Valérie Proulle

Increased levels of circulating microparticles in primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity

IntroductionCell stimulation leads to the shedding of phosphatidylserine (PS)-rich microparticles (MPs). Because autoimmune diseases (AIDs) are characterized by cell activation, we investigated level of circulating MPs as a possible biomarker in primary Sjögrens syndrome (pSS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).MethodsWe measured plasma levels of total, platelet and leukocyte MPs by prothrombinase capture assay and flow cytometry in 43 patients with pSS, 20 with SLE and 24 with RA and in 44 healthy controls (HCs). Secretory phospholipase A2 (sPLA2) activity was assessed by fluorometry. Soluble CD40 ligand (sCD40L) and soluble P-selectin (sCD62P), reflecting platelet activation, were measured by ELISA.ResultsPatients with pSS showed increased plasma level of total MPs (mean ± SEM 8.49 ± 1.14 nM PS equivalent (Eq), P < 0.0001), as did patients with RA (7.23 ± 1.05 n PS Eq, P = 0.004) and SLE (7.3 ± 1.25 nM PS Eq, P = 0.0004), as compared with HCs (4.13 ± 0.2 nM PS Eq). Patients with AIDs all showed increased level of platelet MPs (P < 0.0001), but only those with pSS showed increased level of leukocyte MPs (P < 0.0001). Results by capture assay and flow cytometry were correlated. In patients with high disease activity according to extra-glandular complications (pSS), DAS28 (RA) or SLEDAI (SLE) compared with low-activity patients, the MP level was only slightly increased in comparison with those having a low disease activity. Platelet MP level was inversely correlated with anti-DNA antibody level in SLE (r = -0.65; P = 0.003) and serum β2 microglobulin level in pSS (r = -0.37; P < 0.03). The levels of total and platelet MPs were inversely correlated with sPLA2 activity (r = -0.37, P = 0.0007; r = -0.36, P = 0.002, respectively). sCD40L and sCD62P concentrations were significantly higher in pSS than in HC (P ≤ 0.006).ConclusionsPlasma MP level is elevated in pSS, as well as in SLE and RA, and could be used as a biomarker reflecting systemic cell activation. Level of leukocyte-derived MPs is increased in pSS only. The MP level is low in case of more severe AID, probably because of high secretory phospholipase A2 (sPLA2) activity, which leads to consumption of MPs. Increase of platelet-derived MPs, sCD40L and sCD62P, highlights platelet activation in pSS.

Spectrum of the Mutations in Bernard–Soulier Syndrome

Bernard–Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb‐IX‐V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbβ), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.

Thromboxane synthase mutations in an increased bone density disorder (Ghosal syndrome)

Studying consanguineous families with Ghosal hematodiaphyseal dysplasia syndrome (GHDD), a disorder of increased bone density, we identified mutations in TBXAS1, which encodes thromboxane synthase (TXAS). TXAS, an enzyme of the arachidonic acid cascade, produces thromboxane A2 (TXA2). Platelets from subjects with GHDD showed a specific deficit in arachidonic acid–produced aggregation. We also found that TXAS and TXA2 modulated expression of TNFSF11 and TNFRSF11B (encoding RANKL and osteoprotegerin (OPG), respectively) in primary cultured osteoblasts.

Two novel mutations, Q1053H and C1060R, located in the D3 domain of von Willebrand factor, are responsible for decreased FVIII-binding capacity

Summary. In type 2N von Willebrand disease (VWD), von Willebrand factor (VWF) is characterized by a markedly decreased affinity for Factor VIII (FVIII), and the mutations responsible are essentially located in the D′ domain of VWF. We report the identification, in seven unrelated French families, of two novel type 2N VWD mutations, Q1053H and C1060R (Gln290His and Cys297Arg in mature VWF sequence), in exon 24 of the VWF gene. These missense mutations have been identified in the heterozygous, homozygous or hemizygous states. Using site‐directed mutagenesis and transient expression in COS‐7 cells, we showed that both mutations, although located in the D3 domain of VWF, outside the tryptic fragment containing the FVIII domain, dramatically decrease the binding of VWF to FVIII. In contrast, the R924Q substitution, which was identified in a patient who was heterozygous for C1060R, was shown to be a polymorphism.

Protein A Sepharose immunoadsorption can restore the efficacy of platelet concentrates in patients with Glanzmann's thrombasthenia and anti-glycoprotein IIb-IIIa antibodies

Summary. Type I Glanzmanns thrombasthenia is a rare congenital platelet function disorder, characterized by undetectable platelet membrane glycoprotein IIb–IIIa (GPIIb–IIIa). Severe bleeding is controlled by transfusion of normal platelets, leading in some cases to the occurrence of anti‐GPIIb–IIIa isoantibodies, which induces a loss of transfused platelet efficacy. We used immunoadsorption on protein A Sepharose (IA‐PA), which has been shown to be efficient in decreasing the titre of antibodies in several immune diseases, in three patients with Glanzmanns thrombasthenia and anti‐GPIIb–IIIa isoantibodies on five different occasions. IA‐PA was well tolerated with no deleterious side‐effects reported. It induced a dramatic decrease of total immunoglobulin (Ig)G, including anti‐GPIIb–IIIa isoantibody levels, as assessed by the monoclonal antibody‐specific immobilization of platelet antigens test and the ex vivo inhibition of normal platelet aggregation induced by the patients platelet‐rich or platelet‐poor plasma. Elimination of the antibody was associated with a correction of the bleeding time following platelet transfusion. IA‐PA combined with platelet transfusion made it possible to control two life‐threatening haemorrhages, and allowed two surgical procedures and one bone marrow transplantation to be performed safely. Our experience suggests that IA‐PA, which restores the haemostatic efficacy of platelet transfusion, is a valuable therapeutic strategy in patients with Glanzmanns thrombasthenia and anti‐GPIIb–IIIa isoantibodies.

Protein A sepharose immunoadsorption: immunological and haemostatic effects in two cases of acquired haemophilia

Acquired haemophilia is a life‐threatening disorder caused by circulating auto‐antibodies that inhibit factor VIII coagulant activity (FBIII:C). Immunoadsorption on protein A sepharose (IA‐PA) was performed in two bleeding patients with acquired haemophilia: we observed a dramatic and quick decrease in the anti‐FVIII:C inhibitor titre leading to a normal, albeit transient, haemostatic status. In one case, IA‐PA was the only procedure which succeeded in stopping massive haemorrhage. In the second case, IA‐PA reinforced the haemostatic effect of recombinant activated factor VII by increasing the endogenous plasma factor VIII level. The efficacy of IA‐PA was sustained with immunosuppressive treatment introduced, respectively, 10 and 15 d before the IA‐PA procedures. Our experience with IA‐PA suggests that this extracorporeal anti‐FVIII:C removal procedure is a valuable therapeutic tool for acquired haemophilia and can alleviate life‐threatening haemorrhages.

Multiplate whole blood impedance aggregometry: a new tool for von Willebrand disease

The diagnosis and characterization of von Willebrand disease (VWD) is a well-known challenge. Besides the clinical bleeding tendency, its diagnosis and subtyping require laboratory data, including measurement of PFA-100 closure time (CT), factor VIII:C, von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and ristocetininduced platelet aggregation (RIPA). RIPA with low doses of ristocetin is crucial for the diagnosis of subtype 2B, and is most commonly performed with light transmission aggregometry (LTA). LTA has remained the reference standard since its introduction in 1962 by Born [1], mostly because of its validated applications, including the diagnosis of VWD. However, LTA remains a technically challenging and timeconsuming method, even with extensive experience, as it requires centrifugation steps, which can lead to artefactual platelet subpopulation selection and activation. It also requires large blood volumes, is difficult to perform in cases of thrombocytopenia, and does not take into account interactions between platelets and other blood cells. In contrast, there is less experience of determining platelet aggregation in whole blood with whole blood impedancemetry (WBI) (a method introduced by Cardinal and Flower in 1980 [2]), although it lacks some of the major drawbacks of LTA [3,4]. WBI is a fast method, and allows the omission of centrifugation steps and the performance of platelet function studies under more physiologic conditions with small sample sizes. It is based on the change of impedance proportional to the number of platelets sticking to two electrodes through which an alternating current is passed. Multiplate (for multiple electrode aggregometry ; Dynabyte, Munich, Germany) is a new generation of WBI aggregometer, using diluted blood and single-use test cells containing twin electrodes that reduce the variation in results. This analyzer has been widely used to evaluate patient responses to platelet aggregation inhibitors [5–7]. However, RIPA assessment with WBI has not been evaluated to date. Using a previously characterized VWD patient population, we aimed to compare the WBI aggregometry Multiplate analyzer and the classic LTA (ThromboAggregometer, Affibio, Nancy, France) RIPA methods. We consecutively tested 30 healthy volunteers (HVs) and 30 patients with VWD. All VWD patients had been previously characterized by extensive laboratory workup, and subtyped according to international criteria [8]. Twenty-six patients had inherited VWD: 12 had type 1, 11 had type 2A or type 2M, and 3 unrelated patients had confirmed type 2B with distinct and recognized mutations in exon 28 of the VWF gene [9]. Patient 26 was classified as type 2B without the use of LTA RL (see below) on the basis of her abnormal laboratory results, which included thrombocytopenia and the same Arg1306 fi Trp mutation [10] as her father (who exhibited an increased LTA RL result). Three patients from a single family, initially diagnosed as type 2B, had a platelet-type (PT) VWD (Gly233 fi Ser mutation in the GPIba gene [11]). One patient had acquired VWD. Table 1 shows the patients results obtained by laboratory measurements performed on the same day as their WBI. The values are indicative of the patients laboratory variables, and were not used for diagnostic purposes (for example, patient 1 had a transient thrombocytopenia and patient 24 had a severe infectious disease on the day of the testing). All HVs and patients gave their written informed consent, and were comparable according to age, sex ratio, and blood group O (data not shown). Coagulation studies were performed on citrated platelet-poor plasma obtained after centrifugation at 2500 · g for 10 min. FVIII:C and VWF:RCo were assessed with an automated BCS coagulometer (Siemens Healthcare Diagnostics, Marburg, Germany), using a one-stage clotting assay and APTT-SP HemosIL (Instrumentation Laboratory, Milan, Italy) and FVIII-deficient plasma (Diagnostica Stago, Asnieres, France), or using BC von Willebrand Reactif (Siemens). VWF:Ag was assessed with an automated STAR coagulometer and Liatest VWF:Ag (Diagnostica Correspondence: Valerie Proulle, Service Hematologie Biologique, Hopital Bicetre, 94275 Le Kremlin Bicetre Cedex, France. Tel.: +33 1 45 21 36 12; fax: +33 1 45 21 28 47. E-mail: valerie.proulle@bct.aphp.fr

Heparin-induced thrombocytopenia: Successful biological and clinical management with lepirudin despite severe renal impairment

Heparin-induced thrombocytopenia: Successful biological and clinical management with lepirudin despite severe renal impairment -

Risk factors for thrombosis in an african population.

Little is known about the biological, epidemiological, and clinical risk factors for thrombosis and venous thromboembolism (VTE) among Black Africans. We undertook a study of the prevalence of VTE risk factors for thrombosis in a Senegalese population. A three-year cross-sectional and case-control study involving 105 cases and 200 controls was conducted in various hospitals in Dakar (Senegal). Our results demonstrate that oral contraception, immobilization by casts, surgery, and blood group were significantly associated with VTE occurrence. Additionally, 16 cases and 2 controls had protein S (PS) values of less than 48.4% (M-2SD), exhibiting a highly significant difference (P < 1 x 10−4). The number of cases with a low protein C (PC) level was significantly higher than the respective number of controls. Using logistic regression methods, we established a correlation between significantly associated variables and deep venous thrombosis (DVT) occurrence. Age, obesity, sickle cell disease, and PC deficiency were not significantly associated with thrombosis. In contrast, gender, PS deficiency, varicose veins, surgery, non-O blood type, and the presence of anti-phospholipid antibodies were significantly and independently associated with DVT. These findings are extremely useful for clinical management of patients suffering from DVT and can help to reduce the high recurrence rate observed in our study.