High Neutrophil-Lymphocyte Ratio and Low Lymphocyte-Monocyte Ratio Combination after Thrombolysis Is a Potential Predictor of Poor Functional Outcome of Acute Ischemic Stroke.

(1) Background: Ischemic stroke is one of the leading causes of death and disability. An inflammatory response is observed in multiple stages of cerebral ischemia, particularly in the acute phase. Recent publications revealed that the neutrophil−lymphocyte ratio (NLR) and lymphocyte−monocyte ratio (LMR) may be used to predict long-term prognosis in acute ischemic stroke (AIS) after thrombolysis. To test whether there is a relationship between the combination of these parameters and long-term prognosis, we analyzed the NLR−LMR combination in AIS patients treated with intravenous recombinant tissue plasminogen activator (rtPA); (2) Methods: The study included 285 adults with a diagnosis of AIS and rtPA treatment within a 4.5 h time window. Blood samples were obtained at admission and 24 h after thrombolysis to calculate pre- and post-thrombolysis NLR and LMR. Clinical data, including NIHSS was registered on admission and day 1. The long-term outcome was defined 90 days post-event by the modified Rankin Scale (mRS). Therapy-associated intracranial hemorrhage (ICH) was classified according to ECASS II. Receiver operating characteristic curve (ROC) analysis was performed to determine optimal cutoffs of NLR and LMR as predictors of therapy outcomes; (3) Results: Patients were stratified by cutoffs of 5.73 for NLR and 2.08 for LMR. The multivariate logistic regression model, including all possible confounders, displayed no significant association between NLR or LMR with 3-months functional prognosis. The combination of high NLR−low LMR vs. low NRL−high LMR as obtained 24 h after thrombolysis was found to be an independent predictor of poor 3-months functional outcome (mRS ≥ 2; OR 3.407, 95% CI 1.449 to 8.011, p = 0.005). The proportion of patients between low NLR−high LMR and high NLR−low LMR groups from admission to day 1 showed no significant change in the good outcome group. On the other hand, in the poor outcome group (mRS ≥ 2), low NLR−high LMR and high NLR−low LMR groups displayed a significant shift in patient proportions from 67% and 21% at admission (p = 0.001) to 36% and 49% at 24 h after thrombolysis (p < 0.001), respectively; (4) Conclusions: Our study demonstrated for the first time that a high NLR−low LMR combination as observed at 24 h after thrombolysis can serve as an independent predictor of 3-months poor outcome in AIS patients. This simple and readily available data may help clinicians to improve the prognostic estimation of patients and may provide guidance in selecting patients for personalized and intensified care post-thrombolysis.

The Effect of Activated FXIII, a Transglutaminase, on Vascular Smooth Muscle Cells.

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.

Cellular Factor XIII, a Transglutaminase in Human Corneal Keratocytes.

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.

Characteristics of platelet count and size and diagnostic accuracy of mean platelet volume in patients with venous thromboembolism. A systematic review and meta-analysis.

The purpose of this study was to evaluate the association between platelet (PLT) count and mean platelet volume (MPV) and venous thromboembolism (VTE). Thus, this study reviewed and performed a quantitative synthesis on data from the literature. This meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. 18 studies were included in this paper. A random-effect meta-analysis was conducted for the assessment of heterogeneity using thrombosis place, type of analyzer, type of anticoagulant and incubation time of samples as covariates. A mixed-effect meta-regression was performed based on the subgroup for the whole samples using thrombosis place and method of measurement as moderators for MPV and PLT, respectively. The cumulative estimates and 95% confidence interval (95%CI) of specificity, sensitivity, area under the receiver operator characteristic curve (AUC) and diagnostic odds ratio (DOR) for MPV were calculated using a random effect model. The quality assessments were evaluated according to the quality assessment and diagnostic accuracy tool-2 (QUADAS-2). The primary outcome was the occurrence of VTE. Secondary outcomes included PLT and MPV. Patient with deep vein thrombosis is likely to have a higher value of MPV than control group (P < 0.001). The presence of pulmonary embolism (PE) had no significant effect on the standardized mean difference of MPV between patients and controls. Patients are likely to have less PLT than the control group regarding all studies. However, subgroup analysis demonstrated that this effect was significant for patients with PE (P < 0.05). The summary receiver operating characteristic (SROC) curve indicated that AUC was 0.745 (95% CI: 0.672-0.834). The DOR for MPV was 4.76 (95%CI: 2.3-9.85), with diagnostic accuracy of 0.66.

The lack of aspirin resistance in patients with coronary artery disease.

BACKGROUND: Aspirin resistance established by different laboratory methods is still a debated problem. Using COX1 specific methods no aspirin resistance was detected among healthy volunteers. Here we tested the effect of chronic aspirin treatment on platelets from patients with stable coronary artery disease. The expression of COX2 mRNA in platelets and its influences on the effect of aspirin was also investigated. METHODS: One hundred and forty four patients were enrolled in the study. The direct measurement of COX1 acetylation was carried out by monoclonal antibodies specific to acetylated and non-acetylated COX1 (acCOX1 and nacCOX1) using Western blotting technique. Arachidonic acid (AA) induced TXB2 production by platelets was measured by competitive immunoassay. AA induced platelet aggregation, ATP secretion and VerifyNow Aspirin Assay were also performed. COX2 and COX1 mRNA expression in platelets were measured in 56 patients by RT-qPCR. RESULTS: In 138 patients only acCOX1 was detected, in the remaining six patients nacCOX1 disappeared after a compliance period. AA induced TXB2 production by platelets was very low in all patients including the 6 patients after compliance. AA induced platelet aggregation, secretion and with a few exceptions the VerifyNow Assay also demonstrated the effect of aspirin. Smoking, diabetes mellitus and inflammatory conditions did not influence the results. The very low amount of COX2 mRNA detected in 39 % of the investigated platelets did not influence the effect of aspirin. CONCLUSIONS: No aspirin resistance was detected among patients with stable coronary artery disease. COX2 expression in platelets did not influence the effect of aspirin.

Validation of reference genes for the determination of platelet transcript level in healthy individuals and in patients with the history of myocardial infarction.

RT-qPCR is the standard method for studying changes in relative transcript level in different experimental and clinical conditions and in different tissues. No validated reference genes have been reported for the normalization of transcript level in platelets. The very low level of platelet RNA and the elimination of leukocyte contamination represented special methodological difficulties. Our aims were to apply a simple technique to separate platelets for transcript level studies, and select the most stable reference genes for platelets from healthy individuals and from patients with the history of myocardial infarction. We developed a simple, straightforward method of platelet separation for RNA isolation. Platelet activation was inhibited by using acid-citrate-dextrose for anticoagulation and by prostaglandin E1. Leukocyte contamination was eliminated by three consecutive centrifugations. Samples prepared by this method were free of leukocytes, showed no inhibition in PCR reaction and no RNA degradation. The assay demands low blood volume, which complies with the requirements of everyday laboratory routine. Seventeen potential reference genes were investigated, but eight of them were excluded during optimization. The stability of the remaining genes, EEF2, EAR, ACTB, GAPDH, ANAPC5, OAZ1, HDGF, GNAS, and CFL1, were determined by four different descriptive statistics. GAPDH, GNAS, and ACTB were shown to be the most stable genes in platelets of healthy individuals, while HDGF, GNAS, and ACTB were the most stable in platelets of patients with the history of myocardial infarction. The results confirm that data normalization needs assessment of appropriate reference genes for a particular sample set.

The effect of metoprolol alone and combined metoprolol-felodipin on the digital microcirculation of patients with primary Raynaud's syndrome.

OBJECTIVES: Calcium channel inhibitors have beneficial impact on microcirculation, but beta-blocker effect is controversial. Clinicians still do not agree on beta-blocker combination with other treatments in the management of impaired microcirculation. The aim of the present study was to describe the effects of beta-blocker metoprolol monotherapy and combined with calcium channel inhibitor felodipin on digital microcirculation in primary Raynaud's syndrome. METHODS: We enrolled in this study 46 patients suffering from both hypertension and primary Raynaud's syndrome. Fifteen patients were treated with beta-blocker monotherapy (metoprolol), 13 received combined beta-blocker and calcium channel blocker therapy (felodipin and metoprolol), while 18 patients without any medications served as controls. Measurement of digital microcirculation was carried out with laser Doppler scanner. RESULTS AND CONCLUSIONS: Our investigation concludes that the concurrent administration of beta-blockers with calcium channel inhibitors positively reduces symptoms in patients suffering from Raynaud's syndrome.

Factor XIII A subunit Val34Leu polymorphism in patients suffering atherothrombotic ischemic stroke.

INTRODUCTION: Factor XIII (FXIII) is a key 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 subunit (FXIII-A) Val34Leu polymorphism on the risk of ischemic stroke (IS) has been investigated in a few studies with contradictory results. In spite of their fundamental difference in pathogenesis and hemostatic pathomechanism, only four small studies investigated the effect of FXIII-A Val34Leu polymorphism on the risk of atherothrombotic IS (AIS) separately from cardioembolic IS. Gender specific effect of the polymorphism on the risk of AIS has not been explored. In the present study we investigated the effect of FXIII-A Val34Leu polymorphism on the risk of AIS on a large patient population. MATERIALS AND METHODS: A population control group of 1,146 randomly selected individuals, 496 patients surviving AIS and their age and sex-matched controls selected from the population control group were included in the study. FXIII-A Val34Leu genotype was determined on DNA samples, obtained from peripheral blood leukocytes, by fluorescence resonance energy transfer detection using melting curve analysis. RESULTS: Neither sex nor age affected the distribution of FXIII-A Val34Leu genotypes in population control group. No association was revealed between the risk of AIS and FXIII-A Leu34 carriership and homozygous or heterozygous presentation of Leu34 allele in either gender. CONCLUSION: FXIII-A Val34Leu polymorphism fails to influence the risk of AIS.

Factor XIII and atherothrombotic diseases.

Factor XIII (FXIII) is a protransglutaminase that, after activation, cross-links fibrin chains and several plasma proteins, most importantly alpha (2) plasmin inhibitor, to fibrin. FXIII strengthens the fibrin clot by covalent bonds and protects fibrin from the prompt elimination by the fibrinolytic system. In the last two decades, FXIII has emerged as a key regulator of fibrinolysis. FXIII is also present in platelets, monocytes, and macrophages, but this cellular form does not contribute significantly to maintaining hemostasis. FXIII deficiency is a life-threatening bleeding diathesis whose clinical consequences are well studied. In contrast, the involvement of FXIII in thrombotic disorders and its association with the risk of such diseases are less clear. This review gives an account of the data accumulated mainly in the last decade on the association of FXIII with atherothrombotic diseases and presents conclusions and hypotheses drawn from these data as well as exposing the limitations of the published studies and our knowledge on this topic. The involvement of FXIII in atherogenesis, its role in coronary artery disease, atherothrombotic ischemic stroke, and peripheral artery disease are discussed, with particular reference to the association of FXIII levels and polymorphisms with the risk of these diseases.

Decreased factor XIII levels in factor XIII A subunit Leu34 homozygous patients with coronary artery disease.

INTRODUCTION: The effect of factor XIII A subunit (FXIII-A) Val34Leu polymorphism on the risk of coronary artery disease (CAD) has been extensively studied. In this study we investigated how FXIII-A Val34Leu genotypes influence plasma factor XIII levels in patients with coronary sclerosis (CS) and myocardial infarction (MI) and how fibrinogen level modulates this effect. PATIENTS AND METHODS: 955 consecutive patients admitted for coronary angiography were categorized according to the presence or absence of significant CS and the history of MI. The frequency of FXIII-A Val34Leu polymorphism, fibrinogen, FXIII activity and antigen levels were determined. RESULTS AND CONCLUSIONS: CS or MI decreased FXIII levels in patients homozygous for FXIII-A Leu34 allele, but not in heterozygous or wild type patients. In the subgroup of patients with CS, but without the history of MI no significant effect was detected, which suggests that MI has a more prominent role. The specific activity of plasma FXIII was independent of FXIII-A Val34Leu genotype. FXIII and fibrinogen levels significantly correlated in CS+ and MI+ patients. In MI+ patients of Leu/Val or Leu/Leu genotypes and with fibrinogen levels in the lowest quartile, FXIII levels were lower than in the same patient groups, but with higher fibrinogen level. The low-scale continuous activation of blood coagulation in CAD patients could lead to parallel FXIII and fibrinogen consumption. As the same amount of thrombin activates more Leu34 FXIII than Val34 FXIII, increased FXIII consumption might be responsible for the decreased FXIII levels in Leu34 homozygous CAD patients.

The combined effect of fibrin formation and factor XIII A subunit Val34Leu polymorphism on the activation of factor XIII in whole plasma.

The first step in the activation of blood coagulation factor XIII (FXIII) is the proteolytic cleavage of the potentially active A subunit (FXIII-A) by thrombin at Arg37-Gly38. Both fibrin formation and FXIII-A Val34Leu polymorphism influence the rate of proteolytic activation of purified factor XIII, however their relative importance and interaction in determining the time of onset and the rate of FXIII activation in whole plasma have not yet been explored. In the present study it was shown that in plasma, fibrin formation preceded the truncation of FXIII-A by thrombin, the activation process took place exclusively on the surface of newly formed fibrin and activated FXIII remained associated with the fibrin clot. The time of fibrin formation closely correlated with the time of FXIII activation, while there was no significant relationship between the time of FXIII activation and FXIII-A Val34Leu genotype. However, in the case of Leu34 variant the lag phase between fibrin formation and FXIII-A truncation was significantly shorter than in the case of Val34 variant. The results suggest that in whole plasma the onset of FXIII activation is determined by fibrin formation, while the rate of activation is modulated by Val34Leu polymorphism.

Rapid detection of the factor XIII Val34Leu (163 G-->T) polymorphism by real-time PCR using fluorescence resonance energy transfer detection and melting curve analysis.

The Val34Leu polymorphism in the A subunit of blood coagulation factor XIII (FXIII-A) is located in the activation peptide, just three amino acids upstream of the thrombin cleavage site. The Val-->Leu replacement accelerates the rate of the proteolytic activation of FXIII and it seems to provide protection against myocardial infarction. Methods available for the assessment of the FXIII-A Val34Leu polymorphism are rather time-consuming, laborious and not easily applicable for large-scale studies. In this study a new method based on real-time PCR with fluorescence resonance energy transfer (FRET) detection and melting curve analysis was developed. The rapid, simple method was adapted to the widely used real-time PCR instrument, LightCycler (Roche Diagnostics). The results showed 100% coincidence with those obtained by the traditional PCR-restriction fragment length polymorphism (RFLP) assay and fluorescent DNA sequencing. Using this method, an allele frequency of 24.2% was obtained (n=113), which well agrees with the allele frequency obtained by PCR-RFLP on a different group of the same ethnic Hungarian population (25.9%).