Agnès Veyradier

IgG subclass distribution of anti‐ADAMTS13 antibodies in patients with acquired thrombotic thrombocytopenic purpura

Summary.u2002 Background:u2002ADAMTS13‐neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). Objective:u2002To analyze the IgG subclass distribution of anti‐ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. Methodology:u2002An enzyme‐linked immunosorbent assay‐based method was used to quantify the relative amounts of IgG subclasses of anti‐ADAMTS13 antibodies in acquired TTP plasma. Results:u2002IgG4 (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG1 (52%), IgG2 (50%), and IgG3 (33%). IgG4 was found either alone (17/52) or with other IgG subclasses (35/52). IgG4 was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG4 and IgG1 antibodies (Pu2003<u20030.01). Patients with high IgG4 levels and undetectable IgG1 are more prone to relapse than patients with low IgG4 levels and detectable IgG1. Conclusions:u2002All IgG subclasses of anti‐ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG4, followed by IgG1, antibodies dominating the anti‐ADAMTS13 immune response. Levels of IgG4 could be useful for the identification of patients at risk of disease recurrence.

Ten candidate ADAMTS13 mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw–Schulman syndrome)

Summary.u2002 ADAMTS13, the specific von Willebrand factor (VWF)‐cleaving metalloprotease, prevents the spontaneous formation of platelet thrombi in the microcirculation by degrading the highly adhesive ultralarge VWF multimers into smaller forms. ADAMTS13 severe enzymatic deficiency and mutations have been described in the congenital thrombotic thrombocytopenic purpura (TTP or Upshaw–Schulman syndrome), a rare and severe disease related to multivisceral microvascular thrombosis. We investigated six French families with congenital TTP for ADAMTS13 enzymatic activity and gene mutations. Six probands with congenital TTP and their family were tested for ADAMTS13 activity in plasma using a two‐site immunoradiometric assay and for ADAMTS13 gene mutations using polymerase chain reaction and sequencing. ADAMTS13 activity was severely deficient (<u20035%) in the six probands and one mildly symptomatic sibling but normal (>u200350%) in all the parents and the asymptomatic siblings. Ten novel candidate ADAMTS13 mutations were identified in all families, showing either a compound heterozygous or a homozygous status in all probands plus the previous sibling and a heterozygous status in the parents. The mutations were spread all over the gene, involving the metalloprotease domain (I79M, S203P, R268P), the disintegrin domain (29u2003bp deletion in intron/exon 8), the cystein‐rich domain (acceptor splice exon 12, R507Q), the spacer domain (A596V), the 3rd TSP1 repeat (C758R), the 5th TSP1 repeat (C908S) and the 8th TSP1 repeat (R1096stop). This study emphasizes the role of ADAMTS13 mutations in the pathogenesis of congenital TTP and suggests that several structural domains of this metalloprotease are involved in both its biogenesis and its substrate recognition process.

Thrombotic thrombocytopenic purpura and its diagnosis.

Thrombotic thrombocytopenic purpura (TTP) is a lifethreatening illness whose mortality rate exceeds 90% in the absence of rapid appropriate treatment. Empirical plasmatherapy instituted in the 1970s has reduced the death rate to approximately 25% and both plasma infusions and plasma exchanges remain the only efficient treatments so far. TTP prevalence is about four per one million with a preference for women of childbearing age [1]. To diagnose TTP implies to face a first challenge: to define it. The definition criteria for TTP have significantly evolved as a result of a 20-year retrospective accurate analysis of North American [2,3], European [4–8] and Japanese [9] patients registries and cohorts and a better understanding of TTP pathogenesis. Historically, TTP was originally described in 1924 by Eli Moschcovitz in a 16-year-old girl who presented with fever, anemia, central nervous system impairment, renal insufficiency and both respiratory and cardiac failure related to hyaline platelet thrombi in the terminal arterioles and capillaries of most organs [10]. Several case-reports and reviews of the literature have followed the initial description [11] leading to a definition concept focused on the following pentad: fever, microangiopathic hemolytic anemia, thrombocytopenia, central nervous system abnormalities and renal impairment. TTP, also called Moschcovitz syndrome , was described as a disease affecting primarily adults, but, interestingly, a rare pediatric form of the disease was also reported as early as 1960 [12] and named Upshaw-Schulman syndrome in 1978 [13]. However, further large retrospective analysis of patients clinical features showed that neither fever nor neurologic abnormalities and renal impairment were constant, especially during the early stage of the disease [14]. This observation led to the proposal that the association of microangiopathic hemolytic anemia and unexplained thrombocytopenia are sufficient and essential criteria to suspect TTP at an early stage in order to institute timely plasmatherapy [15,16]. In parallel to the evolution of the standard clinical and biological criteria required for a solid TTP suspicion, the pathophysiological mechanisms for TTP were further elucidated recently. Until the 1980s, the pathogenesis for TTP remained very unclear although numerous candidates like endothelium, platelets or plasma proteins were suggested to participate in the triggering of the disease [17]. The role of von Willebrand factor (VWF), a plasma multimeric protein essential for platelet adhesion and aggregation at the high shear rates of blood flow present in the microvessels, was first put forward in 1982 by Moake et al. [18] who found abnormally large VWF multimers in the plasma of TTP patients. These hyperadhesive ultralarge multimers of VWF were suspected to be directly responsible for spontaneous platelet clumping in the microcirculation leading to ischemic visceral dysfunction. This hypothesis was further supported in 1985 by Asada et al. [19] who demonstrated that platelet thrombi in TTP were enriched in VWF (and not in fibrin) using an immunohistochemistry study of vascular lesions. In 1996, Furlan et al. [20] and Tsai [21] independently isolated and partially characterized a new metalloprotease from human plasma that specifically cleaves VWF at Tyr842Met843, the peptide bond known to be cleaved in vivo. This enzyme was identified as the 13th member of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 repeats) family of metalloproteases and the corresponding gene was cloned in 2001 [22]. In 1998, Furlan et al. [23] and, Tsai and Chun-Yet Lian [24] revealed that most adult patients with acute TTP had a severe functional deficiency of ADAMTS-13 in plasma (< 5% of the activity of a normal pooled plasma), most often related to inhibitory IgG autoantibodies. Further studies of patients with miscellaneous diseases, including other thrombotic microangiopathies like hemolytic uremic syndrome (HUS), confirmed that ADAMTS-13 severe deficiency was about 90% sensitive and specific for TTP [8,25–27]. In addition, Levy et al. [22] found that patients with hereditary TTP associated with a severe ADAMTS-13 functional deficiency were doubly heterozygous or homozygous carriers of mutated ADAMTS-13 alleles. Thus, in 2005, ADAMTS-13 severe deficiency (< 5%), either acquired via circulating autoantibodies or more rarely inherited via recessive ADAMTS-13 mutations, appears as a major specific risk factor for TTP. However, this observation does not allow TTP to be redefined by an undetectable ADAMTS-13 activity in plasma because other Correspondence: Dominique Meyer, Inserm Unité 143, 80, rue du Général Leclerc, Hôpital de Bicêtre, 94276 Le Kremlin-Bicêtre Cedex, France. Tel.: +33 149 595 600; fax: +33 146 719 472; e-mail: dmeyer@ kb.inserm.fr Journal of Thrombosis and Haemostasis, 3: 2420–2427

Cancer-related thrombotic microangiopathy secondary to Von Willebrand factor-cleaving protease deficiency

Cancer-related thrombotic microangiopathy (TM) is a serious complication with a short-term life-threatening prognosis. This complication shares certain similarities with thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, both characterized by circulating platelet aggregates containing ultralarge multimers of Von Willebrand factor (VWF). We report a case of cancer-related thrombotic microangiopathy secondary to disseminated metastatic cancer with undetectable serum Von Willebrand factor-cleaving protease activity and no evidence of serum inhibitory antibody. A concomitant decrease of Ca 19-9 level and hemolysis was observed during chemotherapy, in parallel with normalization of Von Willebrand factor-cleaving protease activity. The role of ultralarge multimers of Von Willebrand factor in platelet aggregation in the context of metastatic disease is discussed with respect to our findings in this case of cancer-related thrombotic microangiopathy.

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

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).

The prognostic value of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency in septic shock patients involves interleukin-6 and is not dependent on disseminated intravascular coagulation

IntroductionADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency has been reported in patients with sepsis but its clinical relevance and pathophysiology remain unclear. Our objectives were to assess the clinical significance, prognostic value and pathophysiology of ADAMTS13 deficiency in patients with septic shock with and without disseminated intravascular coagulation (DIC).MethodsThis was a prospective monocenter cohort study of patients with septic shock. Von Willebrand Factor, ADAMTS13-related parameters and plasma IL-6 concentration were measured at inclusion to the study. Patients were categorized into three groups according to the presence of ADAMT13 deficiency (<30%) or DIC.ResultsThis study included 72 patients with a median age of 59xa0years (interquartile range (IQR) 50 to 71). Each of the included patients received vasopressors; 55 (76%) were under mechanical ventilation and 22 (33%) underwent renal replacement therapy. Overall, 19 patients (26%) had DIC, and 36 patients had ADMTS13 deficiency (50%). Patients with DIC, ADAMTS13 deficiency or both were more severe at ICU admission. Mortality was higher in septic shock patients from group one. By multivariate analysis, Simplified Acute Physiology Score 2 (SAPS2) score (odds ratio (OR) 1.11/point; 95% CI 1.01 to 1.24) and ADAMTS13 activity <30% (OR 11.86; 95% CI 1.36 to 103.52) were independently associated with hospital mortality. There was no correlation between ADAMTS13 activity and the International Society for Thrombosis and Haemostasis (ISTH) score (rs = -0.97, Pu2009=u20090.41) suggesting that ADAMTS13 functional deficiency and DIC were independent parameters. IL-6 level was higher in patients with ADAMTS13 activity <30% [895 (IQR 330 to 1843) pg/mL versus 83 (IQR 43 to 118), Pu2009=u20090.0003).ConclusionsSeptic shock was associated with a functional deficiency of ADAMTS13, independently of DIC. ADAMTS13 functional deficiency is then a prognostic factor for mortality in septic shock patients, independently of DIC.

Terminal platelet production is regulated by von Willebrand factor.

It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s−1) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf −/− mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf −/− and Vwf +/+ blood, exposed to VWF at 1800 s−1 in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf−/− mice than from Vwf+/+ mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf−/− mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s−1 on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.

Screening for bleeding disorders in women with menorrhagia using a platelet function analyzer.

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Desmopressin, an unexpected link between nocturnal enuresis and inherited thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome)

In July 2001, a 6-year-old female Haitian child was referred for treatment of a seventh relapse of a familial thrombotic microangiopathy (TMA). The usual treatment by plasma infusions (10 mL kg) was efficient allowing a clinical remission in 2 weeks. The onset of the disease occurred as soon as birth with jaundice, distress, severe hemolytic anemia and thrombocytopenia requiring exchange transfusions. Relapses were unpredictable with a free interval varying from 5 weeks to 2 years. During relapses, severe mechanical hemolytic anemia with schistocytosis and severe thrombocytopenia were constant although visceral ischemic manifestations were not systematic. Two relapses were characterized by the presence of stage I coma with cerebral ischemic lesions on magnetic resonance imaging angiography. Further biological testing, focused on both von Willebrand factor (VWF), a multimeric glycoprotein released in plasma from endothelial cells and essential for platelet aggregation in the microcirculation, and its specific cleaving-protease, ADAMTS-13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats), allowed to classify this familial TMA as an inherited thrombotic thrombocytopenic purpura (TTP) or Upshaw-Schulman syndrome (USS) [1,2]. Indeed, in our patient, USS was confirmed by an undetectable ADAMTS-13 activity in plasma related to a homozygous Ala596Val ADAMTS-13 mutation inherited from both her mother and her father who are healthy heterozygous carriers [3]. Although patients with USS have a constant severe ADAMTS-13 enzymatic deficiency in plasma, about half of them do not exhibit a chronic disease but only sporadic TTP relapses with symptom-free intervals. In these cases, the identification of triggering factors for acute TTP episodes is essential to try to prevent relapses. Physiological or pathological conditions known to stimulate the release of large VWF multimers from endothelial cells in plasma (infections, inflammation, estroprogestative contraception, pregnancy) are wellestablished triggering contexts for TTP relapses. In our patient, all TTP previous acute episodes were associatedwith infections. Surprisingly, no obvious clinical context could be identified during the current relapse that led us to screen more systematically the recent events potentially involved in the trigger of this acute TTP episode. In addition, we were puzzled by the failure to create a subcutaneous radial arteriovenous fistula (in order to administer preventive monthly plasma infusions) because of recurrent local thrombosis. Unexpectedly, the patient’s mother indicated that she had been giving her daughter who was enuretic, a specific intranasal treatment every evening for several weeks, including during hospitalization. The drug was identified as desmopressin (a synthetic form of anti-diuretic hormone, 1-desamino-8-D-arginine or DDAVP), a classical treatment for enuresis and diabetes insipidus [4]. The critical piece of the puzzle is that intranasal desmopressin used at 10to 15-fold higher doses is also able to stimulate the release of VWF from endothelial cells into plasma. This property is used in the treatment of most patients with type 1 vonWillebrand disease, a bleeding disorder defined by a partial quantitative deficiency of VWF. In our patient, considering ADAMTS-13 severe deficiency, we can speculate that very low doses of desmopressin were sufficient to pass the threshold of circulating ultralarge multimers of VWF beyond which spontaneous platelet aggregation and thus microvascular thrombus formation occur. In other words, desmopressin was very likely the unexpected triggering factor of the current TTP relapse, and maybe also of vascular access thrombosis. Notice that the child never had a TTP relapse during the 4 years following desmopressin cessation. The involvement of desmopressin in the trigger of USS acute episodes has been reported only once [5]. In this case, desmopressin had been used intentionally because of a misanalysis of the role of VWF in USS pathogenesis and it did aggravate the TTP symptoms. Another patient with acquired TTP was also aggravated by the administration of intravenous desmopressin given to correct the bleeding time [6]. Recently, the better understanding of ADAMTS-13 related role of VWF in USS pathophysiology led to clearly contra-indicate any drug inducing the release of VWF from endothelial cells to plasma (especially estroprogestatives and desmopressin) in patients with USS. In this particular context, Correspondence: Agnès Veyradier, Service d’Hématologie biologique, Hôpital Antoine-Béclère, Assistance Publique-Hôpitaux de Paris, 157, rue de la Porte-de-Trivaux, 92 140 Clamart cedex, France. Tel.: 33 (0)1 45 37 43 05; fax: 33 (0)1 46 32 40 55; e-mail: agnes.veyradier@abc.ap-hop-paris.fr

Macrovascular thrombosis in critically ill patients with thrombotic micro-angiopathies

The purpose of this study is to assess the incidence and describe the clinical and pathological features of macrovascular thrombosis during the course of thrombotic micro-angiopathy (TMA) in a 6xa0year retrospective study of all adults with TMA, admitted to a teaching-hospital ICU. Of the 55 patients identified, all had anaemia and thrombocytopenia and 45 (82xa0%) had renal or neurological impairment. All patients received plasmapheresis, steroids, and strict blood pressure control. Macrovascular venous or arterial thromboses were diagnosed in 28 (51xa0%) patients; among them, 7 had cerebral artery thrombosis and 21 (including 13 with central venous catheters) had deep vein thrombosis. Median time from plasmapheresis initiation to thrombosis was 7 (4–10) days. Clinical findings were suggestive of deep venous thrombosis in 7 of the 21 patients (33xa0%) and only one of the 7 patients with stroke had corresponding clinical signs. By multivariate analysis, factors independently associated with macrovascular thrombosis were undetectable ADAMTS13 activity (odds ratio 7.33, 95xa0% confidence interval 1.3–41.3), cardiac involvement with TMA (odds ratio, 3.46; 95xa0% confidence interval, 1.1–13.9) and TMA flare (odds ratio 9.03; 95xa0% confidence interval 1.03–79.4). In conclusion, half of the patients with TMA experience macrovascular thrombosis. Patients with TTP-related ADAMTS13 deficiency and those with cardiac manifestations of TMA are at higher risk for arterial or deep venous thrombosis.