Type I interferon response and vascular alteration in chilblain-like lesions during the COVID-19 outbreak.

BACKGROUND: The outbreak of chilblain-like lesions (CLL) during the COVID-19 pandemic has been reported extensively, potentially related to SARS-CoV-2 infection, yet its underlying pathophysiology is unclear. OBJECTIVES: To study skin and blood endothelial and immune system activation in CLL in comparison with healthy controls and seasonal chilblains (SC), defined as cold-induced sporadic chilblains occurring during 2015 and 2019 with exclusion of chilblain lupus. METHODS: This observational study was conducted during 9-16 April 2020 at Saint-Louis Hospital, Paris, France. All patients referred with CLL seen during this period of the COVID-19 pandemic were included in this study. We excluded patients with a history of chilblains or chilblain lupus. Fifty patients were included. RESULTS: Histological patterns were similar and transcriptomic signatures overlapped in both the CLL and SC groups, with type I interferon polarization and a cytotoxic-natural killer gene signature. CLL were characterized by higher IgA tissue deposition and more significant transcriptomic activation of complement and angiogenesis factors compared with SC. We observed in CLL a systemic immune response associated with IgA antineutrophil cytoplasmic antibodies in 73% of patients, and elevated type I interferon blood signature in comparison with healthy controls. Finally, using blood biomarkers related to endothelial dysfunction and activation, and to angiogenesis or endothelial progenitor cell mobilization, we confirmed endothelial dysfunction in CLL. CONCLUSIONS: Our findings support an activation loop in the skin in CLL associated with endothelial alteration and immune infiltration of cytotoxic and type I IFN-polarized cells leading to clinical manifestations.

[Evaluation of the impact of a training program on vitamin K antagonists (VKA) implemented by pharmacy students aiming at improving the knowledge of patients receiving vitamin K antagonists during their hospital clinical training course]. / Évaluation de l'impact d'un programme de formation aux antagonistes de la vitamine K (AVK) pour les étudiants en pharmacie sur l'amélioration des connaissances des patients traités par AVK pris en charge au cours de leur stage clinique hospitalier.

INTRODUCTION: We developed a training program for pharmacy students aiming at supporting patients receiving vitamin K antagonists (VKAs). The objective was to estimate how the program impacts VKA-treated patient knowledge acquisition and/or improvement on their anticoagulant treatment. METHOD: Using dedicated tools, pharmacy students received education on VKA treatment. Once appointed to clinical wards of Assistance publique-Hôpitaux de Paris, they were in charge of evaluating patient's knowledge on VKA treatment before and after training. Evaluation was conducted using a face-to-face standardized interview (14-item questionnaire). A global score was calculated for each patient. An univariate and multivariate analysis was performed to identify potential variables influencing score result. RESULTS: One hundred and seventy VKA-treated patients were recruited in seven hospitals for evaluation of their knowledge on VKA treatment and on clinical at risk situations. Before intervention, patients obtained an average score of 12.3±3.2 (maximum: 18). Factors significantly associated with the score were possession of a VKA information booklet, VKA treatment duration, treatment initiation and age. Fifty-two patients with a low score were further trained by the pharmacy student. After intervention, their initial score was improved significantly, from 9.9±3.3 to 13.5±2.3 (P<0.0001). DISCUSSION AND CONCLUSION: Increasing patient knowledge is a way to decrease the rate of adverse effects. This study demonstrates that patients with primary poor knowledge improved it significantly thanks to pharmacy students' intervention. This may contribute to lower the VKA-associated risk of adverse events and consequently to the improvement of patients quality of life and healthcare expenditures.

Influence of genetics and non-genetic factors on acenocoumarol maintenance dose requirement in Moroccan patients.

WHAT IS KNOWN AND OBJECTIVE: Coumarin derivatives such as acenocoumarol represent the therapy of choice for the long-term treatment and prevention of thromboembolic diseases. Many genetic, clinical and demographic factors have been shown to influence the anticoagulant dosage. Our aim was to investigate the contribution of genetic and non-genetic factors to variability in response to acenocoumarol in Moroccan patients. METHODS: Our study included 114 adult Moroccan patients, receiving long-term acenocoumarol therapy for various indications. Tests for VKORC1 -1639G>A promoter polymorphism (rs9923231), CYP2C9*2 rs1799853, CYP2C9*3 rs1057910, and CYP4F2 rs2108622 alleles were undertaken using Taq Man(®) Pre-Developed Assay Reagents for allelic discrimination. The statistical analysis was performed using the SAS V9 statistical package. RESULTS AND DISCUSSION: Genotyping showed that the allele frequencies for the SNPs studied were no different to those found in Caucasians population. A significant association was observed between the weekly maintenance dose and the VKORC1 (P = 0·0027) and CYP2C9 variant genotypes (P = 0·0082). A final multivariate regression model that included the target International Normalized Ratio, VKORC1 and CYP2C9 genotypes explained 36·2% of the overall interindividual variability in acenocoumarol dose requirement. WHAT IS NEW AND CONCLUSION: Our study shows large interindividual variability in acenocoumarol maintenance dose requirement in our population. VKORC1 and CYP2C9 variants significantly affected acenocoumarol dose, in-line with results in other populations. For the Moroccan population, the SNPs that have the largest effect on acecoumarol dose are CYP2C9 rs1799853, CYP2C9 rs1057910 and VKORC1 rs9923231.

Imbalance between procoagulant factors and natural coagulation inhibitors contributes to hypercoagulability in the critically ill COVID-19 patient: clinical implications.

OBJECTIVE: Coronavirus Disease-2019 (COVID-19) predisposes patients to thrombosis which underlying mechanisms are still incompletely understood. We sought to investigate the balance between procoagulant factors and natural coagulation inhibitors in the critically ill COVID-19 patient and to evaluate the usefulness of hemostasis parameters to identify patients at risk of venous thromboembolic event (VTE). PATIENTS AND METHODS: We conducted an observational study recording VTEs defined as deep vein thrombosis or pulmonary embolism using lower limb ultrasound (92% of the patients), computed tomography pulmonary angiography (6%) and both tests (2%). We developed a comprehensive analysis of hemostasis. RESULTS: Ninety-two consecutive mechanically ventilated COVID-19 patients (age, 62 years [53-69] (median [25th-75th percentiles]); M/F sex ratio, 2.5; body-mass index, 28 kg/m2 [25-32]; past hypertension (52%) and diabetes mellitus (30%)) admitted to the Intensive Care Unit (ICU) from 03/11/2020 to 5/05/2020, were included. When tested, patients were receiving prophylactic (74%) or therapeutic (26%) anticoagulation. Forty patients (43%) were diagnosed with VTE. Patients displayed inflammatory and prothrombotic profile including markedly elevated plasma fibrinogen (7.7 g/L [6.1-8.6]), D-dimer (3,360 ng/mL [1668-7575]), factor V (166 IU/dL [136-195]) and factor VIII activities (294 IU/dL [223-362]). We evidenced significant discrepant protein C anticoagulant and chromogenic activities, combined with slightly decreased protein S activity. Plasma D-dimer >3,300 ng/mL predicted VTE presence with 78% (95%-confidence interval (95% CI), 62-89) sensitivity, 69% (95% CI, 55-81) specificity, 66% (95% CI, 51-79) positive predictive value and 80% (95% CI, 65-90) negative predictive value [area under the ROC curve, 0.779 (95%CI, 0.681-0.859), p=0.0001]. CONCLUSIONS: Mechanically ventilated COVID-19 patients present with an imbalance between markedly increased factor V/VIII activity and overwhelmed protein C/S pathway. Plasma D-dimer may be a useful biomarker at the bedside for suspicion of VTE.

[Interference of hemoglobin variants on glycated hemoglobin measurement using Architect ci8200 immunoassay (Abbott Diagnostics)]. / Immunodosage de l'hémoglobine glyquée sur ci8200 (Abbott Diagnostics) en présence d'hémoglobines anormales.

The objective of this study was to compare haemoglobin A(1c) (HbA(1c)) levels measured by the immunoturbidimetric assay on ci8200 Architect (Abbott Diagnostics) to those obtained by the high pressure liquid chromatography (HPLC) assay from Tosoh Bioscience. Firstly, we verified correlation between the two methods in 47 subjects without hemoglobin variants: the coefficient of correlation was 0.968 and the regression equation was as follows: y(Abbott) = 0.928x(Tosoh) - 0.081. We then measured HbA(1c) levels by both methods in blood samples of 23 patients with a structural hemoglobin variant (A/S, A/C or S/C). Analysis of these samples revealed significant discrepancies between results of both methods, as indicated by the higher mean value obtained by immunoturbidimetric assay when compared to HPLC assay (7.17 +/- 2.68% vs. 5.86 +/- 1.41%). Classification of patients according to the HAS (French Haute Autorité de Santé) recommendations HbA(1c) was not modified for 68% of patients with abnormal hemoglobin. However, in 32% of cases, discrepancies between the two methods may have clinical importance, which justifies the occurrence of an abnormal hemoglobin when the HbA(1c) assay is performed by the method immunoturbidimetry Abbott Diagnostics.

Bleeding risk of antiplatelet drugs compared with oral anticoagulants in older patients with atrial fibrillation: a systematic review and meta-analysis.

Essentials Hemorrhagic risk of antiplatelet drugs is generally thought to be lower than anticoagulants. We systematically reviewed trials comparing antiplatelet and anticoagulant drugs in older patients. Overall, the risk of major bleeding was similar with antiplatelet and with anticoagulant drugs. In elderly patients, risks and benefits of antiplatelet drugs should be carefully weighted. SUMMARY: Background The hemorrhagic risk of antiplatelet drugs in older patients could be higher than is usually assumed. Objective To compare the bleeding risk of antiplatelet drugs and oral anticoagulants in elderly patients. Methods We carried out a systematic review and meta-analysis. We searched PubMed, EMBASE and the Cochrane Library up to January 2016 for randomized and non-randomized controlled trials (RCTs) and parallel cohorts comparing antiplatelet drugs and oral anticoagulants in patients aged 65 years or older. Two independent authors assessed studies for inclusion. The pooled relative risk (RR) of major bleeding was estimated using a random model. Results Seven RCTs (4550 patients) and four cohort studies (38 649 patients) met the inclusion criteria. The risk of major bleeding when on aspirin or clopidogrel was equal to that when on warfarin in RCTs (RR, 1.01; 95% confidence interval (95% CI), 0.69-1.48; moderate-quality evidence), lower than when on warfarin in non-randomized cohort studies (RR, 0.87; 95% CI, 0.77-0.99; low-quality evidence) and not different when all studies were combined (RR, 0.86; 95% CI, 0.73-1.01). Bleeding of any severity (RR, 0.70; 95% CI, 0.57-0.86) and intracranial bleeding (RR, 0.46; 95% CI, 0.30-0.73) were less frequent with antiplatelet drugs than with warfarin. All-cause mortality was similar (RR, 0.99). Subgroup analysis suggested that major bleeding might be higher with warfarin than with aspirin in patients over 80 years old. Conclusion Elderly patients treated with aspirin or clopidogrel suffer less any-severity bleeding but have a risk of major bleeding similar to that of oral anticoagulants, with the exception of intracranial bleeding.

Interindividual variability in dabigatran and rivaroxaban exposure: contribution of ABCB1 genetic polymorphisms and interaction with clarithromycin.

Essentials Rivaroxaban and dabigatran are substrates of the P-glycoprotein (P-gp) encoded by the ABCB1 gene. We tested the effect of ABCB1 polymorphisms and of a P-gp inhibitor on both drugs' pharmacokinetics. The ABCB1 genotype was not a clinically relevant determinant of both drugs' pharmacokinetics. Administration of P-gp inhibitors with dabigatran or rivaroxaban should be exercised with caution. SUMMARY: Background The direct oral anticoagulants (DOACs) dabigatran and rivaroxaban are both substrates of the P-glycoprotein (P-gp) transporter, encoded by the ABCB1 gene. Rivaroxaban is metabolized by cytochrome P450 A4 (CYP3A4). Interindividual variability in DOAC exposure and frequent P-gp-associated drug-drug interactions have been described in patients. Objective To assess the influence of ABCB1 polymorphisms on the pharmacokinetics of dabigatran and rivaroxaban, associated or not with clarithromycin, a P-gp and CYP3A4 inhibitor. Methods Sixty healthy male volunteers, selected according to ABCB1 genotype (20 homozygous mutated, 20 heterozygous mutated, and 20 wild-type for haplotype 2677-3435), were included in this randomized, two-center, crossover study. All received sequentially a single dose of dabigatran etexilate (300 mg) and rivaroxaban (40 mg) associated or not with clarithromycin. Peak plasma concentration and area under the curve (AUC) were compared across the three ABCB1 genotypes. The effect of clarithromycin on dabigatran or rivaroxaban pharmacokinetics was assessed. Results Interindividual coefficients of variation for AUC were 77% for dabigatran and 51% for rivaroxaban. ABCB1 genotype did not significantly affect drug pharmacokinetics: AUC ratios between mutant-allele carriers and wild-type volunteers were 1.27 (95% confidence interval [CI] 0.84-1.92) and 1.20 (95% CI 0.96-1.51) for dabigatran and rivaroxaban, respectively. Clarithromycin coadministration led to a two-fold increase in both drugs' AUC, irrespective of ABCB1 genotype: ratios of geometric means were 2.0 (95% CI 1.15-3.60) and 1.94 (95% CI 1.42-2.63) for dabigatran and rivaroxaban, respectively. Conclusions ABCB1 genotype is not a significant determinant of interindividual variability in dabigatran and rivaroxaban pharmacokinetics. The levels of one drug did not predict the levels of the other. Coadministration of a P-gp/CYP3A4 inhibitor with dabigatran or rivaroxaban may warrant caution in patients at risk of overexposure.

[Prescribing guidance pocket chart on the initiation of warfarin therapy in a geriatric hospital: a useful but challenging tool]. / Diffusion d'une fiche d'aide à la prescription pour l'initiation d'un traitement par warfarine dans un hôpital gériatrique: intérêt et difficultés de mise en place.

Elderly patients are at high risk of over-anticoagulation and of haemorrhagic risk when on warfarin, especially during treatment induction. In Charles Foix Hospital, a 800-bed geriatric hospital, we specifically developed for in-patients older than 70 years (target INR 2.5) a simple low-dose warfarin induction regimen. The dosing recommendations were summarized on a prescribing guidance pocket chart. Eighteen months after the distribution of the chart, we conducted a one-year observational study in order to evaluate: i/ the time needed to achieve the warfarin maintenance dose; ii/ the prescriber'adherence to the recommendations; iii/ the benefit for elderly patients receiving warfarin therapy. The mean time needed to achieve the warfarin maintenance dose was 12.3 +/- 7.0 days for the 89 patients included in the study: 10.6 +/- 5.9 days for the 30 patients whose prescribers followed the recommendations versus 13.5 +/- 7.6 days for the 59 patients whose prescribers did not follow the recommendations. There is a trend to a more frequent over-anticoagulation in patients whose prescribers did not follow the recommendations. The duration of the heparin-warfarin overlap was significantly shorter when the recommendations were followed. Finally, the reasons for non-adherence to the recommendations were analyzed. This study illustrates an assessment of practice in an health care institution.

[Plasma cell leukaemia]. / La leucémie à plasmocytes.

We report a case of primary plasma cell leukaemia, with an absolute count of plasma cells of 53 Giga/L, diagnosed in a 83-year-old woman. The patient's condition improved, with no circulating plasma cells after 3 weeks of treatment, in response to the combination of thalidomide and dexamethasone administered for 5 days followed by thalidomide alone. The clinical presentation, the morphological, flow cytometric and pathophysiological characteristics of the plasma cell leukaemia and the treatment are summarised in this paper.

Multimodal assessment of non-specific hemostatic agents for apixaban reversal.

BACKGROUND: Non-specific hemostatic agents, namely activated prothrombin complex concentrate (aPCC), PCC and recombinant activated factor (F) VII (rFVIIa), can be used, off-label, to reverse the effects of FXa inhibitors in the rare cases of severe hemorrhages, as no approved specific antidote is available. We have evaluated the ability of aPCC, PCC and rFVIIa to reverse apixaban. METHODS: Healthy volunteer whole blood was spiked with therapeutic or supra-therapeutic apixaban concentrations and two doses of aPCC, PCC or rFVIIa. Tests performed included a turbidimetry assay for fibrin polymerization kinetics analysis, scanning electron microscopy for fibrin network structure observation, thrombin generation assay (TGA), thromboelastometry, prothrombin time and activated partial thromboplastin time. RESULTS: aPCC generated a dense clot constituting thin and branched fibers similar to those of a control without apixaban, increased fibrin polymerization velocity and improved quantitative (endogenous thrombin potential and peak height) as well as latency (clotting and lag times) parameters. Adding PCC also improved the fibrin and increased quantitative parameters, but fibrin polymerization kinetics and latency parameters were not corrected. Finally, rFVIIa improved latency parameters but failed to restore the fibrin network structure, fibrin polymerization velocity and quantitative parameters. CONCLUSION: aPCC was more effective than PCC or rFVIIa in reversing in vitro the effects of apixaban. aPCC rapidly triggered the development of an apparently normal fibrin network and corrected latency and quantitative parameters, whereas PCC or rFVIIa had only a partial effect.

[Relationship between maintenance dosages of fluindione (Préviscan) and warfarin (Coumadin) for patients 70 years and older]. / Nomogramme d'équivalence entre les posologies de fluindione (Préviscan) et de warfarine (Coumadine) pour des patients de 70 ans et plus.

INTRODUCTION: Switching from fluindione, an indanedione vitamin K antagonist derivative, to warfarin, a coumarin one, or vice versa, requires to know the relationships between dosages of these two molecules. METHODS: We conducted a prospective study in 288 consecutive patients aged 70 years and over, converted from fluindione to warfarin. Patients who were retained for the analysis were those for whom maintenance dosages were obtained for both vitamin K antagonists. RESULTS: Eighty-two patients, mean aged 83 ± 6 years, were analysed. The average daily maintenance dosages were 13.8 ± 6.7 mg (range 5-35) and 3.7 ± 1.7 mg (range 1-8) for fluindione and warfarin, respectively. Using a linear regression model, we built a transition algorithm for the maintenance dosages of warfarin and fluindione. CONCLUSION: This is the first study to propose a conversion algorithm to help prescribers to estimate the maintenance dosage when it is necessary for a patient to switch from fluindione to warfarin or conversely.

Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene.

A de novo insertion of an Alu repeated DNA element was found within exon V of the factor IX gene in a patient with severe haemophilia B. The element interrupts the reading frame of the mature factor IX at glutamic acid 96 resulting in a stop codon within the inserted sequence. The Alu repeat is 322 bp long, and the 5' region is shortened by 38 bp. The insertion created a target site duplication of 15 bp consistent with retroposition, and contains a pure polyadenine tract of at least 78 resides at the 3' end. The nucleotide sequence agrees with a consensus for an Alu subfamily which is evolutionarily the most recently inserted, suggesting that it is an exact copy of a putative source gene. These observations indicate that retroposition of Alu elements is a continual process and a mechanism for generating human genetic defects.

Functional analysis of the Arg91Gln substitution in the factor VIII binding domain of von Willebrand factor demonstrates variable phenotypic expression.

An Arg91Gln substitution in the mature von Willebrand factor (vWF) has been associated with defective binding of vWF to factor VIII (FVIII). We studied four families with members initially classified as having type I von Willebrand disease (vWD) who were either homozygous or heterozygous for the Arg91Gln change. The first family was the original case described by Nishino et al. (1) where three members were homozygous for the Gln91 allele. They had a low FVIII coagulant activity:vWF antigen (VIIIC:vWFAg) ratio, from 0.29 to 0.44, and the ability of their plasma vWF to bind FVIII was markedly decreased. All the heterozygous members had normal vWF and FVIII levels but the capacity of their plasma vWF to bind FVIII was reduced and intermediate between the homozygous members and normals. The affected individual from the second family was heterozygous for the Gln91 allele and demonstrated a VIIIC:vWFAg ratio of 0.98. The FVIII binding assay confirmed the heterozygous status indicating that the moderately low levels of vWF were due to reduced expression of both alleles. The propositus from the third family was also heterozygous and had below normal levels of vWF as well as a low VIIIC:vWFAg ratio of 0.34; however, FVIII binding to her plasma vWF was similar to that of the homozygous individuals suggesting that Gln91-vWF was the major circulating form. Her daughter who has type I vWD inherited the allele without the Gln91 mutation indicating that the expression of this allele was indeed impaired. The heterozygous patient in the fourth family had a vWF level of 24 U/dl but an VIIIC:vWFAg ratio greater than 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Epitope mapping of inhibitory monoclonal antibodies to human von Willebrand factor by using recombinant cDNA libraries.

Two recombinant expression libraries containing small (300-600 base pairs) cDNA fragments of von Willebrand Factor (vWF) were screened in order to map the epitope of monoclonal antibodies (MAbs) to vWF. Among eleven MAbs tested, seven were effectively mapped. The epitopes of MAbs 418 and 522, which inhibit the binding of vWF to Factor VIII (FVIII), were localized between Leu 2 and Arg 53 and between Glu 35 and Ile 81 of the vWF subunit respectively, within the N-terminal trypsin fragment called SpIII-T4 [amino acids (aa) 1-272] which contains a binding domain for FVIII. The epitope of MAb 710, which inhibits the binding of vWF to glycoprotein Ib (GPIb), was identified between Ser 593 and Ser 678 on the tryptic 52/48 kDa fragment (aa 449-728) which contains binding domains for GPIb, collagen, heparin, sulfatides and subendothelium extracellular matrices. The epitope of MAb 723, which does not interfere with any known function of vWF, was localized between Ser 523 and Gly 588. The epitopes of MAb 505 and MAb 400, which inhibit the binding of vWF to collagen, were identified between Leu 927 and Arg 1114 within the SPI fragment (aa 911-1365) corresponding to the central part of the vWF subunit. The epitope of MAb 9, which inhibits the binding of vWF to GPIIb/IIIa, was identified in the C-terminal part of the vWF subunit between Gln 1704 and Asp 1746, the latter being the third aa of the RGD sequence common to adhesive proteins and serving as a recognition site for integrin receptors.

Elderly patients treated with tinzaparin (Innohep) administered once daily (175 anti-Xa IU/kg): anti-Xa and anti-IIa activities over 10 days.

Since low molecular weight heparins (LMWH) are partly eliminated by renal excretion, their pharmacodynamic profile may be modified in very elderly patients with age-related renal impairment. The aim of this prospective study was to determine whether tinzaparin (Innohep) 175 anti-Xa IU/kg administered subcutaneously once daily over 10 days does accumulate in hospital patients greater than 70 years of age. Plasma anti-Xa and anti-IIa amidolytic levels and APTT were determined prior to the first injection (day 0), and then, at peak level i.e. 5 h after the second injection (day 2) and subsequently on days 5, 7 and 10. Thirty consecutive inpatients (6 men, 24 women) requiring LMWHs at a curative dose for acute thromboembolic disease were included. Patients' characteristics (mean +/- SD) were: age 87.0+/-5.9 years (range 71-96), body weight 62.7+/-14.6 kg (range 38-90) and creatinine clearance 40.6+/-15.3 mL/min (range 20-72). The mean actual dose of tinzaparin delivered was 174.8 anti-Xa IU/kg. Since no patient had an anti-Xa activity above 1.5 IU/mL, the dose of tinzaparin remained fixed over 10 days. Anti-Xa and anti-IIa peak levels measured on day 2 were 0.66+/-0.20 IU/mL (range 0.26-1.04) and 0.33+/-0.10 IU/mL (range 0.18-0.55), respectively. Ex vivo anti-Xa/anti-IIa ratios were close to 2.1. APTT ratios (patient/control) were strongly correlated with anti-IIa activity (p <0.01). There was no progressive increase of the anti-Xa and anti-IIa activities after repeated administration of tinzaparin over the 10 day treatment period. No correlation was found between anti-Xa and anti-IIa activities and age, weight, or creatinine clearance. No major bleeding occurred during the study and only one minor haematoma at the injection site was reported. No thrombo-embolic complication or death occurred. Tinzaparin may thus be administered safely at a treatment dose (175 anti-Xa IU/kg) in older patients with age-related renal impairment. Neither dose adjustment, nor serial anti-Xa activity monitoring seems to be required in patients with creatinine clearance above 20 mL/min during the first ten day treatment.

Characterization of recombinant von Willebrand factors mutated on cysteine 509 or 695.

The interacting domain of vWF with platelet GPIb has been shown to overlap the large A1 loop formed by the intra-chain disulfide bond linking Cys 509 to Cys 695. In order to further investigate the role of the conformation of this region, we have expressed in COS-7 cells three mutated full-length recombinant vWFs (rvWFs) in which the substitutions Cys509Gly, Cys509Arg or Cys695Gly have been introduced by site-directed mutagenesis. SDS-agarose gel electrophoresis demonstrated an impaired multimerization of the mutants with undetectable high molecular weight multimers and a decrease of the relative amounts of the intermediate sized multimers. Binding analysis showed that rvWFC509G and rvWFC509R did not interact with botrocetin but spontaneously interacted with GPIb; the latter binding remained unchanged in the presence of ristocetin. This indicates that the substitution of Cys509 by Gly or Arg creates a conformation of vWF that increases its binding to GPIb. In contrast, rvWFC695G which did not react with botrocetin was also unable to interact with GPIb even in the presence of ristocetin, indicating that sequences interacting with GPIb are masked and/or disrupted. In conclusion, the substitution of each of the Cys509 and 695 results in mutant proteins which may be "locked" into active or inactive conformations in regard to the binding to platelet GPIb receptor.

Venous thrombosis in older people: prevalence of the factor V gene mutation Q506.

OBJECTIVES: Old age is usually considered to be a risk factor for venous thromboembolism, in conjunction with other factors such as heart failure, major surgery, cancer, long-term immobilization, and antiphospholipid antibodies. Genetic risk factors, especially inherited deficiencies in coagulation inhibitors, also play a role in the pathogenesis of thrombosis, but these are usually diagnosed in thrombophilic patients before the age of 50. The factor V Q506 mutation, responsible for activated protein C resistance, was recently linked to thromboembolic disease. We therefore investigated the prevalence of biological risk factors in older hospital patients with venous thromboembolism. DESIGN: A 2-year study period. SETTING: Ivry sur Seine (Paris), France. PARTICIPANTS: Seventy-nine geriatric patients (60 women and 19 men, mean age 83+/-6.8 years, range 70-102 years) who had had at least one proven episode of venous thromboembolism were enrolled over a 2-year period. MEASUREMENTS: Lupus anticoagulant and antithrombin (AT), protein C (PC), and protein S (PS) levels were determined in plasma. The factor V Q506 mutation was detected on genomic DNA. RESULTS: Lupus anticoagulant was detected in two women, one of whom also had a high level of anticardiolipin IgG, leading to the diagnosis of an antiphospholipid syndrome. No hereditary deficiency in AT, PC, or PS was found, but one patient had an acquired AT deficiency. Interestingly, nine of the 79 patients (11.4%, six women and three men) were heterozygous for the factor V Q506 mutation, although none were homozygous. The only major risk factor for thrombosis identified in these patients was prolonged immobilization in four cases. Four of the nine patients who were heterozygous for the factor V Q506 mutation had recurrent thromboembolism, and two of these patients had been immobilized for long periods. CONCLUSIONS: This study confirms that hereditary deficiencies in coagulation inhibitors, and the lupus anticoagulant, are rarely involved in the pathogenesis of venous thromboembolism in older subjects. In contrast, the factor V Q506 mutation was frequently associated with thrombosis (11.4% of our patients) and should, therefore, be considered an important risk factor in the older people.

Ruling out acute deep vein thrombosis by ELISA plasma D-dimer assay versus ultrasound in inpatients more than 70 years old.

In geriatric care, deep-vein thrombosis (DVT) is mostly diagnosed by noninvasive techniques. The objectives of this prospective study were: (1) to evaluate the power of ELISA plasma D-dimer assay versus ultrasound (US) in ruling out acute DVT of the lower limbs in symptomatic geriatric inpatients, and (2) to determine the most effective D-dimer cutoff value over the age of 70 years. Over a 10-month period, inpatients with suspected lower limb DVT simultaneously underwent US examination and ELISA plasma D-dimer assay. Noninclusion criteria were comorbid conditions able to modify the D-dimer level. Data were processed by receiver operating characteristic (ROC) curve analysis. In total, 150 patients (125 women, 25 men), average age 86.3 years (range 70-101) were included. A diagnosis of lower limb DVT was established in 53 patients (35.3%). With a 500 ng/mL D-dimer cutoff conventional value, DVT was ruled out in only five patients (3.3%), whereas a 750 ng/mL value ruled out DVT in 19 patients (12.7%) with a sensitivity of 98.1%, and a negative predictive value of 95.0%. The only false negative corresponded to a patient with a 15-mm thrombus in the distal calf. In inpatients above 70, an ELISA plasma D-dimer value smaller than 750 ng/mL is a rapid reliable noninvasive means to rule out lower limb DVT, if color flow Doppler ultrasound is not available on site.

[Biological diagnosis of hemoglobinopathies by phenotype analysis]. / Diagnostic biologique des hémoglobinopathies par analyse du phénotype.

Haemoglobinopathies are defined by the presence of qualitative and/or quantitative globin chains abnormalities. In most cases, laboratory diagnosis relies on phenotype analysis. Ethnic group, clinical data, complete blood count in absence of recent blood transfusion, iron status, should be taken into account for the diagnosis. Electrophoresis using an alkaline pH buffer system is used as an initial technique for the detection of haemoglobin variants, showing abnormal bands as compared with reference samples. Isoelectric focusing is more resolutive and could be preferred for this reason: If the migration pattern differs from normal, additional confirmatory tests, as citrate agar electrophoresis (acid pH), are required. Relative amounts of variant haemoglobin (Hb A2, F and S...) are quantified using chromatographic methods. Other tests, such as solubility tests, stability tests, Kleihauer-Betke test, are useful in specific cases. The diagnosis relies on clinical and laboratory findings. In the Paris area, the most common variant is Hb. S. Taalassemia traits are also very common. Screening of SS. S beta D. SO Arab, SD Punjab, SC, or thalassaemia major should be made early so that patients found to have one of these varieties should be referred to haemoglobinopathy centers.

[Evaluation of hemostasis in venous thromboembolism pathology]. / Exploration de l'hémostase dans la pathologie thromboembolique veineuse.

Thromboembolic disease results from an hypercoagulable state and multifactorial causes may lead to hypercoagulability. Thrombogenic risk factors can be acquired and/or inherited. For each thrombophilic patient, the main clinical features retained are: the patient age, the familial history, the recurrence of thromboembolic events, an unusual site of thrombosis. Anti-phospholipid antibodies, which are considered as acquired thrombogenic risk factors, can be detected with coagulation tests and/or Elisa methods. The association of antiphospholipid antibodies with thrombosis is defined as the anti-phospholipid syndrome. Last decades, genetic risk factors were identified. First of all, antithrombin, protein C and protein S deficiencies were described. These deficiencies are involved in about 10% of patients who develop thrombosis before the age of 50. In 1993, a new genetic risk factor was discovered: activated protein C resistance which is due to the Q506 mutation in factor V. This defect represents the most prevalent abnormality of inherited thrombophilia, affecting 20 to 40% of thrombophilic patients. Interestingly, hyperhomocysteinemia, known as potentially predisposing to arterial disease, was also recognized as a risk factor for venous occlusive disease. Several genes encoding homocystein metabolism enzymes, such as cystathionine beta-synthase or methylenetetrahydrofolate reductase are concerned. Establishment of a causal association between the presence of a biological abnormality and the occurrence of thrombosis may lead to an adapted prophylaxis whatever the risk situation.