Intracardiac Fibrinolysis and Endothelium Activation Related to Atrial Fibrillation Ablation with Different Techniques.

OBJECTIVE: The effect of pulmonary vein isolation (PVI) on fibrinolytic and endothelial activation with currently applied periprocedural anticoagulation has not been explored. We measured markers of fibrinolysis and endothelium activation before and after PVI with the second-generation cryoballoon (Cryo), pulmonary vein ablation catheter (PVAC-Gold), and irrigated radiofrequency (IRF). METHODS: Markers of fibrinolysis and endothelium activation in left atrial (LA) blood samples were measured in 31 patients before and after PVI (Cryo:10, PVAC-Gold: 7, IRF: 14). Periprocedural anticoagulation included uninterrupted vitamin K antagonist and iv heparin (ACT≥300 sec) during LA dwelling. RESULTS: Levels of D-dimer (median; interquartile range, mgFEU/L) increased with all techniques (PVAC: 0.34; 0.24-0.50 versus 0.70; 0.61-1.31; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; PAP complex level (ng/ml) increased after Cryo (247.3, 199.9-331.6 versus 270.9, 227.9-346.7; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; PAI-1 activity (%) decreased with the PVAC (1.931; 0.508-3.859 versus 0.735, 0.240-2.707; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; VWF antigen levels and FVIII activity increased after PVI with all the 3 techniques. The levels of soluble VCAM-1 (ng/ml) did not change after PVAC procedures, but increased after Cryo (542, 6; 428.5-753.1 versus 619.2; 499.8-799.0; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93. CONCLUSION: PVI with contemporary ablation techniques and periprocedural antithrombotic treatment induces coagulation and endothelium activation of similar magnitude with different ablation methods.

Antithrombin Debrecen (p.Leu205Pro) - Clinical and molecular characterization of a novel mutation associated with severe thrombotic tendency.

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 trans­Golgi 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.

Molecular characterization of p.Asp77Gly and the novel p.Ala163Val and p.Ala163Glu mutations causing protein C deficiency.

INTRODUCTION: Protein C (PC) is a major anticoagulant and numerous distinct mutations in its coding gene result in quantitative or qualitative PC deficiency with high thrombosis risk. Homozygous or compound heterozygous PC deficiency usually leads to life-threatening thrombosis in neonates. PATIENTS AND METHODS: The molecular consequences of 3 different missense mutations of two patients have been investigated. The first patient suffered from neonatal purpura fulminans and was a compound heterozygote for p.Asp77Gly and p.Ala163Glu mutations. The second patient had severe deep venous thrombosis in young adulthood and carried the p.Ala163Val mutation. The fate of mutant proteins expressed in HEK cells was monitored by ELISA, by Western blotting, by investigation of polyubiquitination and by functional assays. Their intracellular localization was examined by immunostaining and confocal laser scanning microscopy. Molecular modeling and dynamics simulations were also carried out. RESULTS AND CONCLUSIONS: The 163Val and 163Glu mutants had undetectable levels in the culture media, showed intracellular co-localization with the 26S proteasome and were polyubiquitinated. The 77Gly mutant was secreted to the media showing similar activity as the wild type. There was no difference among intracellular PC levels of wild type and mutant proteins. The 163Val and 163Glu mutations caused significant changes in the relative positions of the EGF2 domains suggesting misfolding with the consequence of secretion defect. No major structural alteration was observed in case of 77Gly mutant; it might influence the stability of protein complexes in which PC participates and may have an impact on the clearance of PC requiring further research.

Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game.

Protein C (PC) and protein S (PS) are vitamin K-dependent glycoproteins that play an important role in the regulation of blood coagulation as natural anticoagulants. PC is activated by thrombin and the resulting activated PC (APC) inactivates membrane-bound activated factor VIII and factor V. The free form of PS is an important cofactor of APC. Deficiencies in these proteins lead to an increased risk of venous thromboembolism; a few reports have also associated these deficiencies with arterial diseases. The degree of risk and the prevalence of PC and PS deficiency among patients with thrombosis and in those in the general population have been examined by several population studies with conflicting results, primarily due to methodological variability. The molecular genetic background of PC and PS deficiencies is heterogeneous. Most of the mutations cause type I deficiency (quantitative disorder). Type II deficiency (dysfunctional molecule) is diagnosed in approximately 5%-15% of cases. The diagnosis of PC and PS deficiencies is challenging; functional tests are influenced by several pre-analytical and analytical factors, and the diagnosis using molecular genetics also has special difficulties. Large gene segment deletions often remain undetected by DNA sequencing methods. The presence of the PS pseudogene makes genetic diagnosis even more complicated.