leu polymorphism may protect against venous thrombosis by reducing clot mass.

The ever-increasing research efforts to develop new antithrombotic therapies have led to the reassessment of the role of alpha-2-plasmin inhibitor (α2-PI) in pathological conditions. In particular, experimental stroke studies have suggested correlation between increased free α2-PI level and mortality. However there are only a small number of well-characterized and specific assays available for the measurements of free α2-PI. In plasma α2-PI undergoes both N- and/or C-terminal cleavages resulting four isoforms with modified susceptibility to FXIII catalyzed cross-linking to fibrin and/or loss of plasmin(ogen) binding. Present paper describes a new sandwich ELISA method for the determination of free total α2-PI in plasma and other body fluids. A newly generated biotinylated monoclonal antibody recognizes and captures all the four N- and/or C-terminally modified isoforms of α2-PI while HRPO-labeled polyclonal anti-α2-PI antibody detects the captured antigen. Performing the 2-step assay in streptavidin-coated microplate can be completed within three hours. The assay is well reproducible, total (within laboratory) imprecision in the normal, pathological and very low ranges were 7.4%, 9.1% and In conclusion, our ELISA enables accurate and fast measurement of total free α2-PI in human body fluids.

Introduction Alpha2-plasmin inhibitor (α2-PI) has a heterogeneous composition in the plasma. Both N- and C-terminal cleavages occur that modify the function of the molecule. C-terminal cleavage converts the plasminogen-binding form (PB-α2-PI) to a non-plasminogen-binding form (NPB-α2-PI). N-terminal cleavage by soluble fibroblast activation protein (sFAP) results in a form shortened by 12 amino acids, which is more quickly cross-linked to fibrin. The p.Arg6Trp polymorphism of α2-PI affects N-terminal cleavage. In this work, we aimed to investigate the association between α2-PI heterogeneity and the risk of venous thromboembolism. Materials and methods Two hundred and eighteen patients with venous thromboembolism (VTE) and the same number of age and sex-matched healthy controls were enrolled. Total-α2-PI, PB-α2-PI and NPB-α2-PI antigen levels, α2-PI activity, sFAP antigen levels and p.Arg6Trp polymorphism were investigated. Results Total-α2-PI and NPB-α2-PI levels were significantly elevated in VTE patients, while PB-α2-PI levels did not change. Elevated NPB-α2-PI levels independently associated with VTE risk (adjusted OR: 9.868; CI: 4.095–23.783). Soluble FAP levels were significantly elevated in the VTE group, however, elevated sFAP levels did not show a significant association with VTE risk. The α2-PI p.Arg6Trp polymorphism did not influence VTE risk, however, in the case of elevated sFAP levels the carriage of Trp6 allele associated with lower VTE risk. Conclusion Our results showed that the elevation of total-α2-PI levels in VTE is caused by the elevation of NPB-α2-PI levels. Elevated sFAP level or p.Arg6Trp polymorphism alone did not influence VTE risk. However, an interaction can be detected between the polymorphism and high sFAP levels.

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Introduction Inherited antithrombin (AT) deficiency is a heterogeneous disease. Due to low prevalence, only a few studies are available concerning genotype-phenotype associations. The aim was to describe the clinical, laboratory and genetic characteristics of AT deficiency in a large cohort including children and to add further laboratory data on the different sensitivity of functional AT assays. Patients and methods Non-related AT deficient patients (n = 156) and their family members (total n = 246) were recruited. Clinical and laboratory data were collected, the mutation spectrum of SERPINC1 was described. Three different AT functional assays were explored. Results Thirty-one SERPINC1 mutations including 11 novel ones and high mutation detection rate (98%) were detected. Heparin binding site deficiency (type IIHBS) was the most frequent (75.6%) including AT Budapest3 (ATBp3), AT Padua I and AT Basel (86%, 9% and 4% of type IIHBS, respectively). Clinical and laboratory phenotypes of IIHBS were heterogeneous and dependent on the specific mutation. Arterial thrombosis and pregnancy complications were the most frequent in AT Basel and AT Padua I, respectively. Median age at the time of thrombosis was the lowest in ATBp3 homozygotes. The functional assay with high heparin concentration and pH 7.4 as assay conditions had low (44%) sensitivity for ATBp3 and it was insensitive for AT Basel and Padua I. Conclusion Type IIHBS deficiencies behave differently in clinical and laboratory phenotypes from each other and from other AT deficiencies. Heparin concentration and pH seem to be the key factors influencing the sensitivity of AT functional assays to IIHBS.

Aims. To identify intracardiac hemostasis or fibrinolysis abnormalities, which are associated with atrial fibrillation (AF) and increase the risk of thromboembolism.Patients and Methods. Patient group consisted of 24 patients with AF and control group included 14 individuals with other supraventricular tachycardia undergoing transcatheter radiofrequency ablation. Blood samples were drawn from the femoral vein (FV), left atrium (LA), and left atrial appendage (LAA) before the ablation procedure. Fibrinogen, factor VIII (FVIII) and factor XIII activity, von Willebrand factor (VWF) antigen, thrombin-antithrombin (TAT) complex, quantitative fibrin monomer (FM), plasminogen,α2-plasmin inhibitor, plasmin-α2-antiplasmin (PAP) complex, PAI-1 activity, and D-dimer were measured from all samples.Results. Levels of FVIII and VWF were significantly elevated in the FV and LA of AF patients as compared to controls. TAT complex, FM, PAP complex, and D-dimer levels were significantly elevated in the LA as compared to FV samples in case of both groups, indicating a temporary thrombotic risk associated with the catheterization procedure.Conclusions. None of the investigated hemostasis or fibrinolysis parameters showed significant intracardiac alterations in AF patients as compared to non-AF controls. AF patients have elevated FVIII and VWF levels, most likely due to endothelial damage, presenting at both intracardiac and systemic level.

Background The regulation of plasma factor XIII (FXIII) levels in healthy individuals has been only partially explored. The identification of major non-genetic and genetic regulatory factors might provide important information on the contribution of FXIII to the risk of cardio/cerebrovascular diseases. Objectives To determine the effect of age, smoking, BMI, fibrinogen concentration on plasma FXIII activity, complex FXIII antigen (FXIII-A 2 B 2 ) and total FXIII-B subunit (tFXIII-B) level, to correlate FXIII-B level with the other two FXIII parameters and to assess the variation of FXIII levels in carriers of major FXIII subunit polymorphisms. Methods 268 healthy individuals were enrolled in the study. FXIII activity was measured by the ammonia release assay; FXIII-A 2 B 2 and tFXIII-B were determined by ELISAs. FXIII-A p.Val34Leu, FXIII-B p.His95Arg and FXIII-B intron K c.1952 + 144 C > G polymorphisms were identified by RT-PCR using melting point analysis with fluorescence resonance energy transfer detection. Results All investigated FXIII parameters showed significant positive correlation with age and fibrinogen level; gender and BMI influenced only tFXIII-B. A highly significant positive correlation was demonstrated between tFXIII-B and the other FXIII parameters. FXIII-A p.Val34Leu polymorphism had only slight, if any effect on FXIII levels. The FXIII-B Arg95 allele moderately increased all three FXIII parameters, but the effect became statistically significant only after adjustment. The FXIII-B intron K G allele drastically decreased FXIII levels, and it seemed to be in synergism with the FXIII-A Leu34 allele. Conclusions Plasma FXIII levels are subjected to multifactorial regulation, in which age, fibrinogen level and FXIII-B intron K polymorphism are major determinants.

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.

The protective/inhibitory B subunits of coagulation factor XIII (FXIII-B) is a ~80 kDa glycoprotein containing two N-glycosylation sites. Neither the structure nor the functional role of the glycans on FXIII-B has been explored.To reveal the glycan structures linked to FXIII-B, to design a method for deglycosylating the native protein, to find out if deglycosylation influences the dimeric structure of FXIII-B and its clearance from the circulation.Asparagine-linked carbohydrates were released from human FXIIII-B by PNGase F digestion. The released N-linked oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis. Structural identification utilized glycan database search and exoglycosidase digestion based sequencing. The structure of deglycosylated FXIII-B was investigated by gel filtration. The clearance of deglycosylated and native FXIII-B from plasma was compared in FXIII-B knock out mice.PNGase F completely removed N-glycans from the denatured protein. Deglycosylation of the native protein was achieved by repeated digestion at elevated PNGase F concentration. The total N-glycan profile of FXIII-B featured nine individual structures; three were fucosylated and each structure contained at least one sialic acid. Deglycosylation did not change the native dimeric structure of FXIII-B, but accelerated its clearance from the circulation of FXIII-B knock out mice.Characterization of the glycan moieties attached to FXIII-B is reported for the first time. Complete deglycosylation of the native protein was achieved by a deglycosylation workflow. The associated glycan structure is not required for FXIII-B dimer formation, but it very likely prolongs the half-life of FXIII-B in the plasma.

The A subunit of blood coagulation factor XIII belongs to the family of transglutaminase enzymes. Its active form (FXIIIa) catalyzes isopeptide bond formation between Glu and Lys residues of specific substrates. Little data are available on the mechanism of this reaction. In this work, the first step of the proposed two-step process was investigated using two different protocols of hybrid QM/molecular mechanics (MM) calculations: an ONIOM-based model as well as QM/MM/molecular dynamics (MD) metadynamics simulations in explicit TIP3P solvent with Gromacs, PLUMED, and a DFTB3 package. Based on calculations involving a truncated system derived from docking of a peptide substrate, our study confirms the higher stability of a zwitterionic form of the catalytic Cys and His residues in the Michaelis complex as well as the "resting" state of the enzyme. Potential energy surfaces, obtained by geometry optimizations with Gaussian, show a two-step reaction mechanism with a zwitterionic tetrahedral intermediate formation in the first and NH

BACKGROUND: Factor XIII (FXIII) is a precursor of the blood plasma transglutaminase (FXIIIa) that is generated by thrombin and Ca(2+) and covalently cross-links fibrin to strengthen blood clots. Inactive plasma FXIII is a heterotetramer with two catalytic A subunits and two non-catalytic B subunits. Inactive A subunits have been characterized crystallographically, whereas the atomic structure of the entire FXIII and B subunits is unknown and the oligomerization state of activated A subunits remains controversial. OBJECTIVES: Our goal was to characterize the (sub)molecular structure of inactive FXIII and changes upon activation. METHODS: Plasma FXIII, non-activated or activated with thrombin and Ca(2+), was studied by single-molecule atomic force microscopy. Additionally, recombinant separate A and B subunits were visualized and compared with their conformations and dimensions in FXIII and FXIIIa. RESULTS AND CONCLUSIONS: We showed that heterotetrameric FXIII forms a globule composed of two catalytic A subunits with two flexible strands comprising individual non-catalytic B subunits that protrude on one side of the globule. Each strand corresponds to seven to eight out of 10 tandem repeats building each B subunit, called sushi domains. The remainder were not seen, presumably because they were tightly bound to the globular A(2) dimer. Some FXIII molecules had one or no visible strands, suggesting dissociation of the B subunits from the globular core. After activation of FXIII with thrombin and Ca(2+), B subunits dissociated and formed B(2) homodimers, whereas the activated globular A subunits dissociated into monomers. These results characterize the molecular organization of FXIII and changes with activation.

The ever-increasing research efforts to develop new antithrombotic therapies have led to the reassessment of the role of alpha-2-plasmin inhibitor (α

BACKGROUND Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. METHODS The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. RESULTS EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. CONCLUSIONS EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society.

In this observational study we investigated whether levels of factor XIII (FXIII) and its major polymorphisms affect the outcome of thrombolysis by recombinant tissue plasminogen activator (rtPA) in acute ischemic stroke (AIS) patients. Study cohort included 132 consecutive AIS patients undergoing i.v. thrombolysis within 4.5 h of symptom onset. Blood samples taken on admission, immediately after and 24 h after therapy were analyzed for FXIII activity and antigen levels. FXIII-A p.Val34Leu, p.Tyr204Phe, FXIII-B p.His95Arg and intron K(IVS11 + 144) polymorphisms were genotyped. Neurological deficit was assessed using the National Institutes of Health Stroke Scale. Intracranial hemorrhage was classified according to ECASSII criteria. Long-term functional outcome was defined at 3 months post-event by the modified Rankin scale. FXIII levels showed a gradual decrease immediately after thrombolysis and 24 h later, which was not related to therapy-associated bleeding. In a multiple logistic regression model, a FXIII level in the lowest quartile 24 h post-lysis proved to be an independent predictor of mortality by 14 days post-event (OR:4.95, 95% CI:1.31–18.68, 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.

Dual antiplatelet therapy with clopidogrel and aspirin is frequently used for the prevention of recurrent ischemic events. Various laboratory methods are used to detect the effect of these drugs administered in monotherapy, however their value in dual therapy has not been explored. Here, we determined which methods used for testing the effect of clopidogrel or aspirin are influenced by the other antiplatelet agent. One arm of the study included 53 ischemic stroke patients being on clopidogrel monotherapy showing effective inhibition of the P2Y12 ADP receptor. Laboratory tests routinely used for the detection of aspirin resistance (arachidonic acid (AA)-induced platelet aggregation/secretion, AA-induced thromboxane B2 (TXB2) production in platelet-rich plasma and VerifyNow Aspirin assay) were carried out on samples obtained from these patients. The other arm of the study involved 52 patients with coronary artery disease being on aspirin monotherapy. Methods used for testing the effect of clopidogrel (ADP-induced platelet aggregation and secretion, flow cytometric analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation and a newly developed P2Y12-specific platelet aggregation (ADP[PGE1] test)) were performed on samples obtained from these patients. Clopidogrel monotherapy significantly inhibited AA-induced platelet aggregation and secretion, moreover, AA-induced TXB2 production was also significantly decreased. VASP phosphorylation and AA-induced platelet aggregation showed fair correlation in patients taking clopidogrel only. Clopidogrel did not inhibit the VerifyNow Aspirin test significantly. Aspirin monotherapy influenced ADP-induced platelet aggregation and secretion, but did not have an effect on VASP phosphorylation and on the ADP[PGE1] platelet aggregation test.

In a large proportion of patients with mild bleeding disorders (MBDs) no diagnosis can be established by routine coagulation tests. We investigated whether alterations in plasma clot properties account for MBDs of unknown cause. Ninety-five patients with MBDs of unknown origin and 98 age- and sex-matched healthy controls were investigated. Furthermore, data of 25 patients with a deficiency of factor VIII were analyzed. Plasma clot characteristics in the absence and presence of recombinant tissue plasminogen activator (rtPA) represented by the lag phase, rate of protofibril formation (Vmax), fibrin structure (ΔAbs), time to peak (TTP), half lysis time (t50 and area under the curve (AUC) were measured in turbidometric clot formation and lysis assays. In the fibrinolysis assay, Vmax was lower in patients than in healthy controls. No differences in the other parameters of clot formation and lysis were detected between the groups. There was no clear association of plasma clot properties with the clinical severity of bleeding in patients with MBDs. Patients with known decreased factor VIII levels also showed a lower Vmax. Fibrinogen levels were positively associated with each of the assessed parameters in both groups, with the strongest association with ΔAbs, indicating altered fibrin structure. Factor VIII activity correlated with altered clot characteristics similar to fibrinogen, especially in patients, with the strongest positive correlation to Vmax. This cohort of patients with MBDs of unknown origin showed a lower rate of fibrin formation in the fibrinolysis assay, but otherwise similar plasma clot properties compared to healthy controls.

The aim of the case-control study was to explore the effect of coagulation factor XIII (FXIII) B subunit (FXIII-B) polymorphisms on the risk of coronary artery disease, and on FXIII levels. In the study, 687 patients admitted for coronary angiography to investigate suspected coronary artery disease and 994 individuals representing the Hungarian population were enrolled. The patients were classified according to the presence of significant coronary atherosclerosis (CAS) and history of myocardial infarction (MI). The F13B gene was genotyped for p.His95Arg and for intron K nt29756 C&gt, G polymorphisms, the latter results in the replacement of 10 C-terminal amino acids by 25 novel amino acids. The p.His95Arg polymorphism did not influence the risk of CAS or MI. The FXIII-B intron K nt29756 G allele provided significant protection against CAS and MI in patients with a fibrinogen level in the upper tertile. However, this effect prevailed only in the presence of the FXIII-A Leu34 allele, and a synergism between the two polymorphisms was revealed. Carriers of the intron K nt29756 G allele had significantly lower FXIII levels, and FXIII levels in the lower tertile provided significant protection against MI. It is suggested that the protective effect of the combined polymorphisms is related to decreased FXIII levels.

Blood coagulation factor XIII (FXIII) is essential for maintaining hemostasis. The absence of FXIII results in severe bleeding diathesis, which without prophylaxis frequently leads to fatal bleeding. As the usual hemostasis screening tests remain normal, the diagnosis of FXIII deficiency needs specific tests. Here, we describe FXIII activity determination by the ammonia release assay, which is the first-line test in the diagnostic algorithm for FXIII deficiency. The method for another activity test, the undeservedly rarely used fibrin cross-linking assay, is also presented. Further tests used for the classification of FXIII deficiencies, measurement of FXIII activity in platelets, ELISAs for the measurement of complex plasma FXIII (FXIII-A

Background The association of plasma factor XIII (FXIII) level with venous thromboembolism (VTE) is still controversial and the effect of sex and FXIII B subunit (FXIII-B) polymorphisms in this respect have not been explored. Objectives 1/ To determine FXIII activity and antigen levels in patients with a history of VTE and how they are influenced by sex and FXIII-B polymorphisms. 2/ To explore the association of FXIII levels and FXIII-B polymorphisms with the risk of VTE. Methods 218 VTE patients and equal number of age and sex matched controls were enrolled in the study. FXIII activity was measured by ammonia release assay; FXIII-A2B2 and FXIII-B levels were determined by ELISAs. FXIII-B polymorphisms were identified by RT-PCR using melting point analysis. Results Adjusted FXIII activity and FXIII-A2B2 antigen levels were significantly higher in females with a history of VTE than in the respective controls. FXIII-B levels were significantly lower in male VTE patients than in controls. FXIII-A2B2 antigen levels in the upper tertile increased the risk of VTE in females (adjusted OR: 2.52; CI: 1.18–5.38). Elevated FXIII-B antigen level had a protective effect only in males (adjusted OR: 0.19; CI: 0.08–0.46). FXIII-B Intron K c.1952 + 144 C > G polymorphism significantly lowered FXIII activity, FXIII-A2B2 and FXIII-B antigen levels in both groups. FXIII-B polymorphisms did not influence the risk of VTE. Conclusions In VTE patients the changes of FXIII level and their effect on the risk of VTE show considerable sex-specific differences. Intron K polymorphism results in decreased FXIII levels, but does not influence the risk of VTE.

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.

The overall incidence of thromboembolic events in the neonatal period is 5 per 100 000 births, wherein more than 40% of all such manifestations are symptomatic renal vein thromboses. We describe the case of a newborn female who developed extensive thrombosis, which filled the inferior vena cava and renal vein and was diagnosed in the first weeks of life. A homozygous type II heparin-binding site antithrombin deficiency (c. 391C>T, p. Leu131Phe) was detected in the background. Despite the timely diagnosis and appropriate treatment, clinical signs of renal insufficiency, because of left kidney atrophy and arterial hypertension, were observed. Our case demonstrates the seriousness of the consequences arising after early onset of venous thrombosis caused by homozygous type II heparin-binding site antithrombin deficiency. In addition to prompt diagnosis, of huge importance is the determination of inherited thrombophilia, as it significantly affects therapeutic treatment and indicates that long-term follow-up is mandatory.

Background Aspirin, a commonly used antiplatelet agent, blocks platelet thromboxane A 2 (TXA 2 ) formation from arachidonic acid (AA) by acetylating platelet cyclooxygenase-1 (COX-1). Laboratory methods currently used to detect this antiplatelet effect of aspirin provide variable results. We have reported three methods that assess platelet COX-1 acetylation (inactivation) by aspirin and its direct consequences. The first and second assays use monoclonal anti-human-COX-1 antibodies that only detect acetylated (inactivated) COX-1 and active (non-acetylated) COX-1, respectively. The third method measures platelet production of TXB 2 (the stable metabolite of TXA 2 ) in vitro in response to AA. We compared the results of these three reference methods with other routinely used methods for assessing the functional consequences aspirin treatment. Methods 108 healthy volunteers were treated with low-dose aspirin for 7days. On day 7 following aspirin treatment COX-1 in the platelets was fully acetylated whereas only non-acetylated COX-1 was present in the day 0 platelets. Further, TXB 2 production by day 7 platelets was completely blocked. The following tests were performed on the samples obtained from study participants before and after seven days of aspirin treatment: PFA-100 closure time with collagen/epinephrine cartridge, VerifyNow® (VN) Aspirin Assay, platelet aggregation and ATP secretion using AA, ADP, epinephrine and collagen as agonists. Results Comparing the pre-treatment and day 7 values, methods that use AA as platelet agonist (AA-induced platelet aggregation/secretion and VN Aspirin Assay) showed high discriminative power. In contrast, results of the other tests showed considerable overlap between day 7 and day 0 values. Conclusions Only assays that clearly distinguish between acetylated and non-acetylated platelet COX-1 are useful for establishing the antiplatelet effect of aspirin. The other tests are not suitable for this purpose.

Introduction Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. Materials and methods Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. Results Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans‑Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. Conclusions AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

Background Aspirin is widely used in the prevention of acute atherothrombotic complications. It acetylates Ser529 residue in cyclooxygenase-1 (COX-1) and prevents thromboxane A2 (TXA2) formation from arachidonic acid (AA) in platelets. Laboratory methods used for the detection of aspirin effect provide inconsistent results. Methods Two new methods were developed for the direct and indirect detection of COX-1 acetylation by aspirin in 108 healthy volunteers treated daily with 100 mg enteric-coated aspirin for 7 days. Monoclonal antibodies were raised against acetylated and non-acetylated nonapeptides corresponding to the amino acid sequence of human COX-1 525–533 residues. Using Western blotting technique the antibodies clearly distinguished between acetylated and non-acetylated COX-1 in platelet lysate. The second method measures AA-induced TXB2 production of platelets in diluted platelet rich plasma. Results No acetylated COX-1 was detected in platelets before aspirin treatment. At the same time antibodies raised against non-acetylated peptide gave intense reaction with COX-1 on the Western blot. In contrast, after 7 days of aspirin treatment, with a single exception, only acetylated COX-1 could be detected in the platelet lysate. The non-responding volunteer showed full response to aspirin after controlled drug intake. In parallel experiments aspirin treatment for 7 days practically completely inhibited AA-induced TXB2 production by platelets. Conclusions Chemical (“true”) aspirin resistance, if it exists, must be a rarity among healthy individuals. The new methods could be used for detecting the acetylation of COX-1 by aspirin in patients on preventive aspirin therapy and for evaluating methods routinely used for such purpose.

Acetylsalicylic acid (aspirin) suppresses the generation of prostaglandin H2, which is the precursor of thromboxane A2. Aspirin acts as an acetylating agent in which its acetyl group is covalently attached to a serine residue (S530) in the active site of the cyclooxygenase-1 enzyme. The exact reaction mechanism has not been revealed by experimental methods. In this study the putative structure of human cyclooxygenase-1 was constructed from ovine cyclooxygenase-1 by homology modeling, and the acetylsalicylic acid was docked into the arachidonic acid binding cavity of the enzyme. To characterize the shape of the potential energy surface of the acetylating reaction and to determine the relative energies of the stationary points on the surface, a series of ONIOM-type quantum mechanical/molecular mechanical (QM/MM) calculations were carried out at different QM levels of theories applying electronic embedding approximations. The acetylsalicylic acid and the surrounding amino acids were included in these calculations. Frequency analyses were performed to prove the existence of first order saddle points (representing transition states) and local minima on the potential energy surface. It was found that all levels of theories predicted similar transition state geometries. The activation energy values, however, demonstrated significant dependence on the methods that were applied. All the applied "dependable" ab initio and DFT methods predicted that the breakage of the S530 Oγ--Hγ and formation of the Oγ--C(acetylsalicylic acid carbonyl) bonds occur in a single elementary step.

Inherited deficiency of FXIII A subunit (FXIII-A) is a rare (1:2,000,000) but very severe bleeding diathesis. The incidence is much higher in communities where the practice of consanguineous marriage is combined with founder effect mutation. Because of the high risk of intracranial bleeding, life-long prophylaxis, preferably using FXIII concentrate, is mandatory. In FXIII-B subunit deficiency the bleeding diathesis is mild to moderate. FXIII deficiency is frequently associated with impaired wound healing. Women suffering from FXIII deficiency cannot carry pregnancies to term; in severe cases spontaneous abortion occurs in the first trimester. Plasma-derived heat-inactivated FXIII concentrate and recombinant FXIII-A are available for prophylaxis; a 4 weekly dose of 35 to 40 U/kg is recommended and a trough level of greater than 5% FXIII activity should be aimed for. During pregnancy, 2 weekly prophylaxis with a target trough level of greater than 10% is recommended, and during labor FXIII activity should exceed 30%. During surgical procedures, the target should be higher than 50% FXIII activity. Alloantibodies make FXIII deficiency difficult to manage, but fortunately they are extremely rare. Acquired FXIII deficiency may involve both subunits. Autoantibodies against FXIII subunits also manifest in severe bleeding complication with a relatively high mortality rate. The first-line test in the diagnosis of FXIII deficiency should be a quantitative functional assay based on the measurement of ammonia release or amine incorporation. The sensitivity of the traditional clot solubility assay is not sufficiently robust to enable proper screening. Antigen assays are needed for the classification of FXIII deficiencies. In the case of anti-FXIII antibodies, the diagnostic armory should be supplemented by a mixing test/Bethesda-type inhibitor assay and by assays that detect/measure the binding of antibodies to FXIII and to its subunits.

Background Antithrombin (AT) is a key regulator of the coagulation. In type II deficiency, the heparin-binding-site defect (type II HBS) is considered to be relatively low thrombosis risk. Objectives Our aims were to search for SERPINC1 mutation(s) and to describe the clinical and laboratory phenotype of a large number of AT Budapest3 (ATBp3, p.Leu131Phe) carriers and confirm the presence of a founder effect. Patients/methods AT-deficient patients were recruited and carriers of ATBp3, n = 102 (63 families) were selected. To investigate the founder effect, eight intragenic single nucleotide polymorphisms, a 5'-length dimorphism, and five microsatellite markers were detected. Clinical and laboratory data of the patients were collected and analyzed. Results In AT deficiency, 16 different causative mutations were found, and the great majority of patients were of type II HBS subtype. Most of them (n = 102/118, 86.5%) carried the ATBp3 mutation. The ATBp3 mutant allele was associated with one single haplotype, while different haplotypes were detected in the case of normal allele. The anti-factor Xa-based AT activity assay that we used could detect all ATBp3 patients with high sensitivity in our cohort. ATBp3 homozygosity (n = 26) was associated with thrombosis at a young age and conferred a high thrombotic risk. Half of the heterozygotes (n = 41/76, 53.9%) also had venous and/or arterial thrombosis, and pregnancy complications were also recorded. Conclusion In Hungary, the founder mutation, ATBp3, is the most common AT deficiency. Our study is the first in which the clinical characterization of ATBp3 mutation was executed in a large population.

Introduction It has been shown that thrombomodulin (TM) considerably delays factor XIII (FXIII) activation and this effect is abrogated by Factor V Leiden (FV Leiden ) mutation. The aim of the study was to explore the effect of TM on the cross-linking of α 2 -plasmin inhibitor (α 2 -PI) to fibrin in plasma samples of different FV genotypes and how this effect is related to the impaired fibrinolysis of FV Leiden carriers. Methods In the plasma samples of fifteen individuals with different FV genotypes and in FV deficient plasma supplemented with wild type FV or FV Leiden coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human TM (rhTM). In the recovered clots the extent of α 2 -PI-fibrin cross-linking was evaluated by Western blotting and quantitative densitometry. The effect of rhTM on tissue plasminogen activator (tPA) induced clot lysis was measured by turbidimetric method. Results rhTM significantly delayed the formation of α 2 -PI-fibrin α-chain heterodimers/oligomers in plasma samples containing wild type FV. This effect of rhTM was impaired in the presence of FV Leiden . rhTM delayed tPA-induced clot lysis and this effect of rhTM was more pronounced in plasma containing FV Leiden . When TAFIa was inhibited by potato carboxypeptidase inhibitor, rhTM accelerated clot lysis in the presence of wild type FV, which is explained by the delayed α 2 -PI-fibrin cross-linking. This effect of rhTM did not prevail in the presence of FV Leiden . Conclusion FV Leiden abrogates the delaying effect of rhTM on α 2 -PI-fibrin cross-linking, which contributes to the impaired fibrinolysis observed in FV Leiden carriers.

Leukemic cells often express markers, which are not characteristic of their particular cell lineage. In this study, we identified the “A” subunit of coagulation factor XIII (FXIII-A) in leukemic promyelocytes in de novo AML M3 cases. The cytoplasmic presence of factor XIII-A has previously been shown only in platelets/megakaryocytes and monocytes/macrophages. Furthermore, more recently we described the presence of FXIII-A in leukemic lymphoblasts. We studied 14 patients with this rare type of acute leukemia in a period of 4 years and investigated their bone marrow samples by 3-color flow cytometry upon diagnosis, mainly focusing on FXIII-A expression of leukemic cells. We detected FXIII-A also by ELISA, Western-blot, and confocal laser scanning microscopy. This was a homogenous group of AML M3 patients with translocation t(15;17)(q22;q21) detected by fluorescence in situ hybridization (FISH). In 10 out of 14 samples, FXIII-A was detectable by flow cytometry and was coexpressed with markers characteristic for leukemic promyleocytes (CD45dim/CD13+/CD33+/CD117+/cyMPO+ and HLA-DR-/CD34−/CD14−/CD15−). Staining for the markers GPIIb and GPIX were negative, and FXIII-A was identified in the cytoplasm of the cells by confocal microscopy in a relatively high quantity, as measured by ELISA. By Western blot analysis we could identify FXIII-A in the native 82 kDa form and in cleaved forms corresponding to cleavage products observed when purified FXIII-A was treated by human neutrophil elastase. This novel expression site of FXIII-A in AML M3 can be considered as a leukemia associated immunophenotype and may have pathophysiological significance. © 2012 International Clinical Cytometry Society