Enhancing HLA-B27 antigen detection: Leveraging machine learning algorithms for flow cytometric analysis.

As the association of human leukocyte antigen B27 (HLA-B27) with spondylarthropathies is widely known, HLA-B27 antigen expression is frequently identified using flow cytometric or other techniques. Because of the possibility of cross-reaction with off target antigens, such as HLA-B7, each flow cytometric technique applies a "gray zone" reserved for equivocal findings. Our aim was to use machine learning (ML) methods to classify such equivocal data as positive or negative. Equivocal samples (n = 99) were selected from samples submitted to our institution for clinical evaluation by HLA-B27 antigen testing. Samples were analyzed by flow cytometry and polymerase chain reaction. Features of histograms generated by flow cytometry were used to train and validate ML methods for classification as logistic regression (LR), decision tree (DT), random forest (RF) and light gradient boost method (GBM). All evaluated ML algorithms performed well, with high accuracy, sensitivity, specificity, as well as negative and positive predictive values. Although, gradient boost approaches are proposed as high performance methods; nevertheless, their effectiveness may be lower for smaller sample sizes. On our relatively smaller sample set, the random forest algorithm performed best (AUC: 0.92), but there was no statistically significant difference between the ML algorithms used. AUC values for light GBM, DT, and LR were 0.88, 0.89, 0.89, respectively. Implementing these algorithms into the process of HLA-B27 testing can reduce the number of uncertain, false negative or false positive cases, especially in laboratories where no genetic testing is available.

[Beta-thalassemia and vitamin B12 deficiency and associated complement-mediated hemolysis]. / Béta-thalassaemia, B12-vitamin-hiány és társuló komplementmediált hemolízis.

We present the case of a 67-year-old male patient admitted to our clinic due to weakness and repeated dizziness. Due to his severe microcytic anemia in his laboratory tests, he needed a transfusion of 6 units of selected blood in the days following admission. Our patient was diagnosed with beta-thalassemia minor, which was accompanied by a severe deficiency of vitamin B12. Surprisingly, parallel to vitamin B12 deficiency, we detected laboratory abnormalities indicating complement-mediated autoimmune hemolysis. After correcting the vitamin B12 deficiency, the patient's blood count improved, and the observed immunological abnormalities disappeared. Genetic testing of the hemoglobin gene confirmed the c.118C>T (p.Gln40STOP) variant in heterozygous form. Beta-thalassemia is a relatively common hematological disease, although rarely encountered in Hungary. Genetic testing of patients is possible at the Laboratory Medicine Institute of the Clinical Center in Debrecen. Unfortunately, we do not have accurate information about published domestic epidemiological data. Furthermore, establishing a diagnosis can be difficult if the disease is combined with other hematological disorders, such as the lack of vitamin B12, which can clinically mimic hemolytic anemia in certain features. Our case is considered a rarity in the literature, so in the case of a positive family history, it is recommended to screen immediate family members, which may facilitate the accurate establishment of a later diagnosis. Orv Hetil. 2023; 164(24): 954-960.

Gender-dependent frequency of unconventional T cells in a healthy adult Caucasian population: A combinational study of invariant NKT cells, γδ T cells, and mucosa-associated invariant T cells.

This study tested the hypothesis of gender bias in frequency of unconventional T cells. Unconventional T cells exist as minor subsets of T cells in peripheral blood. Despite their low number, they play a crucial role in various immune-mediated diseases such as inflammation, autoimmunity, allergy, and cancer. Gender-based frequency of these cells altogether on large number of healthy individuals are unestablished creating hurdles to manifest association with various immune-mediated pathologic conditions. In this study, we used a multicolor flow cytometric panel to identify iNKT cells, γδ T cells, and MAIT cells altogether in the peripheral blood samples of 93 healthy adult males and 109 healthy adult females from the Caucasian population. The results revealed iNKT cell median value (% T cells) in females was higher: 0.114% ranging from 0.011 to 3.84%, than males: 0.076% (p value 0.0292), ranging from 0.007 to 0.816% and found to be negatively correlated with age in females (p value 0.0047). However, γδ T cell median value in males was higher: 2.52% ranging from 0.31 to 16.09%, than females: 1.79% (p value 0.0155), ranging from 0.078 to 12.49% and each gender was negatively correlated with age (male p value 0.0003 and female p value 0.0007). MAIT cell median values were 3.04% ranging from 0.11 to 10.75% in males and 2.67% ranging from 0.2 to 18.36% in females. MAIT cells did not show any statistically significant difference between genders and found to be negatively correlated with age (p value < 0.0001). Our results could be used for further gender-wise investigations of various pathologic conditions such as cancer and their prognosis, autoimmune diseases, allergies, and their pathogenicity.

Age-dependent frequency of unconventional T cells in a healthy adult Caucasian population: a combinational study of invariant natural killer T cells, γδ T cells, and mucosa-associated invariant T cells.

Unconventional T cells show distinct and unique features during antigen recognition as well as other immune responses. Their decrease in frequency is associated with various autoimmune disorders, allergy, inflammation, and cancer. The landscape frequency of the unconventional T cells altogether (iNKT, γδ T, and MAIT) is largely unestablished leading to various challenges affecting diagnosis and research in this field. In this study, we have established the age group-wise frequency of iNKT, γδ T, and MAIT cells altogether on a total of 203 healthy adult samples of the Caucasian population. The results revealed that iNKT cells were 0.095%, γδ T cells were 2.175%, and MAIT cells were 2.99% of the total T cell population. γδ and MAIT cell frequency is higher in younger age groups than elderly; however, there is no statistically significant difference in the frequency of iNKT cells. Furthermore, γδ and MAIT cells were negatively correlating with age, supporting immunosenescence, unlike iNKT cells. Our finding could be used for further age-wise investigation of various pathological conditions such as cancer and their prognosis, autoimmune diseases and their pathogenicity.

Combined use of different antibody clones improves the efficiency of human leukocyte antigen B27 detection by flow cytometry.

BACKGROUND: Human leukocyte antigen B27 (HLA-B27) is an major histocompatibility complex Class I cell surface antigen that shows strong association with spondylarthropathies. Although polymerase chain reaction (PCR) is the gold standard method for HLA-B27 detection, monoclonal antibodies, and flow cytometric analysis is also frequently used. We aimed to compare the efficiency of two commercially available monoclonal antibody clones and the DuraClone kit that uses simultaneously these clones. METHODS: Blood samples drawn from 63 patients were analyzed by flow cytometry and PCR. For flow cytometry analysis ABCm3 and FD705 clones were used for flow cytometry as well as the DuraClone Reagent Kit. Results of flow cytometric analysis were confirmed by PCR. RESULTS: Numbers of false-positive and equivocal samples were high when ABCm3 or FD705 clones were used separately: 34 out of 63 and 27 out of 63, respectively. Simultaneous use of the two antibody clones and pre-selection of CD3+ T cells, in the DuraClone kit, significantly decreased the number of these samples. Using the DuraClone kit, only 11 out of 63 samples were inconclusive. Influence of HLA-B7 expression was detectable in some cases when ABCm3 and FD705 were used separately. CONCLUSIONS: Our results show that simultaneous use of HLA-B27 monoclonal antibodies and pre-selection of T cells significantly increased the specificity of the flow cytometric assay, however it did not reach the specificity of PCR. Nevertheless, based on our results, performing the PCR test exclusively in equivocal cases by DuraClone kit reduces the burden of PCR assays and the turn-around time.

Biochemical and Clinical Effects of Vitamin E Supplementation in Hungarian Smith-Lemli-Opitz Syndrome Patients.

Smith-Lemli-Opitz syndrome (SLOS) is a severe monogenic disorder resulting in low cholesterol and high 7-dehydrocholesterol (7-DHC) levels. 7-DHC-derived oxysterols likely contribute to disease pathophysiology, and thus antioxidant treatment might be beneficial because of high oxidative stress. In a three-year prospective study, we investigated the effects of vitamin E supplementation in six SLOS patients already receiving dietary cholesterol treatment. Plasma vitamin A and E concentrations were determined by the high-performance liquid chromatography (HPLC) method. At baseline, plasma 7-DHC, 8-dehydrocholesterol (8-DHC) and cholesterol levels were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The clinical effect of the supplementation was assessed by performing structured parental interviews. At baseline, patients were characterized by low or low-normal plasma vitamin E concentrations (7.19-15.68 µmol/L), while vitamin A concentrations were found to be normal or high (1.26-2.68 µmol/L). Vitamin E supplementation resulted in correction or significant elevation of plasma vitamin E concentration in all patients. We observed reduced aggression, self-injury, irritability, hyperactivity, attention deficit, repetitive behavior, sleep disturbance, skin photosensitivity and/or eczema in 3/6 patients, with notable individual variability. Clinical response to therapy was associated with a low baseline 7-DHC + 8-DHC/cholesterol ratio (0.2-0.4). We suggest that determination of vitamin E status is important in SLOS patients. Supplementation of vitamin E should be considered and might be beneficial.

Effect of α2-plasmin inhibitor heterogeneity on the risk of venous thromboembolism.

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.

Age and Origin of the Founder Antithrombin Budapest 3 (p.Leu131Phe) Mutation; Its High Prevalence in the Roma Population and Its Association With Cardiovascular Diseases.

Background: Antithrombin (AT) is one of the most important regulator of hemostasis. AT Budapest 3 (ATBp3) is a prevalent type II heparin-binding site (IIHBS) deficiency due to founder effect. Thrombosis is a complex disease including arterial (ATE) and venous thrombotic events (VTE) and the Roma population, the largest ethnic minority in Europe has increased susceptibility to these diseases partly due to their unfavorable genetic load. We aimed to calculate the age and origin of ATBp3 and to explore whether the frequency of it is higher in the Roma population as compared with the general population from the corresponding geographical area. We investigated the association of ATBp3 with thrombotic events in well-defined patients' populations in order to refine the recommendation when testing for ATBp3 is useful. Methods and Results: Prevalence of ATBp3, investigated in large samples (n = 1,000 and 1,185 for general Hungarian and Roma populations, respectively) was considerably high, almost 3%, among Roma and the founder effect was confirmed in their samples, while it was absent in the Hungarian general population. Age of ATBp3-as calculated by analysis of 8 short tandem repeat sequences surrounding SERPINC1-was dated back to XVII Century, when Roma migration in Central and Eastern Europe occurred. In our IIHBS cohort (n = 230), VTE was registered in almost all ATBp3 homozygotes (93%) and in 44% of heterozygotes. ATE occurred with lower frequency in ATBp3 (around 6%); it was rather associated with AT Basel (44%). All patients with ATE were young at the time of diagnosis. Upon investigating consecutive young (<40 years) patients with ATE (n = 92) and VTE (n = 110), the presence of ATBp3 was remarkable. Conclusions: ATBp3, a 400-year-old founder mutation is prevalent in Roma population and its Roma origin can reasonably be assumed. By the demonstration of the presence of ATBp3 in ATE patients, we draw the attention to consider type IIHBS AT deficiency in the background of not only VTE but also ATE, especially in selected populations as young patients without advanced atherosclerosis. We recommend including the investigation of ATBp3 as part of thrombosis risk assessment and stratification in Roma individuals.

A DNA pool of FLT3-ITD positive DNA samples can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation - Testing several different ITD sequences and rates, simultaneously.

Internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene is one of the most frequent genetic alteration in acute myeloid leukemia (AML), and it is associated with worse clinical outcome. Not only the presence but also the size, localization and the rate of this variant or the presence of multiple ITDs has prognostic information. The traditional PCR based diagnostic methods cannot provide information about all of these parameters in one assay, however the application of next generation sequencing (NGS) technique can be a reliable solution for this diagnostic problem. In order to evaluate the analytical properties of an NGS-based FLT3-ITD detection assay a quality control sample was prepared from DNA of AML patients containing 19 different FLT3-ITD variants identified by NGS. The higher the total read count was in a certain sample of the NGS run, the more ITD variant types could be detected. The maximal sensitivity of FLT3-ITD detection by NGS technique was as low as 0.007% FLT3-ITD/total allele rate, however, below 0.1% rate, the reproducibility of the quantitation was poor (CV > 25%). DNA pools with several FLT3-ITDs can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation testing several different ITD sequences and rates, simultaneously.

[Pernicious anemia in a thalassemic patient - difficulties of the diagnosis]. / Thalassaemiás betegen észlelt anaemia perniciosa ­ a diagnózis nehézségei.

The bone marrow aspiration, which was done in a leukopenic, hypochromic, microcytic, progressive anemic, thalassemic patient, revealed megaloblastic morphology. The low level of vitamin B12 and the reticulocytosis following the B12 supportation strenghtened the diagnosis of pernicious anemia. The set of the right diagnosis has been delayed by the fact that even in severe anemia one could not obtain the typical signs of B12 deficiency, having a hypochromic, microcytic erythrocyte morphology, due to the thalassemia minor disorder. Orv Hetil. 2018; 159(33): 1368-1371.

Low factor XIII levels after intravenous thrombolysis predict short-term mortality in ischemic stroke patients.

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 < 0.05). No association was found between the investigated FXIII polymorphisms and therapeutic outcomes. In conclusion, our findings indicate that FXIII levels 24 h after thrombolysis might help to identify patients at increased risk for short-term mortality.

Effect of factor XIII levels and polymorphisms on the risk of myocardial infarction in young patients.

Factor XIII (FXIII) stabilizes and protects the fibrin network. Its role in myocardial infarction (MI) is still to be clarified. To evaluate the association of FXIII levels with MI in young patients and to investigate how the FXIII-A p.Val34Leu, FXIII-B p.His95Arg, and IVS11, c.1952 + 144 C>G (Intron K) polymorphisms influence FXIII levels and MI risk. Patients with ST elevation MI below 40 years of age (MI, n = 119), age-matched clinical controls (CC, n = 101) without MI and coronary artery disease, and healthy controls (HC, n = 120) were investigated for FXIII activity, FXIII-A2B2, FXIII-B concentrations and for the polymorphisms. FXIII activity and FXIII-A2B2 antigen were significantly elevated in MI. FXIII activity and antigen were significantly elevated in Arg95, while decreased in Intron K "G" carriers. Smoking had an independent increasing effect on FXIII activity and FXIII-A2B2 antigen. Intron K C>G polymorphism significantly decreased the risk of MI in patients with elevated fibrinogen. Among the investigated factors Intron K C>G polymorphism and smoking have the most powerful effect on FXIII levels and on the risk of MI in the young. The effect of smoking on coronary thrombus formation may partially be attributed to its FXIII increasing effect.

The Murine Factor H-Related Protein FHR-B Promotes Complement Activation.

Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

Factor XIII levels and factor XIII B subunit polymorphisms in patients with venous thromboembolism.

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.

Browning deficiency and low mobilization of fatty acids in gonadal white adipose tissue leads to decreased cold-tolerance of transglutaminase 2 knock-out mice.

During cold-exposure 'beige' adipocytes with increased mitochondrial content are activated in white adipose tissue (WAT). These cells, similarly to brown adipose tissue (BAT), dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). We investigated the effect of tissue transglutaminase (TG2) ablation on the function of ATs in mice. Although TG2+/+ and TG2-/- mice had the same amount of WAT and BAT, we found that TG2+/+ animals could tolerate acute cold exposure for 4h, whereas TG2-/- mice only for 3h. Both TG2-/- and TG2+/+ animals used up half of the triacylglycerol content of subcutaneous WAT (SCAT) after 3h treatment; however, TG2-/- mice still possessed markedly whiter and higher amount of gonadal WAT (GONAT) as reflected in the larger size of adipocytes and lower free fatty acid levels in serum. Furthermore, lower expression of 'beige' marker genes such as UCP1, TBX1 and TNFRFS9 was observed after cold exposure in GONAT of TG2-/- mice, paralleled with a lower level of UCP1 protein and a decreased mitochondrial content. The detected changes in gene expression of Resistin and Adiponectin did not provoke glucose intolerance in the investigated TG2-/- mice, and TG2 deletion did not influence adrenaline, noradrenaline, glucagon and insulin production. Our data suggest that TG2 has a tissue-specific role in GONAT function and browning, which becomes apparent under acute cold exposure.

Regulation of plasma factor XIII levels in healthy individuals; a major impact by subunit B intron K c.1952+144 C>G polymorphism.

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-A2B2) 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-A2B2 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.

Factor XIII B subunit polymorphisms and the risk of coronary artery disease.

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>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.

Factor XIII-A subunit Val34Leu polymorphism in fatal atherothrombotic ischemic stroke.

Factor XIII (FXIII) is a regulator of fibrinolysis and clot firmness. Val34Leu polymorphism of its potentially active A subunit (FXIII-A) leads to faster activation of FXIII, influences clot structure and provides a moderate protection against coronary artery disease. The effect of FXIII-A Val34Leu polymorphism on the risk of atherothrombotic ischemic stroke (AIS) has been investigated in a few studies with contradictory results. In all previous studies, only patients surviving AIS were enrolled and sex-specific effects were not explored. In this retrospective multicenter cohort, we investigated the effect of FXIII-A Val34Leu polymorphism on the risk of fatal AIS in women and men. DNA isolation and genetic determinations in the case of 316 patients who died of AIS were carried out on paraffin-embedded tissue specimens. Genetic analyses for population controls, patients with history of AIS and sex-matched controls were performed on extracted genomic DNA from peripheral blood leukocytes. The prevalence of homozygous wild-type, and heterozygous genotypes, Leu34 carriers and Leu34 allele was not different significantly between the patients with fatal AIS and their respective controls. Logistic regression analysis with age as co-variant demonstrated that in women, homozygous presentation of Leu34 allele represented a more than three-fold increased risk of AIS with fatal outcome. The results demonstrate that FXIII-A Val34Leu polymorphism does not influence the occurrence of AIS, but has an effect on the severity of its outcome. This effect is sex-specific and in homozygous women, the prothrombotic/antifibrinolytic effects of FXIII-A Val34Leu polymorphism seem to prevail.

Thrombomodulin-dependent effect of factor V Leiden mutation on the cross-linking of α2-plasmin inhibitor to fibrin and its consequences on fibrinolysis.

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.

Thrombomodulin-dependent effect of factor VLeiden mutation on factor XIII activation.

INTRODUCTION: Factor V Leiden mutation (FV(Leiden)) is associated with impaired down-regulation of activated FV procoagulant activity and loss of FV anticoagulant function that result in an increased risk of venous thromboembolism. As the downstream effects of FV(Leiden) on clot formation and fibrinolyis have only partially been revealed, we investigated its effect on the activation of factor XIII (FXIII) and the cross-linking of fibrin. METHODS: In the plasma samples of fifteen healthy individuals with known FV genotypes coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human thrombomodulin (rhTM). FV deficient plasma supplemented with purified wild type FV or FV(Leiden) were also investigated. Clots were recovered and analyzed by SDS-PAGE and quantitative densitometric evaluation of Western blots. RESULTS: rhTM considerably delayed the activation of FXIII in the plasma from FV wild type individuals. This effect of rhTM was significantly impaired in the plasma from FV(Leiden) carriers. The results were confirmed in experiments with FV deficient plasma supplemented by FV prepared from wild type individuals or FV(Leiden) homozygotes. Fibrin γ-chain dimerization was also considerably delayed by rhTM in plasma samples from individuals without Leiden mutation, but not in plasma samples from FV(Leiden) heterozygotes or homozygotes. The difference between heterozygotes and homozygotes was not statistically significant. CONCLUSION: The highly diminished delaying effect of TM on FXIII activation and on the cross-linking of fibrin in FV(Leiden) carriers might represent a novel mechanism contributing to the increased thrombosis risk of these individuals.