Nathalie Hézard

FACTOR V LEIDEN : DETECTION IN WHOLE BLOOD BY ASA PCR USING AN ADDITIONAL MISMATCH IN ANTEPENULTIMATE POSITION

Factor V Leiden mutation was initially detected in thrombophilic patients and relatives by PCR RFLP (Restriction Fragment Length Polymorphism) according to Bertina (1). This technique presents some drawbacks and the current trend is to simplify the diagnosis. We describe a technique of Allele Specific Amplification (ASA) which is optimized in terms of reliability: an additional mismatch in antepenultimate position enables to obtain the same specificity as PCR RFLP. Furthermore, coamplification of internal control warrants an optimal sensitivity. All the PCR have been simplified: the DNA extraction improvement allows to analyse the genotype with only a few microliters of whole blood whatever the anticoagulant and the procedure of preservation (freezing, dried blood spots, storage at +4 degrees C for several days). This technique saves time. Moreover, full automation of the ASA technique may be shortened thanks to the lack of extraction and the positive/negative reading of the PCR signal.

AlphaIIbbeta3 integrin: new allelic variants in Glanzmann thrombasthenia, effects on ITGA2B and ITGB3 mRNA splicing, expression, and structure-function.

Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation due to defects in integrin αIIbβ3 (ITGA2B, ITGB3), a fibrinogen receptor. Mutations from 24 GT patients and two carriers of various origins, Caucasian, North‐African and Asian were characterized. Promoter and exon sequences of αIIb and β3 genes were amplified and directly sequenced. Among 29 identified mutations, 17 new allelic variants resulting from nonsense, missense and deletion/insertion mutations were described. RNA alterations were evaluated by using Web servers. The αIIb p.S926L, p.V903F, and β3 p.C38Y, p.M118R, p.G221D substitutions prevented complex expression at the surface of COS‐7 cells by altering the αIIb or the β3 subunit structure. As shown by free energy analyses applied on the resolved structure of αIIbβ3 and structural modeling of the mutant, the p.K253M substitution of β3 helped to define a key role of the K253 in the interaction of the αIIb β‐propeller and the β3 β‐I domains. finally, the αIIb p.Q595H substitution allowed cell surface expression of the complex but its corresponding c.2800G>T mutation is predicted to alter normal RNA splicing. In conclusion, our study yielded the discovery of 17 new GT allelic variants, revealed the key role of K253 of αIIb for the αIIbβ3 complex formation and provides an additional example of an apparently missense mutation causing a splicing defect. Hum Mutat 30:1–10, 2010.

Interleukin-10 promoter polymorphism and venous thrombosis. A case-control study

The human interleukin-10 promoter gene is highly polymorphic. IL-10 polymorphisms have been associated with various autoimmune and lymphoproliferative disorders. Although IL-10 has been shown to modulate thrombin generation in several experimental models, it is not known whether IL-10 polymorphisms could be a risk factor for venous thrombosis. We therefore conducted a case-control study comparing 74 consecutive patients who experienced at least one episode of documented venous thromboembolic event and 100 healthy controls. All subjects were Caucasians. Five polymorphisms of the IL-10 promoter gene were studied: two highly polymorphic dinucleotide repeats, IL-10 R and IL-10G, and three single nucleotide polymorphisms at position -1082, -819 and -592. Factor VG1691C Leiden mutation was systematically determined. Multivariate logistic regression analysis showed that IL-10 G13 and G10 alleles are independent risk factors for venous thrombosis (Odds ratio:OR = 3.33, p = 0.003 and OR = 2.83, p = 0.03, respectively). Furthermore, IL-10 G10 allele is more frequent in recurrent disease.

Elastin-Derived Peptides Are New Regulators of Thrombosis

Objective— Elastin is the major structural extracellular matrix component of the arterial wall that provides the elastic recoil properties and resilience essential for proper vascular function. Elastin-derived peptides (EDP) originating from elastin fragmentation during vascular remodeling have been shown to play an important role in cell physiology and development of cardiovascular diseases. However, their involvement in thrombosis has been unexplored to date. In this study, we investigated the effects of EDP on (1) platelet aggregation and related signaling and (2) thrombus formation. We also characterized the mechanism by which EDP regulate thrombosis. Approach and Results— We show that EDP, derived from organo-alkaline hydrolysate of bovine insoluble elastin (kappa-elastin), decrease human platelet aggregation in whole blood induced by weak and strong agonists, such as ADP, epinephrine, arachidonic acid, collagen, TRAP, and U46619. In a mouse whole blood perfusion assay over a collagen matrix, kappa-elastin and VGVAPG, the canonical peptide recognizing the elastin receptor complex, significantly decrease thrombus formation under arterial shear conditions. We confirmed these results in vivo by demonstrating that both kappa-elastin and VGVAPG significantly prolonged the time for complete arteriole occlusion in a mouse model of thrombosis and increased tail bleeding times. Finally, we demonstrate that the regulatory role of EDP on thrombosis relies on platelets that express a functional elastin receptor complex and on the ability of EDP to disrupt plasma von Willebrand factor interaction with collagen. Conclusions— These results highlight the complex nature of the mechanisms governing thrombus formation and reveal an unsuspected regulatory role for circulating EDP in thrombosis.

Evaluation of dabigatran, rivaroxaban and apixaban target-specific assays in a multicenter French study

Dabigatran etexilate, rivaroxaban and apixaban (DOACs) are widely used and measurement of their concentration is desirable in certain clinical situations. Target-specific assays are available but limited information exists on their performance especially in their ability to accurately measure low and high concentrations. AIMS To define, in a multicenter study, the precision and accuracy of DOAC measurements in daily practice. METHODS 15 plasma samples (kindly provided by Hyphen-Biomed) spiked with 5 blinded concentrations of dabigatran, rivaroxaban or apixaban (targeted 0-40-100-250-500ng/mL, actual concentrations measured by HPLC-MS/MS), were sent to 30 haemostasis laboratories. DOAC concentration, PT and aPTT were measured once in each sample using local reagents. Interlaboratory precision was determined by its coefficient of variation (CV) and accuracy by its bias. RESULTS 464 DOAC measurements were performed in the 30 laboratories using 4 dabigatran and 5 rivaroxaban/apixaban calibrated assays on 3 analysers. Inter-laboratory CVs were below 18% for concentrations ≥100ng/mL, and higher for concentrations ~40ng/mL; biases were below 8% for all drugs and concentrations. In DOAC-free samples, concentrations were all below the lower limit of quantification except for one value (dabigatran: 35ng/mL). Depending on the concentrations, significant differences were found between reagents in rivaroxaban and apixaban concentration values. PT and aPTT ratios displayed a low sensitivity to apixaban. CONCLUSION Our results suggest that calibrated DOAC assays allow the reliable measurement of a wide range of drug concentrations, even though improvement of their performances is necessary, especially for measuring low concentrations.

Is flow cytometry accurate enough to screen platelet autoantibodies

BACKGROUND: The diagnosis of immune thrombocytopenic purpura (ITP) is a diagnosis of exclusion, as stated by international guidelines. Nevertheless, the assessment of platelet (PLT) antibodies has been reported as helpful for the diagnosis and the follow‐up of ITP patients. PLT antibodies are detected by highly specialized assays, such as monoclonal antibody–specific immobilization of PLT antigen (MAIPA) test. Flow cytometry for PLT‐associated immunoglobulin G (PAIgG) detection has been described more recently. This study was meant to evaluate the utility of flow cytometry to screen accurately patients needing further MAIPA testing.

Platelet VASP phosphorylation assessment in clopidogrel-treated patients: Lack of agreement between Western blot and flow cytometry

Vasodilator-stimulated phosphoprotein (VASP) 239 phosphorylation flow cytometric assessment has been reported as a tool to evaluate the responsiveness to clopidogrel in coronary heart disease (CHD) patients. We report for the first time the comparison between flow cytometry and two challenger assays, aggregometry and Western blot. We studied 21 clopidogrel-treated CHD patients, and 28 healthy volunteers. Aggregometry showed platelet function inhibition in patients. VASP 239 phosphorylation was assessed using flow cytometry and Western blot. ADP receptor response index (RI) were calculated using the formula (PGE1) – (PGE1 + ADP)/(PGE1) × 100. Flow cytometry was not able to detect clopidogrel intake, as RI were 99 ± 10% [68–130] in healthy volunteers, and 91 ± 17% [66–127] in treated patients (ns). On the contrary, RI mean in Western blot was 91 ± 8% [76–127] in healthy volunteers, and 37 ± 25% [4–80] in patients (p<0.05). The extreme values in Western blot revealed inter-individual variability in response to treatment. The comparison between both tests showed a total lack of agreement. Flow cytometric VASP 239 phosphorylation assay lacks sensitivity to detect clopidogrel intake, contrary to Western blot and aggregometry. Caution is required before classifying patients as ‘low-responders’ to thienopyridines using such method.

Differential coagulation inhibitory effect of fondaparinux, enoxaparin and unfractionated heparin in cell models of thrombin generation.

Anticoagulants, including unfractionated heparin (UFH), enoxaparin and fondaparinux, are approved drugs in acute coronary syndrome (ACS). Monocytes and monocyte-derived microparticles (MMPs) play an important procoagulant role in ACS by expressing high tissue factor (TF) levels, which in turn triggers thrombin generation. The objective of our study is to compare the in-vitro inhibitory effect of UFH, enoxaparin and fondaparinux in monocytes and MMP models. Human-elutriated monocytes were activated for 5 and 18 h by lipopolysaccharide to obtain activated monocytes (ac-M) or MMPs, respectively. Thrombin generation inhibition was assessed using ac-M or MMPs mixed with platelet-poor plasma containing increased concentrations of anticoagulants. Thrombin generation inhibition was dose-dependent with a differential effect according to the drug: the highest for UFH, the lowest for fondaparinux. Rate index was the most sensitive parameter. For fondaparinux, its IC50 values (anti-Xa IU/ml) were 0.59 ± 0.05 for ac-M and 0.17 ± 0.03 for MMPs. For enoxaparin, rate index IC50 values were 0.27 ± 0.03 for ac-M and 0.19 ± 0.02 for MMPs. Our data support the notion that cell-induced thrombin generation assay may be a reliable alternative to anti-Xa assessment in determining patient anticoagulation level.