Diagnostic value of clot formation parameters determined by rotational thromboelastometry in 63 patients with congenital dysfibrinogenemia.

Rotational thromboelastometry (ROTEM) is a global hemostasis assay. The diagnosis added value of ROTEM in congenital dysfibrinogenemia remains to be established. The aim of this study was to analyze clot formation by ROTEM in a cohort of dysfibrinogenemic patients and to establish correlations with genotype, clinical features, and coagulation parameters. The study included genetically confirmed congenital dysfibrinogenemia cases (n = 63) and healthy controls ( n  = 50). EXTEM, INTEM, FIBTEM tests were used to measure ROTEM parameters, that is, clotting time (CT), clot formation time (CFT), maximal clot firmness (MCF) and amplitude 10 min after CT (A10). The ISTH bleeding assessment tool was used to determine bleeding episodes. CT (INTEM) was statistically significantly shorter in congenital dysfibrinogenemia patients compared to controls while CFT (EXTEM) was prolonged. Patients's MCF in EXTEM, INTEM, and FIBTEM were similar to controls while A10 (FIBTEM) was statistically significantly lower. Fibrinogen activity was positively correlated with fibrinogen antigen, A10 and MCF in all three assays. Bleeding phenotypes were observed in 23 (36.5%) patients. Only CFT in EXTEM and CT in INTEM were statistically different in patients with bleeding phenotype versus controls. Carriers of the FGA mutation p.Arg35His had a CT (EXTEM) slightly prolonged and a reduced A10 (FIBTEM) compared to controls. Some ROTEM parameters were able to distinguish congenital dysfibrinogenemia patients from controls, and patients with a bleeding phenotype. Prolonged CFT in EXTEM were associated with congenital dysfibrinogenemia and bleeding phenotype. Bleeding episodes in most patients were generally mild and prevalence of thrombosis was very low.

High On-Treatment Platelet Reactivity in Patients Undergoing Complex Percutaneous Coronary Interventions.

Patient response to P2Y12 inhibitor therapy is heterogeneous, and those with high on-treatment platelet reactivity (HTPR) are at an increased risk of thrombotic complications. The aim of our study was to determine whether selecting a high-risk patient group of individuals after complex percutaneous coronary intervention (PCI) would show the clinical benefit of HTPR testing for preventing thrombotic complications. Blood samples of patients after complex PCI were acquired 1 day and 1 month after the intervention. The samples were tested using vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) and platelet function assay (PFA). The occurrence of clinically significant stent thrombosis with repeated revascularization of the target vessel was observed over a 1-year period. One day after PCI, 37% of patients had HTPR as established by VASP-P. One month after PCI, the percentage of patients with HTPR decreased to 30.9%. According to PFA, 1 day after PCI, 33.3% of patients had HTPR. This percentage declined to 19.8% after 1 month. All measurements identified a significantly higher proportion of HTPR in patients on clopidogrel compared to ticagrelor and prasugrel. Two cases of early stent thrombosis and 1 case of late stent thrombosis were identified. Further study of adenosine diphosphate receptor blocker on-treatment response in patients undergoing complex PCI is necessary.

DNA Polymorphisms in Pregnant Women with Sticky Platelet Syndrome.

Sticky platelet syndrome (SPS) is a thrombophilia caused by the increased aggregability of platelets in response to the addition of low concentrations of epinephrine (EPI) and/or adenosine diphosphate (ADP). Some of the single nucleotide polymorphisms (SNP), alleles and haplotypes of platelet glycoprotein receptors were proved to have a role in the etiology of thrombotic episodes When comparing SPS and the control group, in VEGFA rs3025039, the p value for both CC vs. TT and CT vs. TT analyses was <0.001. Interestingly, no minor TT genotype was present in the SPS group, suggesting the thrombotic pathogenesis of recurrent spontaneous abortions (RSA) in these patients. Moreover, we found a significant difference in the presence of AT containing a risky A allele and TT genotype of ALPP rs13026692 (p = 0.034) in SPS patients when compared with the controls. Additionally, we detected a decreased frequency of the GG (CC) genotype of FOXP3 rs3761548 in patients with SPS and RSA when compared with the control group (p value for the CC (GG) vs. AA (TT) 0.021). This might indicate an evolutionary protective mechanism of the A (T) allele in the SPS group against thrombotic complications in pregnancy. These results can be used for antithrombotic management in such pregnant patients.

Use of Fibrinogen Determination Methods in Differential Diagnosis of Hypofibrinogenemia and Dysfibrinogenemia.

BACKGROUND: Fibrinogen plays an important role in hemostasis. The normal concentration of fibrinogen in blood plasma is between 1.8 - 4.2 g/L. Decreased fibrinogen levels are observed in congenital afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, disseminated intravascular coagulation, fibrinolytic therapy, some more severe hepatic parenchymal disorders, and increased blood loss. Elevated fibrinogen levels occur in inflammatory diseases and neoplastic diseases, in pregnancy, and postoperative conditions. Functional fibrinogen measurement is also one of the basic coagulation screening tests. The fibrinogen antigen assay is used to distinguish between qualitative and quantitative fibrinogen disorders. METHODS: The aim of the study was the use of fibrinogen determination methods in differential diagnosis of hypofibrinogenemia and dysfibrinogenemia, statistical evaluation and determine the relationship of fibrinogen Clauss assay, prothrombin time (PT) derived fibrinogen assay, and fibrinogen antigen in the group of 60 patients with congenital fibrinogen disorders (n = 40 dysfibrinogenemia; n = 20 hypofibrinogenemia). RESULTS: The results measured by the PT-derived fibrinogen assay were approximately four times higher compared to the fibrinogen Clauss assay in the group of patients with dysfibrinogenemia. In patients with hypofibrinogenemia, there is a correlation (r = 0.9016) between the fibrinogen Clauss assay and PT-derived fibrinogen assay with a statistical significance of p < 0.0001. Using a linear or quadratic interpolation function, we were able to determine the fibrinogen Clauss assay and the fibrinogen antigen assay before analysis. CONCLUSIONS: The higher level of the PT-derived fibrinogen assay compared to the fibrinogen Clauss assay in the group of patients with dysfibrinogenemia may pose a greater risk to asymptomatic patients who require diagnosis and treatment in case of bleeding. The fibrinogen value using the PT-derived fibrinogen assay could erroneously give a normal level. The use of the interpolation function is important to estimate the value of fibrinogen activity and antigen before the analysis itself by the Clauss assay or analysis by the fibrinogen antigen assay.

Impact of Dabigatran Treatment on Rotation Thromboelastometry.

A rapid and reliable assessment of the dabigatran effect is desirable in dabigatran treated patients with uncontrolled bleeding or before acute surgery. The aim of this study was to study the anticoagulant effects of dabigatran in patients with atrial fibrillation (AF) as assessed by the whole blood assays ROTEM, and how data from these methods correlate to plasma dabigatran concentrations measured by Hemoclot. ROTEM was performed with ROTEM Gamma (Pentapharm GmbH, Munich, Germany). The assays used in our study were Ex-tem and In-tem assay. Plasma dabigatran concentrations were determined by hemoclot thrombin inhibitor assay (Hyphen BioMed, France) at trough and post-dose in 27 patients on dabigatran 150 mg BID. Median plasma dabigatran concentrations at trough were 74 ng/mL (11.2-250) and post-dose (2 h after ingestion) 120 ng/mL (31-282). The ROTEM clotting time (CT) and maximum clot firmnes (MCF) correlated strongly with dabigatran concentrations when activated with the reagents Ex-tem (p < 0.0001) and In-tem (p < 0.0001). In summary, in our study, we have found that the ROTEM variable CT and MCF, when activated with triggers Ex-tem and In-tem, has a strong and highly significant correlation with the plasma dabigatran concentration in a real-life population of AF-patients and could thereby be an alternative to estimate dabigatran concentration in emergency situations. However, additional studies are needed to further validate these findings.

Genetic Variants in the FGB and FGG Genes Mapping in the Beta and Gamma Nodules of the Fibrinogen Molecule in Congenital Quantitative Fibrinogen Disorders Associated with a Thrombotic Phenotype.

Fibrinogen is a hexameric plasmatic glycoprotein composed of pairs of three chains (Aα, Bß, and γ), which play an essential role in hemostasis. Conversion of fibrinogen to insoluble polymer fibrin gives structural stability, strength, and adhesive surfaces for growing blood clots. Equally important, the exposure of its non-substrate thrombin-binding sites after fibrin clot formation promotes antithrombotic properties. Fibrinogen and fibrin have a major role in multiple biological processes in addition to hemostasis and thrombosis, i.e., fibrinolysis (during which the fibrin clot is broken down), matrix physiology (by interacting with factor XIII, plasminogen, vitronectin, and fibronectin), wound healing, inflammation, infection, cell interaction, angiogenesis, tumour growth, and metastasis. Congenital fibrinogen deficiencies are rare bleeding disorders, characterized by extensive genetic heterogeneity in all the three genes: FGA, FGB, and FGG (enconding the Aα, Bß, and γ chain, respectively). Depending on the type and site of mutations, congenital defects of fibrinogen can result in variable clinical manifestations, which range from asymptomatic conditions to the life-threatening bleeds or even thromboembolic events. In this manuscript, we will briefly review the main pathogenic mechanisms and risk factors leading to thrombosis, and we will specifically focus on molecular mechanisms associated with mutations in the C-terminal end of the beta and gamma chains, which are often responsible for cases of congenital afibrinogenemia and hypofibrinogenemia associated with thrombotic manifestations.