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Severe ADAMTS13 Deficiency in Adult Idiopathic Thrombotic Microangiopathies Defines a Subset of Patients Characterized by Various Autoimmune Manifestations, Lower Platelet Count, and Mild Renal Involvement

Coppo, Paul; Bengoufa, Djaouida; Veyradier, Agnès; Wolf, Martine; Bussel, Annette; Millot, Gaël Armel; Malot, Sandrine; Heshmati, Farhad; Mira, Jean-Paul; Boulanger, Emmanuelle; Galicier, Lionel; Durey-Dragon, Marie-Agnès; Frémeaux-Bacchi, Véronique; Ramakers, Michel; Pruna, André; Bordessoule, Dominique; Gouilleux, Valérie; Scrobohaci, Marie-Lorraine; Vernant, Jean-Paul; Moreau, Delphine; Azoulay, Elie; Schlemmer, Benoît; Guillevin, Loïc; Lassoued, Kaïss for the Réseau d'Etude des Microangiopathies Thrombotiques de l'Adulte

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doi: 10.1097/01.md.0000133622.03370.07
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Abstract

INTRODUCTION

In adults, thrombotic microangiopathies (TMAs) represent a heterogeneous group of rare diseases, characterized by microangiopathic hemolytic anemia with fragmented red cells (schistocytes) and peripheral thrombocytopenia. TMAs encompass thrombotic thrombocytopenic purpura (TTP), typically characterized by fever and neurologic manifestations33, and hemolytic uremic syndrome (HUS), in which acute renal failure is the prominent abnormality13. Despite a growing body of evidence suggesting that these 2 entities have different pathophysiologic mechanisms, their clinical and standard laboratory manifestations are not specific, and distinguishing them clearly remains challenging14,45,51.

The pathophysiology of TMA is a multifactorial process that frequently includes an endothelial cell injury and the subsequent release of pro-aggregant factors in the plasma, particularly high molecular weight von Willebrand factor (vWF) multimers5,25,26,58. In some instances, endothelial cell injury is thought to be mediated by various associated medical conditions, such as transplantation, drug intake, cancer, and human immunodeficiency virus (HIV) infection45, and leads to a microangiopathic process also termed TTP-like disease by some authors44. However, in most cases TMA is considered to be idiopathic since no such associated condition is identified, and the factors that initiate the disease in this context remain unclear. In the last few years, 2 groups have presented evidence that a protease specifically involved in the cleavage of high molecular weight vWF multimers (termed ADAMTS13), was deficient in TTP12,54. Indeed, a failure to process these multimers may lead to the accumulation of high molecular weight vWF multimers in plasma, and then to platelet aggregation and consumption that cause ischemia in the brain and other organs. A decrease in ADAMTS13 activity was also reported in various physiologic or pathologic conditions, but this decreased activity was usually mild4,23,30. It is noteworthy that values as low as 15% were occasionally found in these conditions, without evidence of TTP. These levels are in contrast to the extremely low levels (<5% of normal values) in patients with episodes of TTP, and it is likely that severe ADAMTS13 deficiency is required to develop TTP4,27. This enzymatic deficiency was related to mutations of the encoding gene in pediatric cases22, whereas in most adult cases it was associated with a plasmatic IgG inhibitor12,54. These findings represent an important step in the understanding of TMA pathophysiology, and led some authors to propose ADAMTS13 as a stringent tool for the diagnosis of TTP12,54. However, the relevance of ADAMTS13 in clinical practice remains unclear and controversial, and whether to discriminate TTP from HUS on the basis of ADAMTS13 activity is still a matter of debate41,42,55. Indeed, patients with neurologic involvement suggestive of TTP may display detectable ADAMTS13 activity. On the other hand, some patients with a diagnosis of HUS may have undetectable ADAMTS13 activity41. In an attempt to specify the relationship between ADAMTS13 status and clinical and laboratory presentation of adult TMA, we studied extensively a large number of adult patients diagnosed as having idiopathic TMA. Our results suggest that adult TTP, as defined by severe ADAMTS13 deficiency, is a specific form of TMA characterized by a wide spectrum of autoimmune manifestations, and also more severe thrombocytopenia, and mild renal involvement.

PATIENTS AND METHODS

Study Design

In an attempt to identify nosologic subsets of adult patients with TMA on the basis of exhaustive clinical and laboratory investigations, we initiated a large retrospective study that was first at a single center (October 1997 to August 2000), and then at multiple centers (September 2000 to April 2003). Some of these patients have been previously reported10.

Patients

All adult (>18 years old) patients fulfilling the following diagnosis criteria for TMA and treated with plasma manipulation were included in the study. TMA diagnosis criteria were the presence of acute thrombocytopenia (defined as a platelet count <150 × 109/L), and a negative Coomb test microangiopathic hemolytic anemia (as assessed by the presence of schistocytes on a peripheral-blood film). Exclusion criteria consisted of eclampsia, disseminated intravascular coagulation (as defined by a decreased fibrinogen value associated with positive D-dimers detected by enzyme-linked immunosorbent assay [ELISA]), and valvular and/or vascular prosthesis10,43. Patients with TTP-like diseases for whom organ failure and cytopenias may be directly related to the associated condition and not only to the microangiopathic process, that is, solid or hematopoietic cell transplantation, cancer and chemotherapy, and Centers for Disease Control and Prevention (CDC) stage C HIV infection, were not included.

For each patient, a detailed clinical examination was systematically performed on admission, with particular attention to the central nervous system, skin, and joints. Patients' age, sex, past medical history, and ethnic group were also recorded. Extensive laboratory investigations were performed in all patients. Renal function was assessed by plasmatic urea and creatinine levels, as well as by estimation of glomerular filtration rate (GFR) using Gault and Cockroft's method. Antinuclear antibodies (ANA), anticardiolipin and anti-β2 glycoprotein 1 (β2GP1) antibodies, and ADAMTS13 activity were systematically investigated on admission, and for some patients during complete remission. Diagnosis of systemic lupus erythematosus (SLE) was made according to the revised American Rheumatism Association (ARA) criteria16.

Patients were treated intensively with high volumes of viro-inactivated plasma, either associated or not with plasma exchanges, steroids, and antiplatelet agents, as previously described10. Complete remission was defined as the reversal of clinical manifestations and thrombocytopenia. Relapse was defined as the reappearance of clinical and/or laboratory parameters after 30 consecutive days of complete remission. Flare-up was defined as a worsening of clinical manifestations and/or thrombocytopenia during treatment or during the 30 consecutive days following complete remission.

Immunologic Investigations

All samples were referred to the reference center (Hôpital Saint-Louis, Paris) for detection of ANA, which were detected by both indirect immunofluorescence on HEp2 cells and air-dried rat liver tissue sections (Biomedical Diagnostics, Marne-la-Vallée, France), and ELISA (Anaprofil Varelisa, Pharmacia Diagnostics, Freiburg, Germany). The threshold positivity for dilution was 1:80. Anti-double-stranded DNA (dsDNA) antibodies were detected by ELISA (normal < 100 IU) and Farr assay (normal < 10%)40. Anti-extractable nuclear antigens (ENA) antibodies (anti-Sm, -U1-snRNP, -Ro/SSA, -La/SSB, -Scl 70, and Jo1) were detected by both ELISA and counter immunoelectrophoresis with reference sera8,31, using purified antigens obtained from rabbit and bovine thymus powder (Pel Freez, AR), and from human spleen extract (Laboratoire d'Immunopathologie, Hôpital Larrey, Angers, France). Anticardiolipin antibodies (Biomedical Diagnostics) and anti-β2gp1 antibodies (Varelisa, Pharmacia Diagnostics) were detected by ELISA (IgG isotype). Threshold positivity was ≥15 GPL units (anticardiolipin antibodies) and ≥10 GPL units (anti-β2gp1 antibodies). Lupus anticoagulant was sought in all patients using kaolin clotting time.

Evaluation of ADAMTS13 Activity

We collected blood by atraumatic venipuncture into citrate. Plasma was immediately prepared by centrifugation at 4000 rpm for 15 minutes twice at 4 °C, aliquoted, and stored at −20 °C. We measured ADAMTS13 activity in plasma and searched for an associated inhibitor at the reference laboratory for the investigation of ADAMTS13 in France (Hematology Laboratory of Hôpital Antoîne Béclère, Clamart), as previously described57. ADAMTS13 deficiency was considered severe in patients with an undetectable enzymatic activity (<5% of normal activity).

Statistical Analysis

Statistics were performed using the JMP software (SAS Institute Inc., Cary, NC) and the R environment. Medians and ranges were determined for all continuous variables. Patients with severe ADAMTS13 deficiency were compared to patients with detectable ADAMTS13 activity by a Wilcoxon test for continuous variables and by a 2-tailed Fisher exact test for categorical variables.

RESULTS

General Characteristics (Table 1)

Forty-six patients were included in the study from 12 national centers. Severe ADAMTS13 deficiency (<5% of normal activity) was disclosed in 31 patients (67.4%), whereas the remaining 15 (32.6%) had a detectable ADAMTS13 activity >25% (Figure 1). Clinical and laboratory characteristics of each group are reported in Tables 1 and 2.

F1-3
FIGURE 1:
ADAMTS13 activity on admission. We identified patients with severe ADAMTS13 deficiency (<5% of normal activity) and patients with detectable enzymatic activity. Black symbols correspond to patients with an ADAMTS13 inhibitor. Gray area delimits ADAMTS13 values of normal subjects.
T1-3
TABLE 1:
Clinical Characteristics According to ADAMTS13 Activity
T2-3
TABLE 2:
Standard Laboratory Data According to ADAMTS13 Activity*

The prevalence of Afro-Caribbean origin was high in patients with severe ADAMTS13 deficiency (15 of 31 patients; 48.4%). These patients were included from 5 different centers, and originated from North Africa (9 patients), West Africa (5 patients), or the Caribbean (1 patient). Conversely, only 2 patients with detectable ADAMTS13 activity originated from West Africa (1 case) or the Caribbean (1 case) (13.3%; p = 0.03) (see Table 1).

Age, sex, fever, and the prevalence of the different neurologic manifestations were not statistically different between patients with a severely decreased ADAMTS13 activity and those with detectable enzymatic activity. Previous episodes of TMA were recorded in 4 patients with severe ADAMTS13 deficiency (13%) and in 1 patient with detectable ADAMTS13 activity (6.7%). These occurred 5 years (range, 4-9 yr) and 17 years before the current episode, respectively (see Table 1).

Patients With Severe ADAMTS13 Deficiency Have a Lower Platelet Count and a Less Severe Renal Failure (Table 2)

Standard laboratory values were compared between the 2 groups. Patients with severe ADAMTS13 deficiency had a lower platelet count than those with detectable ADAMTS13 activity (12 × 109/L [range, 2-69 × 109/L], vs 49.5 × 109/L [range, 6-103 × 109/L], respectively; p = 0.0004). On the other hand, patients with detectable ADAMTS13 activity had more severe renal failure than patients with severe ADAMTS13 deficiency, as assessed by plasmatic urea (25.8 mmol/L [range, 12.1-49.8 mmol/L] vs 9.7 mmol/L, [range, 3.3-37 mmol/L], respectively; p < 0.0001), creatinine (329 μmol/L [range, 95-1269 μmol/L] vs 98 μmol/L [range, 58-448 μmol/L], respectively; p < 0.0001), and estimated GFR (15.8 mL/min [range, 5.6-80 mL/min] vs 78 mL/min [range, 9-157 mL/min], respectively; p < 0.0001).

No difference was distinguished among patients in terms of hemoglobin value and lactate dehydrogenase (LDH) level (see Table 2).

Patients With Severe ADAMTS13 Deficiency Display Various Autoimmune Manifestations (Table 3)

T3-3
TABLE 3:
Immunologic Findings According to ADAMTS13 Activity*

Ten patients with severe ADAMTS13 deficiency (32.2%) presented with features suggesting autoimmunity; specifically, nondestructive polyarthritis (4 cases); diffuse cutaneous lesions histopathologically consistent with the diagnosis of discoid lupus (3 cases); and autoimmune thyroiditis associated with type I diabetes mellitus, Raynaud phenomenon, and sarcoidosis-like disease (1 case each). This latter was revealed by chronic lymphocytic alveolitis associated with a nodular erythema and a chronic fibrosing hepatopathy with Mycobacterium tuberculosis-negative liver epithelioid granulomas. In addition to nondestructive polyarthritis, 1 patient had a past history of malar rash and extramembranous glomerulonephritis, suggestive of SLE. By contrast, only 1 patient with detectable ADAMTS13 activity had a history of autoimmune disease, specifically rheumatoid arthritis with psoriasis.

ANA were positive in 22 patients with severe ADAMTS13 deficiency (71%), whereas no patient with detectable ADAMTS13 activity displayed such antibodies (p = 0.0001). This statistical association gives a specificity and a positive predictive value of 100% for ANA in predicting severe ADAMTS13 deficiency in TMA. Of note, ANA were positive in all 10 patients with a history of autoimmune disease. They displayed a homogeneous, and/or speckled, and/or nucleolar fluorescence on both human Hep2 cells and rat liver tissue sections at dilutions ≥1:80. By ELISA and counter immunoelectrophoresis, anti-ENA antibodies specificity was Ro/SSA in 2 patients. In a third patient, ELISA was found positive for Ro/SSA specificity, whereas indirect immunofluorescence was negative. One patient had anti-U1 snRNP antibodies. In the remaining patients, no specificity could be determined. Anti-dsDNA antibodies were present in 3 patients, in addition to ANA. Conversely, anti-dsDNA antibodies could not be found in any patient with detectable ADAMTS13 activity. Anticardiolipin antibodies were positive in 1 patient with severe ADAMTS13 deficiency. Anti-β2gp1 antibodies were negative in the 20 patients tested in this group. Two patients with detectable ADAMTS13 activity had anticardiolipin antibodies. In both groups, patients had a normal kaolin clotting time, suggesting the absence of lupus anticoagulant.

Severe ADAMTS13 deficiency was related to a plasmatic inhibitor in 17 cases (55%). In the remaining patients, no plasmatic inhibitor could be identified (see Figure 1).

One patient with severe ADAMTS13 deficiency fulfilled enough criteria for the diagnosis of SLE (Table 4, Patient 10). In the remaining patients, autoimmune manifestations evidenced on admission failed to define a nosologically well-defined connective tissue disease.

T4-3
TABLE 4:
Clinical and Laboratory Follow-up in 10 Patients with Severe ADAMTS13 Deficiency and Autoimmune Manifestations

Detailed clinical and laboratory long-term follow-up was available in 10 patients with severe ADAMTS13 deficiency (see Table 4). Within a median period of 25 months (range, 1-60 mo), ANA, anti-dsDNA antibodies, and anticardiolipin antibodies remained positive in 8 patients, whereas in 1 patient ANA disappeared (Patient 8). In 1 patient (Patient 5), ANA appeared after 25 months of follow-up. Three patients (Patients 1-3) developed anti-dsDNA antibodies within a median time of 9 months (range, 9-13 mo). A transient left hemiparesia with anti-dsDNA antibodies occurred in Patient 2 in association with anticardiolipin antibodies, 14 and 29 months after the onset of TMA, respectively. Neither of the 2 patients with anticardiolipin antibodies (Patients 2 and 9) developed deep venous thrombosis. The appearance of additional autoimmune manifestations during follow-up led to the diagnosis of TTP associated with SLE-like disease in 3 patients, in association with antiphospholipid syndrome in 1 case. For the remaining patients with no defined connective tissue disease but with some manifestations of autoimmunity, the final diagnosis of pauci-immune TTP was retained (see Table 4).

In 13 patients with initial severe ADAMTS13 deficiency, ADAMTS13 activity was investigated after a median follow-up of 79 days (range, 6 d to 46 mo) from diagnosis (Figure 2). ADAMTS13 activity completely recovered in 8 patients. Among them, 6 had a detectable inhibitor at diagnosis, whereas in the other 2 patients, no inhibitor could be identified. In another patient, ADAMTS13 activity partially recovered (20% of normal activity). By contrast, in 4 other patients, ADAMTS13 activity remained persistently undetectable. In 1 of these patients, a plasmatic inhibitor persisted during complete remission, leading to persistent ADAMTS13 inhibition and multiple relapses.

F2-3
FIGURE 2:
Outcome of ADAMTS13 activity during remission in patients with severe ADAMTS13 deficiency at diagnosis. Two patients fully recovered ADAMTS13 activity despite the absence of plasmatic inhibitor at diagnosis. Black symbols correspond to patients with an ADAMTS13 inhibitor. Gray area delimits ADAMTS13 values of normal subjects.

Renal Function Prognosis May Be Worse in Cases of Detectable ADAMTS13 Activity (Table 5)

All patients were treated with plasma manipulation until durable complete remission. Six patients with severe ADAMTS13 deficiency and 2 patients with detectable ADAMTS13 activity received high-dose plasma infusion with no plasmapheresis (19.3% vs 13.3%, respectively; p = not significant). In the remaining patients, plasma exchange was performed as the first treatment or following an initial treatment with plasma infusion10. The frequency of administering steroids and antiplatelet agents was comparable between the groups (data not shown). Hemodialysis was performed more frequently in patients with detectable ADAMTS13 activity (7/15 patients, 46.7%) than in patients with severe ADAMTS13 deficiency (3/31 patients, 9.7%) (p = 0.008). One patient with detectable ADAMTS13 activity was lost to follow-up. Three patients of this group (21.4%) experienced end-stage renal failure after the first episode (2 cases) or after 3 episodes (1 case) of TMA. By contrast, this complication occurred in only 1 patient with severe ADAMTS13 deficiency (3.2%; p = 0.08). It is important to note, however, that this patient also had a previous history of severe type 2 diabetes mellitus with renal involvement. Time to durable platelet count and LDH level recovery was comparable in the 2 groups. Patients with severe ADAMTS13 deficiency had 1 (7 patients) or more (3 patients) episodes of flare-up. Three patients in this group relapsed within a median of 9 months (range, 76 d to 26 mo) after TMA diagnosis. Patients with detectable ADAMTS13 activity experienced 1 (1 patient) or 2 (2 patients) episodes of flare-up of the disease, and 1 patient relapsed 9 months after TMA diagnosis. These differences did not reach the 0.05 significance level (see Table 5).

T5-3
TABLE 5:
Outcome According to ADAMTS13 Activity*

DISCUSSION

We conducted the current study to assess whether ADAMTS13 activity could be used as a tool to distinguish different groups of patients with apparently idiopathic TMA with particular characteristics. Our findings support the view that adult TTP and HUS, as defined by ADAMTS13 activity12,27,54,57, represent 2 clearly distinct clinical and pathophysiologic variants of TMA. Based on the hypothesis that TTP is pathophysiologically related to severe ADAMTS13 deficiency12,22,27,54, we suggest that adult TTP, known as idiopathic, may represent a specific syndrome characterized by 1) a more frequent occurrence in Afro-Caribbean individuals, 2) a more profound thrombocytopenia, 3) mild renal involvement, and 4) various autoimmune manifestations. In contrast with these findings, patients with detectable enzymatic activity had mainly severe renal failure, consistent with the diagnosis of HUS. These patients required hemodialysis in half of the cases, and up to 21.4% of them experienced end-stage renal failure. It is noteworthy, however, that we found no difference between patients with severe ADAMTS13 deficiency and those with detectable ADAMTS13 activity in term of prevalence of fever and neurologic involvement. Particularly, episodes of seizure and focal deficiency were observed in both groups. These results therefore straighten the view that classical clinical presentation (that is, fever and neurologic involvement), as opposed to renal function14, platelet count, and ADAMTS13 activity12, may represent a less stringent manner to distinguish between TTP and HUS. This finding may be explained at least in part by the exclusion of patients with microangiopathic processes related to various associated conditions (namely, transplantation, cancer and chemotherapy, and CDC stage C HIV infection), for whom the diagnosis of TMA is sometimes uncertain44. Another result that emerges from the current study is that ADAMTS13 activity had no prognostic value in terms of response to treatment, episodes of flare-up, relapses, and survival. Though patients with detectable ADAMTS13 activity seemed to experience end-stage renal failure more frequently than patients with severe ADAMTS13 deficiency, this difference did not reach a significant level (p = 0.08) in this study with a relatively limited number of patients, and further studies will be required to confirm this tendency. It must be emphasized that as a result of our recruitment, our findings concern specifically adult TMA. Therefore, clinical but also standard laboratory presentation of this form may differ from others, such as familial and recurrent forms. In these latter, ADAMTS13 deficiency is more frequently related to mutations of the encoding gene than to autoantibodies, and it is likely that familial and/or recurrent TMA with congenital ADAMTS13 deficiency may represent another specific subset of TMA distinct from TMA with immune-mediated ADAMTS13 deficiency. As underlined by Remuzzi and coworkers41, ADAMTS13 activity in these forms of TMA may fail to distinguish TTP from HUS. This may explain the discrepancy between the different studies in literature concerning the specificity of ADAMTS13 for the diagnosis of TTP.

It is noteworthy in the current study that a large number of patients with severe ADAMTS13 deficiency (48.4%) shared a common ethnic and geographic origin (Afro-Caribbean), suggesting a particular immunogenetic background that may predispose such patients to generate autoantibodies against ADAMTS13. Although we are unable to exclude recruitment bias totally, the fact that these patients originated from up to 5 different centers largely distant from each other makes this hypothesis unlikely. Moreover, this feature has been emphasized previously by other groups15,56.

Different findings in the current study are consistent with the fact that severe ADAMTS13 deficiency in our patients occurred within a particular context of autoimmunity. First, various past histories of autoimmune manifestations or autoimmune diseases were found in this group. Particularly, we found that an unexpectedly large number of patients displayed ANA. Anti-ENA antibodies were found in a minority of patients, suggesting that ANA in TTP recognize yet unknown epitopes. We note that 3 patients developed additional autoimmune manifestations during follow-up, while TTP was in complete remission. These consisted of anti-dsDNA antibodies, in association with transient neurologic manifestations and anticardiolipin antibodies in 1 case, this latter association suggesting the diagnosis of antiphospholipid syndrome. One additional patient initially negative for ANA developed these antibodies during follow-up. These observations suggest that the pattern of autoimmune manifestations in patients with a history of TTP may be gradually completed within follow-up until, for some cases, the diagnosis of a disease close to SLE. We note that this particular evolution has been reported also for other autoimmune diseases, such as autoimmune cytopenias, antiphospholipid syndrome, or discoid lupus6,28,34,60.

Second, a plasmatic ADAMTS13 inhibitor, reported to be an IgG autoantibody12,54, could be identified in 55% of patients. In the remaining patients however, we cannot rule out that at least in some cases, a low titer inhibitor that may not be detectable by our current assay57 could exist. In regard to this hypothesis, 2 patients with no detectable inhibitor at the time of the disease finally recovered normal ADAMTS13 activity during complete remission, suggesting an acquired enzymatic deficiency. Moreover, in 1 patient with persistent ADAMTS13 deficiency, analysis of genomic DNA did not identify any mutation in the ADAMTS13 gene, ruling out a genetic deficiency in this patient (data not shown). Alternatively, one could hypothesize that a non-neutralizing autoantibody, which could not be evidenced by a functional assay, was present in these patients. Such a hypothesis has been demonstrated recently by Scheiflinger et al48. This group reported an adult patient with severe ADAMTS13 deficiency but no detectable inhibitor, for whom high titers of non-neutralizing IgM and IgG antibodies were found nevertheless, using a newly developed ELISA. The authors concluded that such non-neutralizing autoantibodies may however shorten the half-life of ADAMTS13 and/or its binding to the endothelial cell surface, thereby compromising ADAMTS13 activity in vivo48. Taken together, these findings support evidence that TTP identified as idiopathic is frequently associated with autoimmunity, and may in fact correspond to a specific autoimmune disease. This latter may be characterized by a wide spectrum of manifestations, ranging from ANA-negative patients to patients with SLE or a SLE-like disease, including patients with various nonspecific autoimmune manifestations. The latter patients with some autoimmunity were identified as having a pauci-immune TTP. Importantly, all these manifestations may occur progressively during follow-up, and thus these patients require a careful long-term follow-up. Our results are in agreement with previous studies supporting the clear association between TTP and autoimmunity. Indeed, TTP could be associated with various autoimmune diseases including SLE(18, 35, 36, 57, this study) or other connective tissue diseases1,3,7,9,17,19,20,24,38,47,49,50, autoimmune cytopenias2,11,32 (Table 6), or even autoantibodies with no nosologically determined autoimmune disease52. Therefore, the development of an ADAMTS13 inhibitor may be part of a more general autoimmune process, occurring within a particular immunogenetic background. In that regard, the association of TTP with pregnancy and estrogen intake45 strengthens the concept of TTP as an autoimmune disease, since estrogens have been reported to have a role in the initiation of some connective tissue diseases, especially SLE46,53.

T6-3
TABLE 6:
Autoimmune Diseases Associated with TTP, Previous Studies

The mechanisms of endothelial cell injury remain poorly understood in idiopathic TMA, and a large spectrum of autoantibodies were reported to be possibly involved in this process. Indeed, complement-fixing antiendothelial antibodies may induce vascular injury, and antiplatelet antibodies may induce platelet activation and aggregation21,39,59. These antibodies, however, were found to be equally present in TTP and HUS, and may represent only an immune response directed against endothelial cell and platelet antigens, in relation to a nonspecific initial cellular injury. Other antibodies such as anti-CD36 antibodies also have been found to be largely associated with TTP52, but again, their interpretation, as well as their role in the initiation of the disease, has never been demonstrated37. In TTP, the involvement of ANA in endothelial cell damage remains questionable. However, we found no correlation between the level of serum ANA and the severity of TTP (data not shown), rendering this hypothesis unlikely. In the current study, anticardiolipin antibodies were not commonly found regardless of ADAMTS13 activity, whereas anti-β2GP1 were systematically negative, and kaolin clotting time was normal. These results, in agreement with those of Montecucco29, suggest that these autoantibodies are poorly, if ever, involved in TMA pathophysiology.

An unexpected finding of the current study was the high prevalence of ANA in patients with severe ADAMTS13 deficiency, with both a positive predictive value and specificity of ANA for the diagnosis of severe ADAMTS13 deficiency of 100%, and a sensitivity of 71%. Indeed, this association, to our knowledge hitherto undescribed, strongly suggests that a large number of cases of TTP may be associated with ANA, and that the presence of these antibodies may constitute an additional, rapidly available criterion for TTP diagnosis in patients with apparently idiopathic TMA. This finding may open other interesting perspectives, since ANA could be used to identify rapidly a large number of patients with severe ADAMTS13 deficiency, when specific therapies involving purified or recombinant ADAMTS13 are available.

CONCLUSION

The current study provides evidence that apparently idiopathic TTP in adults, as defined by severe ADAMTS13 deficiency, corresponds to a specific form of TMA characterized by a wide spectrum of autoimmune manifestations that may occur within a particular genetic background. Indeed, the synthesis of an ADAMTS13 plasmatic inhibitor may be part of a more general autoimmune process.

ACKNOWLEDGMENTS

We are grateful to all the physicians from the participating centers for providing clinical and laboratory data and plasma samples: Dr. C. Adrie (Centre Hospitalier Régional Delafontaine, Seine-Saint-Denis); Pr. Baud (Service de Réanimation Médicale, Hôpital Lariboisière); Dr. R. Belhocine (Unité d'Hémaphérèse, Hôpital Hôtel-Dieu, Paris); Dr. S. Bréchignac (Hôpital Avicenne, Seine-Saint-Denis); Pr. J. C. Brouet, Dr. M. Malphettes, Pr. J. P. Clauvel and Pr. E. Oksenhendler, (Service d'Immuno-Hématologie), Dr. G. Thiery and Dr. F. Fieux (Service de Réanimation Médicale), Dr. S. Neuville (Service de Maladies Infectieuses), Dr. N. Parquet (Unité de Thérapie Cellulaire et de Clinique Transfusionnelle), Dr. F. Amesland (Laboratoire d'Immunologie), Dr. F. Agbalika (Laboratoire de Virologie), and Pr. D. Charron (Laboratoire d'Immunologie et d'Histocompatibilité) (Hôpital Saint-Louis, Paris); Dr. P. Hazera (Service de Médecine Interne, Centre Hospitalier Mémorial France Etats-Unis, Saint-Lô); Pr. P. Charbonneau, Dr. C. Daubin, Dr. A. Lesage (Service de Réanimation Médicale, Hôpital de Caen); Dr F. Pène (Service de Réanimation Médicale, Hôpital Cochin, Paris); and Dr. F. Vincent (Service de Néphrologie, Hôpital Tenon, Paris).

We also acknowledge Dr V. Soumelis (Service Hématologie Adultes, Hôpital Necker, Paris) and E. Daugas (Service Néphrologie B, Hôpital Tenon, Paris) for discussion and advice; Dr. J. Dando (INSERM U362, Institut Gustave Roussy, Villejuif) for helpful suggestions for the English version of the manuscript; S. Savigny (Laboratoire d'Hématologie, Hôpital Antoîne Béclère, Clamart) and R. Tessier (ETS), E. Terrier (Laboratoire d'Hémostase), and M. L. Touron (Unité d'Hémaphérèse) (Hôpital Saint-Louis, Paris) for technical assistance.

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