Marie Dreyfus

Efficacy of mycophenolate mofetil in adult refractory auto-immune cytopenias: a single center preliminary study.

Abstract:  Treatment of auto‐immune cytopenia refractory to front line therapy with intravenous immunoglobulins and steroids is a matter of concern. We assessed the efficacy and safety of mycophenolate mofetil in a prospective open preliminary study. Study design: Adult patients with steroid refractory auto‐immune cytopenias were included. Mycophenolate mofetil (MMF) was added to treatment given at the time of inclusion, and efficacy was evaluated in term of improvement of platelet/haemoglobin levels and in term of reduction of previously given drugs, if any. All auto‐immune thrombocytopenic purpura (AITP) patients had serologic assessment for associated auto‐antibodies at the time of inclusion. Cytopenias associated with other auto‐immune diseases, lymphoproliferative diseases or HIV infection were excluded. Results: From November 1999 through November 2003, 13 patients were included (nine AITP, three auto‐immune haemolytic anaemia (AIHA), one Evans’ syndrome; four males, nine females; age: 35–72 yr). For AITP patients, an overall response of 78% was observed. Retrospective analysis showed no significant difference between patients having a short disease duration (<1 yr) and longer disease duration; between patients who previously received more or less than three treatments; and between patients for whom MMF was started as monotherapy or in association with prednisone, However, all AITP patients presenting associated auto‐antibodies responded to MMF, while only 50% of patients without associated antibodies were responders. All patients presenting AIHA and Evans’ syndrome were responders. The drug was well tolerated, with no significant side effects reported. The cumulative data suggest a potential place for MMF in the treatment arsenal of refractory cytopenias.

Willebrand Factor and Ristocetin I. MECHANISM OF RISTOCETIN‐INDUCED PLATELET AGGREGATION

Summary. Ristocetin induces aggregation of normal platelet‐rich plasma over a wide range of concentrations. Low doses induce a biphasic response of which the first wave is not mediated by ADP and proceeds without the initial platelet shape change. The absence of aggregation in von Willebrands disease results from the lack of a component, Willebrand factor, which is eluted together with platelets, factor VIII activity and Willebrand (or factor VIII related) antigen in the void volume fraction when platelet‐rich plasma is gel filtered (Sepharose 2B). Twice washed normal platelets aggregate to ristocetin whereas four times washed platelets only aggregate when low volumes of normal haemophilia A plasma or serum are added. The interaction of ristocetin with platelets and plasmatic components has been investigated. A mechanism is proposed for ristocetin‐induced aggregation.

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.

Prevalence of antiphospholipid-related antibodies in unselected patients with history of venous thrombosis

Antiphospholipid antibodies (aPL) are heterogeneous and are now accepted to be mainly phospholipid-protein-dependent antibodies. Although these antibodies are classically associated with thrombosis, their clinical relevance remains to be established. The subgroups of antibodies characterized by their proteic targets were reported to be more appropriate thrombotic markers. We analysed the prevalence of a large panel of antiphospholipid-related antibodies (aPLR), comprising antibodies directed to phospholipid-protein complexes and to different protein cofactors (β2GPI, prothrombin, annexin V and protein S), in 122 consecutive unselected patients who had experienced at least one venous thrombotic event. The presence of lupus anticoagulants was assessed with an integrated assay using hexagonal phase phospholipids. Two types of aPL (APA and anti-β2GPI-PL) were measured using a mixture of phospholipids containing cardiolipin and goat serum or human β2GPI, respectively, as a source of protein cofactor. Our results show a similar prevalence, close to 15%, of lupus anticoagulants, APA and anti-β2GPI-PL. In contrast, antibodies to β2GPI were detected in only 8% of the patients, and very few patients had antibodies directed to other proteins. Of the 35 patients having at least one positive aPLR, 17 were classified as severe, because they had recurrent or early onset of thrombosis (< 35 years). The distribution of aPLR between severe and mild cases was not significantly different except for lupus anticoagulants. Our results clearly indicate that lupus anticoagulant is the only aPLR test to be strongly associated with the severity of thrombosis.

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.

Injection of recombinant activated factor VII can induce transient increase in circulating procoagulant microparticles

Recombinant activated factor VII (rFVIIa) is an effective haemostatic treatment in haemophiliacs with inhibitors. In vitro, FVIIa concentrations corresponding to those obtained with therapeutic doses of rFVIIa have been shown to induce normal thrombin generation and platelet activation in the absence of factors VIII or IX. To further study the in vivo haemostatic changes induced by rFVIIa, circulating procoagulant microparticles (MP) were measured in patients treated with discontinuous injections of Novoseven. In 6 out of 15 patients, a transient peak of procoagulant MP was observed after injection, occurring 15 min to 2 h after infusion. It was composed primarily of platelet-derived MP and was of very short duration. This peak was not observed in haemophiliacs without inhibitor, who were treated with conventional replacement therapies. Our results provide further in vivo evidence that rFVIIa specifically activates platelets, either directly or as a consequence of a burst of thrombin generation that could account for its haemostatic efficacy.

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

Acquired hemophilia in older people: a poor prognosis despite intensive care.

the start of the study, systemic blood pressure increased to 95/65 mm Hg; after 2 weeks of treatment, the patient was able to leave the bed and to stay in a seat and was found to be amenable to a rehabilitation program. Our results suggest that sildenafil could be effectively and safely used in very old patients with secondary pulmonary hypertension, allowing the daily dose of furosemide to be reduced and thereby making escalating doses of loop diuretics unnecessary. The phosphodiesterase 5 (PDE 5) pathway is highly represented in the lungs, and sildenafil, a selective PDE 5 inhibitor, by making more cyclic guanosine monophosphate available, is able to induce a selective pulmonary vasodilation, which preserves the ventilation:perfusion ratio, as the improvement of arterial blood gases in our patients and in previous series indicates. This finding is consistent with selective or prevalent vasodilation of ventilated lung units; nonselective pulmonary vasodilation would expose the patient to an imbalance between ventilation and perfusion, thus worsening the gas exchanges and, ultimately, PaO2, PaCO2, or both. Baroreflex sensitivity decreases with age in healthy subjects, and deconditioning secondary to chronic diseases could further contribute to worsening vascular reflexes, putting frail elderly people at special risk for sildenafilinduced hypotension. Nevertheless, in a patient with severe pulmonary hypertension and systemic hypotension, sildenafil could improve systemic blood pressure and contribute to making the patient amenable to a rehabilitation program. This favorable result suggests that even severe hypotension coexisting with pulmonary hypertension does not exclude a trial with sildenafil, because relieving the pulmonary vascular barrage could improve left ventricular filling and, subsequently, arterial pressure. Alternatively, it cannot be excluded that sildenafil relieved arterial hypotension by increasing PaO2 and thereby improving heart function. Indeed, findings in hypoxemic patients with chronic obstructive pulmonary disease show that hypoxia impairs left ventricular relaxation and indirectly could depress the systolic function. The present experience cannot guarantee that any older patients with pulmonary hypertension and systemic hypotension will benefit from sildenafil. The risk exists that, in occasional patients, lack of pulmonary vascular effect and retained systemic vascular effect worsen rather than improving health status. Accordingly, it seems safe to monitor vital parameters and SPP at least on the occasion of the first dose of sildenafil. With this precaution in practice, sildenafil may be a valuable tool in the management of severe secondary pulmonary hypertension even in the very old.

A Laboratory Phenotype/Genotype Correlation of 1167 French Patients From 670 Families With von Willebrand Disease: A New Epidemiologic Picture.

Abstractvon Willebrand disease (VWD) is a genetic bleeding disease due to a defect of von Willebrand factor (VWF), a glycoprotein crucial for platelet adhesion to the subendothelium after vascular injury. VWD include quantitative defects of VWF, either partial (type 1 with VWF levels <50 IU/dL) or virtually total (type 3 with undetectable VWF levels) and also qualitative defects of VWF (type 2 variants with discrepant antigenic and functional VWF levels). The most bleeding forms of VWD usually do not concern type 1 patients with the mildest VWF defects (VWF levels between 30 and 50 IU/dL).The French reference center for VWD performed a laboratory phenotypic and genotypic analysis in 1167 VWD patients (670 families) selected by their basic biologic phenotype: type 3, type 2, and type 1 with VWF levels <30 IU/dL. In these patients indeed, to achieve an accurate diagnosis of VWD type and subtype is crucial for the management (treatment and genetic counseling).A phenotype/genotype correlation was present in 99.3% of cases; 323 distinct VWF sequence variations (58% of novel) were identified (missense 67% versus truncating 33%). The distribution of VWD types was: 25% of type 1, 8% of type 3, 66% of type 2 (2A: 18%, 2B: 17%, 2M: 19%, 2N: 12%), and 1% of undetermined type. Type 1 VWD was related either to a defective synthesis/secretion or to an accelerated clearance of VWF. In type 3 VWD, bi-allelic mutations of VWF were found in almost all patients. In type 2A, the most frequent mechanism was a hyper-proteolysis of VWF. Type 2B showed 85% of patients with deleterious mutations (distinct from type 2B New York). Type 2M was linked to a defective binding of VWF to platelet glycoprotein Ib or to collagen. Type 2N VWD included almost half type 2N/3.This biologic study emphasizes the complex mechanisms for both quantitative and qualitative VWF defects in VWD. In addition, this study provides a new epidemiologic picture of the most bleeding forms of VWD in which qualitative defects are predominant.