[Endothelial dysfunction in thrombotic thrombocytopenic purpura: therapeutic perspectives]. / Dysfonction endothéliale au cours du Purpura Thrombotique Thrombocytopénique : un nouvel axe de prise en charge ?

Immune Thrombotic Thrombocytopenic Purpura (iTTP) is a rare but severe disease with a mortality rate of almost 100 % in the absence of adequate treatment. iTTP is caused by a severe deficiency in ADAMTS13 activity due to the production of inhibitory antibodies. Age has been shown to be a major prognostic factor. iTTP patients in the elderly (60yo and over) have more frequent organ involvement, especially heart and kidney failures compared with younger patients. They also have non-specific neurologic symptoms leading to a delayed diagnosis. Factors influencing this impaired survival among older patients remain unknown so far. Alteration of the functional capacity of involved organs could be part of the explanation as could be the consequences of vascular aging. In fact, severe ADAMTS13 deficiency is necessary but likely not sufficient for iTTP physiopathology. A second hit leading to endothelial activation is thought to play a central role in iTTP. Interestingly, the mechanisms involved in endothelial activation may share common features with those involved in vascular aging, potentially leading to endothelial dysfunction. It could thus be interesting to better investigate the causes of mid- and long-term mortality among older iTTP patients to confirm whether inflammation and endothelial activation really impact vascular aging and long-term mortality in those patients, in addition to their presumed role at iTTP acute phase. If so, further insights into the mechanisms involved could lead to new therapeutic targets.

[Venous thrombo-embolism during immune-mediated thrombotic thrombocytopenic purpura is prevalent in patients with a prolonged treatment with therapeutic plasma exchange]. / La maladie thrombo-embolique veineuse au cours du purpura thrombotique thrombocytopénique autoimmun est associée à un traitement prolongé par échanges plasmatiques.

INTRODUCTION: Thrombotic thrombocytopenic purpura (TTP) is a devastating disease characterized by disseminated microvascular thrombosis. Despite pro-thrombotic predisposing conditions, the prevalence of macrovascular venous thrombosis event (VTE) in immune-mediated TTP (iTTP) has rarely been assessed. METHODS: We reviewed data of all iTTP patients of the French reference Center for thrombotic microangiopathies registry prospectively enrolled through a 10-year period, between 2008 and 2018. Venous thrombosis included either thrombosis of central venous catheter, symptomatic deep venous thrombosis of the limbs or pulmonary embolism. RESULTS: Forty-eight (12.7%) VTE were diagnosed. VTE was diagnosed after a median time of 7 [IQR, 3-16] days following the first therapeutic plasma exchange (TPE) and consisted mainly in catheter-related thrombosis (73%), and to a lesser extend symptomatic deep venous thrombosis (16%), proximal pulmonary embolism (8%) and splanchnic vein thrombosis (2%). Cases with VTE (VTE+ cases), required more TPE to achieve remission (P < 0.01), and the total volume of plasma required to achieve remission was larger (P < 0.01) than for VTE- cases. There was also a trend for more rituximab use in the VTE+ cases as compared to the VTE- cases (47% vs 33%; respectively; P = 0.07). Curative anticoagulation was started in 38 cases (79%), while 6 VTE cases did not receive any antithrombotic agents, and catheter was systematically removed when catheter-related thrombosis was diagnosed. VTE+ cases had a higher number of inserted central venous catheters than VTE- cases (P < 0.05). CONCLUSION: VTE is a frequent condition occurring during iTTP management and is observed when patients require a prolonged treatment with daily TPE and multiple catheter insertions. Therapeutic strategies aimed at reducing the duration of TPE treatment in iTTP should substantially reduce this complication.

Caplacizumab to treat immune-mediated thrombotic thrombocytopenic purpura.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare and life-threatening disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and multiorgan failure, resulting from autoantibody-mediated severe A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) deficiency. In spite of treatment with plasma exchange and immunosuppression, patients remain at risk of exacerbations, refractoriness and death. Caplacizumab (Cablivi; Ablynx, a Sanofi company), a nanobody targeting von Willebrand factor (vWF), has been recently approved in the E.U. and the U.S. as the first therapeutic specifically indicated for the treatment of adults experiencing an episode of iTTP. Caplacizumab blocks the interaction of all multimers with platelets and, therefore, has an immediate effect on platelet aggregation and the ensuing formation and accumulation of platelet-rich microthrombi. This immediate effect of caplacizumab has the potential to protect the patient from tissue ischemia and organ dysfunction while the underlying disease process resolves. We detail here the preclinical and clinical data on caplacizumab for iTTP, including the recent studies that led to approval by the U.S. Food and Drug Administration (FDA) in 2019.

An open conformation of ADAMTS-13 is a hallmark of acute acquired thrombotic thrombocytopenic purpura.

Essentials Conformational changes in ADAMTS-13 are part of its mode-of-action. The murine anti-ADAMTS-13 antibody 1C4 discriminates between folded and open ADAMTS-13. ADAMTS-13 conformation is open in acute acquired thrombotic thrombocytopenic purpura (TTP). Our study forms an important basis to fully elucidate the pathophysiology of TTP. SUMMARY: Background Acquired thrombotic thrombocytopenic purpura (aTTP) is an autoimmune disorder characterized by absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies. Recently, it was shown that ADAMTS-13 adopts a folded or an open conformation. Objectives As conformational changes in self-antigens play a role in the pathophysiology of different autoimmune diseases, we hypothesized that the conformation of ADAMTS-13 changes during acute aTTP. Methods Antibodies recognizing cryptic epitopes in the spacer domain were generated. Next, the conformation of ADAMTS-13 in 40 healthy donors (HDs), 99 aTTP patients (63 in the acute phase versus 36 in remission), 12 hemolytic-uremic syndrome (HUS) patients and 63 sepsis patients was determined with ELISA. Results The antibody 1C4 recognizes a cryptic epitope in ADAMTS-13. Therefore, we were able to discriminate between a folded and an open ADAMTS-13 conformation. We showed that ADAMTS-13 in HDs does not bind to 1C4, indicating that ADAMTS-13 circulates in a folded conformation. Similar results were obtained for HUS and sepsis patients. In contrast, ADAMTS-13 of acute aTTP patients bound to 1C4 in 92% of the cases, whereas, in most cases, this binding was abolished during remission, showing that the conformation of ADAMTS-13 is open during an acute aTTP episode. Conclusions Our study shows that, besides absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies, an open ADAMTS-13 conformation is also a hallmark of acute aTTP. Demonstrating this altered ADAMTS-13 conformation in acute aTTP will help to further unravel the pathophysiology of aTTP and lead to improved therapy and diagnosis.

Amplified endogenous plasmin activity resolves acute thrombotic thrombocytopenic purpura in mice.

Essentials Plasmin is able to proteolyse von Willebrand factor. It was unclear if plasmin influences acute thrombotic thrombocytopenic purpura (TTP). Plasmin levels are increased during acute TTP though suppressed via plasmin(ogen) inhibitors. Allowing amplified endogenous plasmin activity in mice results in resolution of TTP signs. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in the absence of ADAMTS-13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role of plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results In mice with an acquired ADAMTS-13 deficiency, plasmin is generated during TTP as reflected by increased plasmin-α2-antiplasmin (PAP)-complex levels. However, mice still developed TTP, suggesting that this increase is not sufficient to control the pathology. As mice with TTP also had increased plasminogen activator inhibitor 1 (PAI-1) levels, we investigated whether blocking the plasmin(ogen) inhibitors would result in the generation of sufficient plasmin to influence TTP outcome in mice. Interestingly, when amplified plasmin activity was allowed (α2-antiplasmin-/- mice with inhibited PAI-1) in mice with an acquired ADAMTS-13 deficiency, a resolution of TTP signs was observed as a result of an increased proteolysis of VWF. In line with this, in patients with acute TTP, increased PAP-complex and PAI-1 levels were also observed. However, neither PAP-complex levels nor PAI-1 levels were related to TTP signs and outcome. Conclusions In conclusion, endogenous plasmin levels are increased during acute TTP, although limited via suppression through α2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a back-up for ADAMTS-13 in mice and resolve TTP signs as a result of an increased proteolysis of VWF.

Assessment of endothelial damage and cardiac injury in a mouse model mimicking thrombotic thrombocytopenic purpura.

Essentials Endothelial injury is thought to be a key event in thrombotic thrombocytopenic purpura (TTP). Endothelial and cardiac damages were assessed in a model of TTP using ADAMTS-13 knockout mice. Damages of cardiac perfusion and function were associated with nitric oxide pathway alteration. Endothelial dysfunction constitutes a critical event in TTP development and cardiac injury. SUMMARY: Background Cardiac alterations represent a major cause of mortality in patients with thrombotic thrombocytopenic purpura (TTP). Endothelial injury remains poorly defined, but seems to be a key initiating event leading to the formation of platelet-rich thrombi in TTP patients. Objectives To assess the changes in endothelial function and the induced cardiac damage in a mouse model of TTP. Patients/methods We used an animal model in which TTP-like symptoms are triggered by injection of 2000 units kg-1 of recombinant von Willebrand factor in ADAMTS-13 knockout mice. Results These mice developed TTP-like symptoms, i.e. severe thrombocytopenia, schistocytosis, and anemia. On day 2, magnetic resonance imaging demonstrated a decrease in left ventricular perfusion associated with alteration of left ventricular ejection fraction, fractional shortening, and cardiac output, suggesting early systolic dysfunction. This was associated with decrease in endothelium-mediated relaxation responses to acetylcholine in mesenteric and coronary arteries, demonstrating severe early endothelial dysfunction. In parallel, we showed decreased cardiac expression of endothelial nitric oxide (NO) synthase and increased expression of antioxidant enzymes, suggesting alteration of the NO pathway. At this time, cardiac immunohistochemistry revealed an increase in the expression of VCAM-1 and E-selectin. Conclusion This study provides evidence that the heart is a sensitive target organ in TTP, and shows, for the first time, strong mesenteric and coronary endothelial dysfunction in an induced-TTP model. The mechanisms incriminated are the occurrence of a pro-oxidant state, and proadhesive and proinflammatory phenotypes. This previously largely unrecognized vascular dysfunction may represent an important contributor to the systemic organ failure occurring in TTP.

[Thrombotic thrombocytopenic purpura in a newborn]. / Purpura thrombotique thrombocytopénique chez un nouveau-né.

We report the case of a newborn presenting with hemolytic anemia, thrombocytopenia, hyperbilirubinemia, and renal failure in the first hours of life. An early plasmatherapy was undertaken, with good outcome. The specific von Willebrand factor-cleaving protease activity (ADAMTS 13 for a disintegrin and metalloprotease with thrombospondin type 1 repeats) was found to be low. This is the specific biologic diagnostic element of congenital thrombotic thrombocytopenic purpura (TTP). This disease of constitutional thrombotic microangiopathy is rare. The prognosis, usually life-threatening, was completely transformed given the better understanding of the pathogenesis of the disease and therapeutic progress.

ADAMTS-13 and von Willebrand factor predict venous thromboembolism in patients with cancer.

UNLABELLED: ESSENTIALS: Cancer patients are at high risk of venous thromboembolism (VTE). In this study, cases and controls were cancer patients who did or did not develop VTE. von Willebrand factor (VWF) levels were higher if compared with controls and correlated with cancer stage. VWF and ADAMTS-13 are associated with the occurrence of VTE in cancer. BACKGROUND: Patients with cancer are at high risk of venous thromboembolism (VTE). ADAMTS-13 regulates von Willebrand factor (VWF) activity, which plays a role in the development of cancer and in VTE. OBJECTIVES: The aim of this study was to search for an association between the levels of VWF and ADAMTS-13 and VTE in patients with cancer and to compare current scoring systems for prediction of VTE before and after addition of these parameters. PATIENTS/METHODS: In a case-control study, in which patients with recently diagnosed cancer were followed-up for 6 months, we compared 20 patients who developed VTE (cases) and 140 patients with cancer without VTE (controls), matched for sex, age, and type and stage of cancer. We measured VWF, ADAMTS-13 (activity and antigen), P-selectin, D-dimer and F1 + 2 levels at baseline, and calculated both the Khorana score and the Khorana score expanded after addition of P-selectin and D-dimer levels. RESULTS: VWF levels were significantly higher in cases when compared with controls (326 ± 185% vs. 242 ± 158%) and correlated with advanced stage of cancer: localized, 185 [142; 222]; locally advanced, 240 [146; 257]; metastatic, 267 [153; 324] (mean [interquartile range]). The addition of two biomarkers, ADAMTS-13 activity and F1 + 2 levels, to the Khorana score improved receiver operating curves. CONCLUSIONS: von Willebrand factor and ADAMTS-13 are associated with the occurrence of VTE in patients with cancer. Moreover, addition of ADAMTS-13 and F1 + 2 levels to the Khorana score considerably increases the predictive value for VTE.

[Telemedicine in thrombotic microangiopathies: A way forward in rare diseases requiring emergency care]. / L'activité de télémédecine dans les microangiopathies thrombotiques : un progrès dans les maladies rares nécessitant une prise en charge en urgence.

Thrombotic microangiopathies (TMA) represent rare diseases requiring a high skill for their management that deserved in France the identification of a dedicated National reference center. TMA are short-term life-threatening diseases; however, with an adapted management, their prognosis can be excellent. It is therefore mandatory to recognize and treat them rapidly according to standard guidelines. Telemedicine is a specialized hub consisting of highly skilled staff trained in a specific domain of medicine. The telemedicine activity of a reference center is an important representative indicator of its expertise and recourse ability. It requires to be accurately evaluated and promoted. In this work, we report the French reference center for TMA telemedicine activity since its setting-up. TMA represent an interesting model of diseases that required a specific organization of telemedicine activity to adapt to clinicians demand, which includes particularly the need to answer resorts in real time 24/7.

[Mild hemophilia A fortuitously discovered during Henoch-Schönlein purpura]. / Découverte fortuite d'une hémophilie A mineure à l'occasion d'un purpura rhumatoïde.

Henoch-Schönlein purpura is a common form of immunological vasculitis in children. Hemophilia A is a genetic disorder, inherited in a X-linked recessive pattern, and characterized by spontaneous hemorrhage or prolonged bleeding due to factor VIII deficiency. The clinical signs depend on the severity of factor VIII deficiency. We herein report the case of a 4-year-old boy admitted to the emergency room for typical rheumatoid purpura, associated with a lengthening of aPTT, whose exploration had uncovered mild hemophilia A. Laboratory assays should explore lengthening of aPTT: firstly the presence of lupus anticoagulant without bleeding risk, in an inflammatory context; secondly a deficiency of VWF and one of the factors involved in the extrinsic coagulation pathway associated with bleeding risk.

Cardiac troponin-I on diagnosis predicts early death and refractoriness in acquired thrombotic thrombocytopenic purpura. Experience of the French Thrombotic Microangiopathies Reference Center.

BACKGROUND: Cardiac involvement is a major cause of mortality in patients with thrombotic thrombocytopenic purpura (TTP). However, diagnosis remains underestimated and delayed, owing to subclinical injuries. Cardiac troponin-I measurement (cTnI) on admission could improve the early diagnosis of cardiac involvement and have prognostic value. OBJECTIVES: To assess the predictive value of cTnI in patients with TTP for death or refractoriness. PATIENTS/METHODS: The study involved a prospective cohort of adult TTP patients with acquired severe ADAMTS-13 deficiency (< 10%) and included in the registry of the French Reference Center for Thrombotic Microangiopathies. Centralized cTnI measurements were performed on frozen serum on admission. RESULTS: Between January 2003 and December 2011, 133 patients with TTP (mean age, 48 ± 17 years) had available cTnI measurements on admission. Thirty-two patients (24%) had clinical and/or electrocardiogram features. Nineteen (14.3%) had cardiac symptoms, mainly congestive heart failure and myocardial infarction. Electrocardiogram changes, mainly repolarization disorders, were present in 13 cases. An increased cTnI level (> 0.1 µg L(-1) ) was present in 78 patients (59%), of whom 46 (59%) had no clinical cardiac involvement. The main outcomes were death (25%) and refractoriness (17%). Age (P = 0.02) and cTnI level (P = 0.002) showed the greatest impact on survival. A cTnI level of > 0.25 µg L(-1) was the only independent factor in predicting death (odds ratio [OR] 2.87; 95% confidence interval [CI] 1.13-7.22; P = 0.024) and/or refractoriness (OR 3.03; 95% CI 1.27-7.3; P = 0.01). CONCLUSIONS: A CTnI level of > 0.25 µg L(-1) at presentation in patients with TTP appears to be an independent factor associated with a three-fold increase in the risk of death or refractoriness. Therefore, cTnI level should be considered as a prognostic indicator in patients diagnosed with TTP.

[The revolution of monoclonal antibodies in the treatment of thrombotic microangiopathy]. / La révolution des anticorps monoclonaux dans la prise en charge des microangiopathies thrombotiques.

Thrombotic microangiopathies (TMA) define a syndrome characterized by the association of microangiopathic haemolytic anaemia with schistocytes, peripheral thrombocytopenia, and organ injury of variable severity. Thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uremic syndrome (HUS) are the main forms of TMA. Recent advances in the pathophysiology of those two diseases, which include in HUS the identification of a deregulation of the alternative complement pathway, and in TTP a severe deficiency in ADAMTS-13, allowed to develop specific, pathophysiology-based therapies. Therefore, rituximab and eculizumab tends to be increasingly used, and there is an urgent need to define consensual modes of administration at the international level, as well as common definitions of response evaluation and follow-up explorations.

Acute renal failure is prevalent in patients with thrombotic thrombocytopenic purpura associated with low plasma ADAMTS13 activity.

BACKGROUND: Among patients with thrombotic microangiopathies, acute kidney injury (AKI) is the hallmark of hemolytic uremic syndrome (HUS) and is largely underestimated in patients with thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: We sought to report AKI features and outcomes in patients with TTP. METHODS: We conducted a retrospective study of 92 patients with TTP assessed by low ADAMTS13 activity (< 10%) between 2001 and 2013. A logistic regression identified variables independently associated with AKI. RESULTS: Among the 92 patients, 54 (58.7%) presented with AKI, including 25 (46.3%) with stage 3 AKI. Fourteen (27.4%) patients had a nephrotic-range proteinuria and 21 (45.6%) had hemoglobinuria. Hematuria and leucocyturia were detected in 19 (41.3%) and 16 patients (36.4%), respectively. Renal replacement therapy (RRT) was required in 14 patients (25.9%). Six months after TTP remission, RRT-free patients had median (IQR) MDRD (Modification of Diet in Renal Disease formula estimating the glomerular filtration rate) of 93 mL min(-1) per 1.73 m(2) (68.8-110) and three patients required long-term dialysis. Mild or moderate chronic renal disease occurred in 23/54 (42.6%) AKI patients. By multivariate analysis, serum level of complement component 3 at admission was the only factor independently associated with AKI (OR per 0.25 unit decrease of C3, 0.85; CI, 1.82-8.33; P = 0.001). CONCLUSIONS: In patients with TTP, AKI is present in more than half the patients, and half of those will have lasting renal effects. Further studies to better understand the pathophysiology of renal involvement in patients with TTP and to identify a subset of patients with TTP syndrome overlapping HUS are warranted.

[Atypical hemolytic and uremic syndrome associated with von Willebrand factor-cleaving protease (ADAMTS 13) deficiency in children]. / Syndrome hémolytique et urémique atypique et déficit en ADAMTS 13.

Hemolytic and uremic syndrome (HUS) is a classical form of thrombotic microangiopathies characterized by the association of hemolytic anemia with schizocytes, thrombocytopenia, and acute renal failure. Two forms of HUS have been described: the typical form that occurs after ingestion of a strain of bacteria, usually Escherichia coli types, which expresses verotoxin (also called shiga-like toxin), typically followed by bloody diarrhea, and atypical HUS, which is rare during childhood and can also be revealed by bloody diarrhea. We report a case of a 25-month-old infant who presented with hematuria and pallor after an episode of diarrhea. Biological tests revealed anemia, thrombocytopenia, and renal failure. The diagnosis of typical HUS was made, but the causal microorganism was not identified. Progression was favorable within 5 days of plasma transfusions. Two months later, the patient presented with the same symptoms and neurological impairment without any diarrhea. Von Willebrand factor-cleaving protease activity (ADAMTS 13) was low. Therefore, the diagnosis of atypical HUS by severe deficiency of ADAMTS 13 was suggested. The treatment was based on plasma transfusions resulting in remission. Atypical HUS associated with severe ADAMTS 13 deficiency rarely occurs in childhood. The prognosis, usually threatening, has been completely transformed thanks to a better understanding of the pathogenesis and to therapeutic progress.

Congenital Thrombotic Thrombocytopenic Purpura: Atypical Presentation and New ADAMTS 13 Mutation in a Tunisian Child.

BACKGROUND: Congenital deficiency of ADAMTS13 is characterized by systemic platelet clumping, hemolytic anemia and multiorgan failure. Although, more than 100 mutations have been reported, atypical clinical presentation may be involved in diagnostic difficulties. CASE REPORT: A 2 year old Tunisian child presented with chronic thrombopenic purpura which failed to respond to corticosteroids. Hemolytic anemia with schistocytes, occurred ten months later, with no previous history of diarrhea or any neurological abnormality. Renal function and coagulation screening tests were normal. The count of platelet improved after fresh frozen infusion (FFP). Extensive investigations revealed a severe deficiency of ADAMTS 13 activity (level< 5%). Gene sequencing identified mutation in exon 18 of ADAMTS 13 gene. Prophylactic regimen with regular infusions of FFP was associated to favorable outcome. CONCLUSION: Early ADAMTS 13 activity testing and gene sequencing associated to precocious plasmatherapy are recommended to reduce morbidity and mortality of congenital TTP.

[Human immunodeficiency virus-associated thrombotic microangiopathies]. / Purpura thrombotique thrombocytopénique et autres syndromes de microangiopathie thrombotique au cours de l'infection par le virus de l'immunodéficience humaine.

Human immunodeficiency virus (HIV) infection represents a risk factor for thrombotic microangiopathy. HIV-associated thrombotic microangiopathies encompass two entities with distinct pathophysiology, clinical presentation, treatment and prognosis. Thrombotic thrombocytopenic purpura associated with human immunodeficiency virus is typically characterized by a sudden onset in a patient with a moderate immune deficiency and a few events of opportunistic diseases, and a profound acquired deficiency in the von Willebrand factor cleaving protease ADAMTS13. This diagnosis requires a well-codified management including daily therapeutic plasma exchanges, a highly active antiretroviral therapy and eventually immunomodulatory drugs. The prognosis is good with a response rate and an overall survival comparable to that of HIV-negative thrombotic thrombocytopenic purpura. On the opposite, HIV-associated thrombotic microangiopathy with a progressive onset that occurs in profoundly immunocompromised patients with past history of multiple opportunistic diseases usually have a detectable ADAMTS13 activity and a worse prognosis. Usual treatment is poorly efficient. Forthcoming studies should assess the role of immunomodulatory drugs such as rituximab in the setting of HIV-associated thrombotic microangiopathy, and identify possible risk factors associated with the occurrence of these diseases.

[Clopidogrel induced thrombotic thrombocytopenic purpura]. / Purpura thrombotique thrombocytopénique secondaire à l'utilisation du clopidogrel.

INTRODUCTION: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder. Drug-induced TTP is uncommon and we report a TTP associated with the use of clopidogrel. CASE REPORT: We report a 50-year-old man who presented with acute myocardial infarction and received clopidogrel therapy. He developed acute TTP ten days after clopidogrel onset. Imputability of the drug was demonstrated during a reintroduction test. Deficiency of ADAMTS 13 was confirmed and autoantibodies against ADAMTS 13 were detected. Complete remission was obtained after 24 plasma exchange sessions and adjunction of corticosteroids. CONCLUSION: Drug-induced TTP are probably immunologic, as was demonstrated in our patient. Clinicians should be aware of this possible uncommon adverse effect of clopidogrel because prompt therapy is imperative for life saving.

Validation of the first commercial ELISA for type 2N von Willebrand's disease diagnosis.

Type 2N von Willebrand's disease (VWD) is characterized by a factor VIII (FVIII) deficiency and a low FVIII/VWF ratio related to a markedly decreased affinity of von Willebrand factor (VWF) to FVIII. Type 2N VWD is diagnosed using assays allowing the measurement of plasma VWF capacity to bind FVIII (VWF:FVIIIB). These assays, crucial in order to distinguish type 2N VWD patients from mild haemophiliacs A and haemophilia A carriers, remain exclusively homemade and limited to laboratories possessing a high level of expertise in VWD. We evaluated the first commercial ELISA (Asserachrom® VWF:FVIIIB; Stago) comparated to a reference method in a multicentric study involving 205 subjects: 60 healthy volunteers, 37 haemophiliacs A, 17 haemophilia A carriers, 37 patients with type 2N VWD, 9 heterozygous carriers for a 2N mutation and 45 patients with miscellaneous other types of VWD (all previously characterized). A diluted plasma sample adjusted to 10 IU dL(-1) of VWF:Ag was incubated with a rabbit antihuman VWF polyclonal antibody. After removing the endogenous FVIII, recombinant FVIII (rFVIII) was added and bound rFVIII was quantified using a peroxydase-conjugated mouse antihuman FVIII monoclonal antibody. The intra-assay and inter-assay reproducibility was satisfactory. In all subgroups, both methods were well correlated. All type 2N VWD patients exhibited a markedly decreased VWF:FVIIIB (lower than 15%) and all heterozygous 2N carriers had a moderately decreased VWF:FVIIIB (between 30% and 65%). All controls (healthy subjects, haemophiliacs A and haemophilia A carriers) had a normal VWF:FVIIIB (higher than 80%) except one healthy volunteer and three haemophiliacs who exhibited a moderately decreased VWF:FVIIIB suggesting a heterozygous status for a 2N mutation. In conclusion, the Asserachrom® VWF:FVIIIB is easy to perform, standardized and accurate for type 2N VWD diagnosis with a 100% sensitivity and specificity.