SERPINE1 and MTHFR genetic variants in patients with embolic stroke of undetermined source: links with fibrin clot properties.

INTRODUCTION: The SERPINE1 c.-820G (4_5), MTHFR gene variants, and unfavourably altered fibrin clot features, have been suspected to be associated with embolic stroke of undetermined source (ESUS). We investigated the SERPINE1 c.-820G (4_5) gene variants alone and coexisting with MTHFR c.665C > T and c.1286A > C gene variants in relation to thrombophilic factors and plasma fibrin clot properties in Polish patients with ESUS. PATIENTS AND METHODS: Unrelated consecutive patients with ESUS (n = 206) were genotyped by TaqMan assay. Thrombophilia screening was performed four weeks or more after a thrombotic event while off oral anticoagulation. Factor VIII (FVIII) activity was determined by a coagulometric assay, while lipoprotein(a) was determined using immunoturbidimetry. We determined fibrin clot permeability (Ks) and clot lysis time (CLT). Apparently healthy individuals without a family history of stroke or venous thromboembolism (n = 30), and patients with a history of atrial fibrillation (n = 25) or carotid artery disease-related stroke (n = 21), served as controls. RESULTS: Among ESUS patients, the SERPINE1 c.-820G (4_5) minor allele frequency was 0.57. There were no differences in common factors associated with thrombophilia among ESUS patients regarding SERPINE1 variants. The overall prevalence of FVIII > 150IU/dL was 26% (n = 53) and elevated FVIII predominated in SERPINE1 variants carriers (n = 45; 84.9%), including 36 (68%) carriers of MTHFR variant. Moreover, 4.3-fold higher Lp(a) levels along with 50% reduced Ks and 46% prolonged CLT were found in patients with mutant SERPINE1 combined with mutant homozygotes in the MTHFR c.665C > T variant compared to the wild type SERPINE1 combined with mutant homozygotes in the MTHFR c.665C >T (P < 0.001). CONCLUSIONS: The SERPINE1 c.-820G (4_5) variants carriers have increased FVIII levels, while the SERPINE1 c.-820G (4_5) mutant homozygotes coexisting with MTHFR c.665C > T have more prothrombotic fibrin clot features and elevated Lp(a). Our study underlines the cumulative effect of genetic risk factors in patients with ESUS that might require specific antithrombotic therapy.

Plasma thiol levels and methylenetetrahydrofolate reductase gene c.665C > T and c.1286A > C variants affect fibrin clot properties in Polish venous thromboembolic patients.

BACKGROUND AND AIMS: Aminothiols, including cysteine (Cys) and glutathione (GSH) in relation to fibrin clot phenotype were not investigated in patients with venous thromboembolism (VTE) and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene variants. We aimed to explore the associations between MTHFR variants and plasma oxidative stress indicators including aminothiols as well as fibrin clot properties with plasma oxidative status and fibrin clot properties in this group of patients. METHODS: In 387 VTE patients the MTHFR c.665C > T and c.1286A > C variants were genotyped, together with chromatographic separation of plasma thiols. We also determined nitrotyrosine levels and fibrin clot properties, including clot permeability (Ks), lysis time (CLT), and fibrin fibers thickness. RESULTS: There were 193 patients with MTHFR c.665C > T (49.9%) and 214 (55.3%) with c.1286A > C variants. Both allele carriers with total homocysteine (tHcy) levels >15 µM (n = 71, 18.3%), compared to patients with tHcy ≤15 µM had 11.5% and 12.5% higher Cys levels, 20.6% and 34.3% higher GSH levels as well as 28.1% and 57.4% increased nitrotyrosine levels, respectively (all P < 0.05). The MTHFR c.665C > T carriers with tHcy levels >15 µM compared to tHcy ≤15 µM had 39.4% reduced Ks and 9% reduced fibrin fibers thickness (both P < 0.05) with no differences in CLT. In the MTHFR c.1286A > C carriers with tHcy levels >15 µM, Ks was decreased by 44.5%, CLT prolonged by 46.1%, and fibrin fibers thickness was reduced by 14.5% compared to patients with tHcy ≤15 µM (all P < 0.05). Nitrotyrosine levels in MTHFR variants carriers correlated with Ks (r = -0.38, P < 0.05) and fibrin fibers diameter (r = -0.50, P < 0.05). CONCLUSIONS: Our study indicates that patients with MTHFR variants and tHcy >15 µM are characterized by elevated Cys and nitrotyrosine levels associated with prothrombotic fibrin clot properties.

Antithrombin Deficiency Is Associated with Prothrombotic Plasma Fibrin Clot Phenotype.

BACKGROUND: Deficiency of antithrombin increases risk of venous thromboembolism. We hypothesized that antithrombin deficiency affects fibrin clot structure and function. METHODS: We evaluated 148 patients (age: 38 [32-50] years; 70% women) with genetically confirmed antithrombin deficiency and 50 healthy controls. Fibrin clot permeability (Ks) and clot lysis time (CLT) along with thrombin generation capacity were assessed before and after antithrombin activity normalization in vitro. RESULTS: Antithrombin-deficient patients had lower antithrombin activity (-39%) and antigen levels (-23%) compared with controls (both p < 0.01). Prothrombin fragment 1 + 2 levels were 26.5% higher in patients with antithrombin deficiency than in controls along with 94% increased endogenous thrombin potential (ETP) and 108% higher peak thrombin (all p < 0.01). Antithrombin deficiency was associated with 18% reduced Ks and 35% prolonged CLT (both p < 0.001). Patients with type I (n = 65; 43.9%) compared with type II antithrombin deficiency (n = 83; 56.1%) had 22.5% lower antithrombin activity (p < 0.001) and despite similar fibrinogen levels, 8.4% reduced Ks, 18% prolonged CLT, and 30% higher ETP (all p < 0.01). Reduced Ks was associated with lower antithrombin antigen level (ß = - 6.1, 95% confidence interval [CI]: -1.7 to -10.5), while prolonged CLT was associated with lower antithrombin antigen (ß = - 69.6, 95% CI: -9.6 to -129.7), activity (ß = - 2.4, 95% CI: -0.3 to -4.5), higher PAI-1 (ß = 12.1, 95% CI: 7.7-16.5), and thrombin-activatable fibrinolysis inhibitor levels (ß = 3.8, 95% CI: 1.9-5.7). Addition of exogenous antithrombin reduced ETP (-42%) and peak thrombin (-21%), and improved Ks (+8%) and CLT (-12%; all p < 0.01). CONCLUSION: Our study suggests that enhanced thrombin generation and prothrombotic plasma fibrin clot phenotype can contribute to increased risk of thrombosis in patients with antithrombin deficiency.

Fibrinogen ß chain and FXIII polymorphisms affect fibrin clot properties in acute pulmonary embolism.

BACKGROUND: Prothrombotic fibrin clot properties, including increased clot density, are in part genetically determined. We investigated whether fibrinogen alpha-chain gene (FGA) c.991A>G (rs6050), fibrinogen beta chain gene (FGB) -455G>A (rs1800790) and factor XIII gene (F13) c.103G>T (rs5985) polymorphisms affect plasma fibrin clot properties in patients with acute pulmonary embolism (PE). METHODS: As many as 126 normotensive patients with PE, free of cancer, were genotyped by TaqMan assay. Fibrin clot permeability (Ks ), clot lysis time (CLT) and endogenous thrombin potential (ETP) were assessed on admission. RESULTS: The minor allele frequencies were as follows: FGA rs6050 (n = 62, 0.31), FGB rs1800790 (n = 40, 0.17) and F13 rs5985 (n = 49, 0.23). There were no differences related to any of the polymorphisms with regard to demographic, clinical and laboratory data, except for fibrinogen concentration, which was higher in carriers of F13 rs5985 polymorphism (p = .024), and PE combined with deep-vein thrombosis, which was less prevalent in FGB rs1800790 polymorphism carriers (p = .004). Carriers of FGB rs1800790 A allele and F13 rs5985 T allele had lower Ks , prolonged CLT and higher ETP compared with major homozygotes (all p < .05). After adjustment for fibrinogen, all differences remained significant (all p < .01). There were no associations between the FGA rs6050 polymorphism and Ks , CLT or ETP. CONCLUSION: Our study showed that FGB rs1800790 and F13 rs5985 polymorphisms contribute to the prothrombotic fibrin clot phenotype and these effects are strong enough to be observed in the acute phase of PE.

Identification of Cellular Factors Required for SARS-CoV-2 Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the recently emerged virus responsible for the COVID-19 pandemic. Clinical presentation can range from asymptomatic disease and mild respiratory tract infection to severe disease with lung injury, multiorgan failure, and death. SARS-CoV-2 is the third animal coronavirus to emerge in humans in the 21st century, and coronaviruses appear to possess a unique ability to cross borders between species and infect a wide range of organisms. This is somewhat surprising as, except for the requirement of host cell receptors, cell-pathogen interactions are usually species-specific. Insights into these host-virus interactions will provide a deeper understanding of the process of SARS-CoV-2 infection and provide a means for the design and development of antiviral agents. In this study, we describe a complex analysis of SARS-CoV-2 infection using a genome-wide CRISPR-Cas9 knock-out system in HeLa cells overexpressing entry receptor angiotensin-converting enzyme 2 (ACE2). This platform allows for the identification of factors required for viral replication. This study was designed to include a high number of replicates (48 replicates; 16 biological repeats with 3 technical replicates each) to prevent data instability, remove sources of bias, and allow multifactorial bioinformatic analyses in order to study the resulting interaction network. The results obtained provide an interesting insight into the replication mechanisms of SARS-CoV-2.

Comparison of Antigen Tests and qPCR in Rapid Diagnostics of Infections Caused by SARS-CoV-2 Virus.

Diagnostics of the coronavirus disease 2019 (COVID-19) using molecular techniques from the collected respiratory swab specimens requires well-equipped laboratory and qualified personnel, also it needs several hours of waiting for results and is expensive. Antigen tests appear to be faster and cheaper but their sensitivity and specificity are debatable. The aim of this study was to compare a selected antigen test with quantitative polymerase chain reaction (qPCR) tests results. Nasopharyngeal swabs were collected from 192 patients with COVID-19 symptoms. All samples were tested using Vitassay qPCR SARS-CoV-2 kit and the Humasis COVID-19 Ag Test (MedSun) antigen immunochromatographic test simultaneously. Ultimately, 189 samples were tested; 3 samples were excluded due to errors in taking swabs. The qPCR and antigen test results were as follows: 47 positive and 142 negative, and 45 positive and 144 negative, respectively. Calculated sensitivity of 91.5% and specificity of 98.6% for the antigen test shows differences which are not statistically significant in comparison to qPCR. Our study showed that effectiveness of the antigen tests in rapid laboratory diagnostics is high enough to be an alternative and support for nucleic acid amplification tests (NAAT) in the virus replication phase in the course of COVID-19.