Whole F8 gene sequencing identified pathogenic structural variants in the remaining unsolved patients with severe hemophilia A.

BACKGROUND: No F8 genetic abnormality is detected in approximately 1% to 2% of patients with severe hemophilia A (HA) using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. OBJECTIVES: The study aimed to identify the causal variation in families with a history of severe HA for whom genetic investigations failed. METHODS: We performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or quantitative polymerase chain reaction. RESULTS: A structural variant disrupting F8 was found in each propositus, so that all the 815 families with a history of severe HA registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, another was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of an Xq28 349 kb gained in F8 intron 5. CONCLUSION: All the genetically unsolved cases of severe HA in this cohort were due to structural variants disrupting F8. This study highlights the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach fails.

Isolation and Characterization of CD39-like Phosphodiesterase (Cc-PDE) from Cerastes cerastes Venom: Molecular Inhibitory Mechanism of Antiaggregation and Anticoagulation.

BACKGROUND: Cerastes cerastes venom contains several bioactive proteins with inhibitory potential of platelet aggregation and blood coagulation. OBJECTIVE: The current study deals with purification, characterization and determination of structural properties of Cc-PDE, the first phosphodiesterase from Cerastes cerastes venom. MATERIAL AND METHODS: The purification process consists of three successive chromatographies including G75-Sephadex size exclusion, DEAE exchange chromatography and affinity using Sildenafil as a main PDEs' specific inhibitor. The amino acid sequence of purified Cc-PDE was determined by liquid chromatography coupled off line to MALDI-TOF/TOF. Modeling and structural features were obtained using several bioinformatics tools. In vivo and in vitro antiplatelet aggregation and anticoagulant assays were performed. RESULTS: Cc-PDE (73 506.42 Da) is a 654-residue single polypeptide with 1-22 signal peptide and it is characterized by the presence of predominant basic amino acids suitable to alkaline pI (8.17). Cc-PDE structure is composed of ß-strands (17%) and α-helices (24%) and it shares a high identity with homologous snake venom PDEs. Cc-PDE hydrolyzes both Bis-p-nitrophenyl phosphate (Km = 2.60 ± 0.95 mM, Vmax = 0.017 ± 0.002569 µmol.min-1) and p-nitrophenyl phosphate (Km = 7.13 mM ± 0.04490 mM, Vmax = 0.053 ±0.012 µmol.min-1). Cc-PDE prevents ADP- and ATP-induced platelet aggregation by hydrolyzing ADP and ATP, reducing surface P-selectin expression and attenuating platelet function. In addition, Cc-PDE inhibits coagulation factors involved in the intrinsic pathway demonstrated by a significant prolongation of activated partial thromboplastin time and in vivo long-lasting anticoagulation. CONCLUSION: The obtained results revealed that Cc-PDE may have a therapeutic potential and could be a remedy for thromboembolic diseases as an alternative of anticoagulant and antiplatelet aggregation chemical origins.

[Platelet activation in lung cancer]. / Activation plaquettaire dans le cancer du poumon.

During primary hemostasis the platelets aggregate to form the platelet thrombus. ADP and thrombin generated by coagulation are the main agonists in platelet aggregation. In a previous study we were able to show that patients with lung cancer had hypercoagulability, hyperfibrinogemia (≥ 6.22 g/L) was predictive of thromboembolic disease at the start of diagnosis before any therapy. In this study, we studied platelet aggregation in these patients in order to demonstrate whether they have hyperaggregability associated with the hypercoagulability demonstrated previously, and this by evaluating abnormalities in primary hemostasis (platelet count and platelet aggregation). One hundred and one patients diagnosed before any therapy and 72 blood donors were included. Agonists used for platelet aggregation are collagen and adenosine diphosphate at low concentrations. Hyperaggregability is observed when blood platelets are stimulated by ADP at different concentrations (p ≤ 0.01). This hyperaggregability is influenced by the histological type and not the development of the cancer, the age of the subjects and the platelet count, it is independent of hyperfibrinogemia and the occurrence of thromboembolic disease. However, an increase in the platelet level is found in patients with hyperfibrinogemia. Patients with lung cancer present platelet activation observed by aggregometry in response to ADP; which is not influenced by hyperfibrinogemia during cancer.

Isolation and Functional Identification of an Antiplatelet RGD-Containing Disintegrin from Cerastes cerastes Venom.

The current report focuses on purification, structural and functional characterization of Cerastategrin from Cerastes cerastes venom, a novel basic disintegrin (pI 8.36) with 128 amino acid residues and a molecular weight of 13 835.25 Da measured by MALDI-MSMS. The 3D structure of Cerastategrin is organized as α-helix (13%), ß-strand (15%) and disordered structure (30%) and presents homologies with several snake venom disintegrins. Structural modeling shows that Cerastategrin presents an RGD motif that connects specifically to integrin receptors. Cerastategrin exhibits the inhibition of ADP induced platelets with an IC50 of 0.88 µg/mL and shows in vivo long stable anticoagulation effect 24 h post-injection of increasing doses ranging from 0.2 to 1 mg/kg, therefore, Cerastategrin maintained irreversibly the blood incoagulable. Moreover, Cerastategrin decreases the amount of bounded αIIbß3 and reduced significantly the quantity of externalized P-Selectin. Cerastategrin acts as a molecule targeting specifically the receptor αIIbß3; therefore, it behaves as a potent platelet activation inhibitor. As a new peptide with promising pharmacological properties, Cerastategrin could have a potential therapeutical effect in the vascular pathologies and may be a new effective treatment approach.

[Activation of coagulation in patients with lung cancer]. / Activation de la coagulation chez des patients atteints du cancer du poumon.

The aim of this study is to evaluate the anomalies of coagulation (by assaying the factor VIII, fibrinogen, D-dimer and resistance to activated protein C) in patients with lung cancer. METHODS: 101 patients newly diagnosed with lung cancer before treatment and 72 control blood donors were included in the study after informed consent. All coagulation tests were performed on Stago STA-Compact. Statistical analyses were performed using the SPSS software version 22. RESULTS: The study of the coagulation showed that plasma levels of all coagulation parameters were significantly higher in patients compared to controls. Coagulation was not influenced by the age of patients. No significant difference was found between the histological types in terms of coagulation. Factor VIII level was significantly elevated in stage IV patients compared to stage I + II + III patients. At the cut-off value of 6.22 g/L, the elevation of fibrinogen had a significant statistical relationship with thromboembolic disease (p=0.014) giving an hazard ratio of 3.868, confidence interval [1.358-11.012]. In multivariate analysis the hazard ratio doubled to 6.398, confidence interval [1,970-20,778]. DISCUSSION: Lung cancer patients showed an increase in coagulation factors that resulted in a state of hypercoagulability that was independent of the histology of lung cancer. The elevation of fibrinogen was predictive of thromboembolic disease at the early diagnosis of lung cancer before any therapy.

Contribution of the collagen binding activity (VWF:CB) in the range of tests for the diagnosis and classification of von Willebrand disease.

Von willebrand disease (VWD) is a common inherited bleeding disorder. The diagnosis may need a large panel of tests that differ in term of sensibility and specificity, and because of the effect of multifactorial modifiers (genetic or environmental); there is difficulty in defining diagnostic limits. We performed a panel of tests on 19 patients suffering from recurrent bleeding, to diagnose and classify VWD subtypes, by introducing the von Willebrand factor (VWF) collagen binding test (VWF:CB), then comparing the results with the activity of VWF risticetin cofactor (VWF:RCo) and multimer pattern. We considered 30% limit rate of VWF, as described by many authors, to make the diagnosis of VWD. The diagnosis of type 1 of VWD has been confirmed in 7patients, subtype 2A in 2 patients, subtype 2M in 2 patients and type 3 in 2 patients. We also defined a new group of 6 patients named "uncertain type 1" that didn't fill into the type 1 diagnostic criterion. The comparison between VWF:CB and VWF:RCo showed good correlation for all types of VWD except for type 2 while comparison between VWF:CB and multimer pattern showed good concordance for all types of VWD diagnosed. In conclusion, VWF:CB can be a good alternative to VWF:RCo for the diagnosis of quantitative deficiencies of VWF. It can also replace the multimer pattern study. However, the introduction of VWF:CB didn't help in the diagnosis of the "uncertain type 1" group of patients and cannot be a replacement for qualitative defect.