Schistocytic anaemia, severe thrombocytopenia, and renal dysfunction: thrombotic microangiopathy due to severe acquired ADAMTS-13 deficiency Case 2

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1 103/ Schattauer GmbH Schistocytic anaemia, severe thrombocytopenia, and renal dysfunction: thrombotic microangiopathy due to severe acquired ADAMTS-13 deficiency Case 2 A. Winkler, J. A. Kremer Hovinga, V. Bianchi, J.-D. Studt, B. Lämmle Central Haematology Laboratory, Inselspital, University Hospital, Bern, Switzerland Keywords ADAMTS-13, microangiopathy, thrombotic thrombocytopenic purpura, von Willebrand factor-cleaving protease Summary We present the case of a woman (age: 64 years) with acute thrombotic microangiopathy due to severe acquired ADAMTS-13 (von Willebrand factor-cleaving protease) deficiency. She was successfully treated with plasma exchange therapy and glucocorticosteroids. She relapsed seven months later, and splenectomy led to lasting remission. Pathomechanisms of thrombotic thrombocytopenic purpura, especially the role of ADAMTS-13, are discussed and therapeutic measures outlined. Awoman (age: 64 years) presented in July 1999 to a peripheral hospital with increasing fatigue and nausea. Upon admission she was somnolent and icteric. Neither fever nor petechiae were detectable. The patient s medical history was significant for a discoid lupus erythematosus diagnosed in 1984 with partial alopecia. She also reported phototoxicity. Schlüsselwörter Mikroangiopathie, thrombotisch-thrombozytopenische Purpura, von-willebrand-faktor-spaltende Protease, ADAMTS-13 Zusammenfassung Wir berichten über eine Frau (Alter: 64 Jahre) mit akuter thrombotischer Mikroangiopathie bei schwerem akquiriertem Mangel an von-willebrand-faktor-spaltender Protease (ADAMTS-13). Sie wurde erfolgreich mit Plasmapheresen und Glukokortikosteroiden behandelt. Sieben Monate später kam es zu einem Rezidiv, eine Splenektomie wurde vorgenommen und führte zu anhaltender Remission. Aktuelle Kenntnisse zur Pathophysiologie der thrombotisch-thrombozytopenischen Purpura werden diskutiert, insbesondere die Rolle von ADAMTS-13. Abschließend werden therapeutische Maßnahmen vorgestellt. Mikroangiopathisch-hämolytische Anämie, schwere Thrombozytopenie und Niereninsuffizienz: thrombotische Mikroangiopathie bei schwerem akquirierten ADAMTS-13-Mangel Hämostaseologie 2003: 23: Diagnosis Laboratory data revealed profound thrombocytopenia ( /l) and normochromic, normocytic anaemia (haemoglobin 78 g/l). The peripheral blood smear showed many schistocytes (Fig. 1). Serum creatinine was 264 µmol/l (reference range: µmol/l), urea was 39 mmol/l (reference range: mmol/l). There was no proteinuria. The erythrocyte sedimentation rate was 130 mm/h and C-reactive protein 15 mg/l (reference value <5 mg/l). Ultrasonography of the abdomen indicated no splenomegaly and computed tomography of the brain was normal. A diagnosis of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) was suspected and the patient was referred to our hospital for further diagnostics and treatment. Additional investigations revealed negative direct Coombs test, elevated reticulocyte count ( /l; reference range: /l), free haemoglobin in plasma (0.09 g/l; reference value: <0.01 g/l), elevated total bilirubin concentration (45 µmol/l; reference range: 0-17 µmol/l), elevated lactate dehydrogenase (4605 U/l; reference range: <480 U/l), elevated hydroxybutyrate dehydrogenase (1325 U/l; reference range: U/l). Haptoglobin was <0.06 g/l (reference range: g/l), haemosiderin was detected in the urine. Coagulation profile turned out as normal. Serologic examinations were negative for human immunodeficiency virus, hepatitis B and C virus infection. Immunologic tests were positive for antinuclear antibody (1 : 320), anti- RNP, and anti-ds DNA. Bone marrow aspiration showed hyperactive haematopoietic activity with increased megakaryopoiesis and erythropoiesis as well as focal lymphocyte clusters.

2 104/12 Fig. 1 Peripheral blood smear of the patient with acquired TTP: numerous fragmented erythrocytes (schistocytes) and severe thrombocytopenia count was /l and haemoglobin 93 g/l. Follow-up at our outpatient clinic 3 months later showed a normal platelet count, no signs of haemolysis, and an ADAMTS-13 activity of 100%. In February 2000, she suffered from a relapse of TTP: Haemoglobin was 73 g/l, platelet count amounted to /l, activity of lactate dehydrogenase was 2931 U/l. She had noted progressing general fatigue over the preceeding 3-4 weeks. The preexisting discoid skin lesion on the cheek had worsened. In January, prednisone had been tapered from 5 mg/d to 5 mg every other day. ANA were slightly elevated (1 : 80), anti-ds DNA antibodies were negative at that time. Plasma exchange therapy with replacement of FFP and glucocorticosteroids were reinitiated. ADAMTS-13 activity upon admission was 15%. After a first plasma exchange session it increased to 50%, after the second to 100%. No ADAMTS-13 inhibitor was detectable. Platelet count after four plasma exchange therapy sessions was /l. Two weeks later the patient underwent splenectomy. Vaccination against pneumococcal bacteria and Haemophilus influenzae type B was performed as soon as glucocorticosteroids were tapered to 5 mg/d (two weeks after splenectomy). On four follow-up visits (after 6, 12, 24, and 36 months), she was in a good general condition without any signs of haemolysis and platelet count within the reference range. ADAMTS-13 activity was 100% on each occasion. Therapy, follow-up Daily plasma exchange therapy of about 3 l using fresh frozen plasma (FFP) replacement was started immediately. Additional FFP was administered between plasma exchange sessions. The patient also received methylprednisolone (200 mg/d). Before each plasma exchange session the von Willebrand factor-cleaving protease (ADAMTS-13) activity, as well as the protease inhibitor were determined. Initially, ADAMTS-13 activity was <3% and a weak inhibitor was detectable (Fig. 2). On day 4 ADAMTS-13 activity increased to about 30%, on day 10 to 100% with no detectable inhibitor. Platelet count and lactate dehydrogenase activity normalized on day 8. By that time, serum creatinine was within the reference range. After 18 days in the hospital the woman was discharged in good general condition with prednisone (dosage: 70 mg/d). Platelet Discussion Our patient suffered from acquired thrombotic thrombocytopenic purpura due to an antibody inhibiting the von Willebrand factor-cleaving protease, now denoted as ADAMTS(a disintegrin and metalloprotease with thrombospondin type 1 motifs)- 13. We here present a short overview of the thrombotic microangiopathies (TMAs), discuss the role of ADAMTS-13 in TTP, and outline therapeutic measures in patients with TMAs. TTP belongs to the thrombotic microangiopathies. It was described by Moschcowitz for the first time in 1924 (27). The concept consisting of the classical disease defining pentad: thrombocytopenia, microangiopathic haemolytic anaemia, neurological symptoms, renal dysfunction, fever evolved over the years. Fig. 2 ADAMTS-13 activity in acute acquired TTP: quantitative immunoblotting of purified vwf substrate degraded by barium chloride-activated ADAMTS-13 in plasma (1 : 20) Lane 1: initially (ADAMTS-13 activity <3% of the normal); lanes 2-11: during plasma exchange therapy (ADAMTS-13 activity 15% on day 2 and 100% on day 10); lanes 12-14: assay calibration by normal plasma dilutions 1 : 20, 1 : 80, 1 : 160 corresponding to ADAMTS-13 activity of 100, 25, and 12.5%, respectively. Haemolytic uraemic syndrome (HUS), first described by Gasser et al. (14), is a similar form of TMA. The diagnosis is often made in cases with predominating renal involvement, while TTP most often is diagnosed when neurological symptoms are present. HUS occurs mostly in young children after infection by E. coli O157 : H7 leading to haemorrhagic diarrhea (typical or D+ HUS), but is also diagnosed in the absence of enteropathogenic E. coli infection (atypical HUS).

3 105/14 Besides idiopathic TTP, E. coli O157 : H7-associated HUS and idiopathic HUS, thrombotic microangiopathies associated with a number of conditions are variably referred to as TTP, HUS, TTP-HUS, TTPlike disease or secondary TTP (Tab. 1). Tab. 1 Thrombotic microangiopathies Role of ADAMTS-13 in TTP Different mechanisms were reported to play a pathogenetic role in idiopathic TTP. Abnormal interaction of endothelial cells and circulating platelets is generally considered to be the crucial defect. High concentrations of thrombomodulin as well as plasminogen activator inhibitor 1 were found, indicating endothelial damage. Another observation was the presence of calpain or cathepsin-like cysteine protease activity in acute TTP, postulated to cause platelet agglutination. Specific platelet aggregating factors (e. g. 2 proteins with molecular mass of 37 and 59 kda, respectively) were described. Deficiencies as well as abnormalities of prostacyclin were also reported (for review see 9, 24, 30). New insights into the pathophysiology of TTP emerged in recent years. Moake et al. (23) observed unusually large von Willebrand factor multimers (ULvWF) in plasma samples of patients with chronic relapsing TTP. vwf is a glycoprotein synthesized and secreted into the plasma by endothelial cells and megakaryocytes. It mediates the initial platelet adhesion to the subendothelial wall of a damaged vessel. The vwf is composed of a variable number of subunits (molecular mass 250 kda) linked by disulfide bonds. The binding affinity for collagen and platelet receptors is enhanced in the large multimeric forms of vwf (Fig. 3). Proteolytic degradation of ULvWF at elevated shear stress is a physiologic event in healthy individuals (6, 37). A specific protease cleaving vwf at the peptide bond 842 Tyr 843 Met was isolated from normal human plasma (11, 38). Further studies showed that this vwfcleaving protease (vwf-cp) is absent in patients with constitutional TTP and blocked by an inhibitor in patients with acquired (non-familial) TTP. Furlan et al. (13) found a complete deficiency of vwf-cp in four a b c * reproduced with kind permission from Springer-Verlag GmbH & Co. KG, Heidelberg: Furlan M. Von Willebrand factor: molecular size and functional activity. Ann Hematol 1996; 72: 341-8, Fig. 1 patients (including two brothers) with relapsing TTP. No inhibitor was detected. The occurrence of relapsing TTP in two brothers suggested a hereditary nature of ADAMTS-13 deficiency. In 1998 an inhibitor of vwf-cp was detected in a patient with TTP who lacked any protease activity (12).The inhibitor was identified as IgG. It disappeared after the initial treatment, its reappearance was followed by a clinical relapse. In 2001 vwf-cp was purified, partially sequenced and identified as a new member of the ADAMTS family of metalloproteases (8, 16, 43). Independently, genome-wide linkage analysis in patients with hereditary TTP and their family members mapped the locus of the responsible gene to chromosome 9q34, showing that it encoded ADAMTS- 13 (21). Double heterozygous or homozygous ADAMTS-13 gene mutations were responsible for severe ADAMTS-13 deficiency in the affected patients. In the meantime several mutations throughout the ADAMTS-13 gene have been found in hereditary TTP (2, 20, 21, 31). Two large studies (10, 36) suggested that most patients diagnosed with TTP had a severe deficiency of ADAMTS-13 activity, most often due to IgG autoantibodies inhibiting enzymatic activity of the protease. Interestingly, patients clinically diagnosed with HUS exhibited most often normal ADAMTS-13 activity (10). This is rather surprising because TTP and HUS are often difficult to distinguish clinically (15). However, a prospective study of patients with TMA (41) confirmed that 47/66 patients (71%) clinically diagnosed with TTP had severe ADAMTS-13 deficiency whereas most patients with the diagnosis HUS had normal or subnormal ADAMTS-13 activity. Fig. 3 Structure of vwf in relation to its function* a) denaturing electrophoresis (1% SDS-agarose) of vwf in normal plasma: large multimers on top, electrophoretic migration towards the bottom of the gel; b) bottom band of dimeric vwf (~500 kda); c) arrow symbolizing the decrease in function (binding affinity to collagen and platelet glycoprotein Ib) from large to small sized vwf multimers

4 106/15 Schistocytic anaemia, thrombocytopenia, renal dysfunction mechanisms are involved in the pathogenesis of TTP (Fig. 4). During the 36 months of follow-up after splenectomy our patient did not experience a relapse. Fig. 4 Established and hypothetical mechanisms in the pathogenesis of acquired TTP A controversial discussion is taking place concerning the specificity of ADAMTS-13 deficiency for TTP. Mannucci et al. (22) reported decreased ADAMTS-13 activity in newborns, pregnant women, and patients with various disease conditions (liver cirrhosis, inflammatory diseases), concluding that ADAMTS-13 deficiency was not specific for TTP. Moore et al. (25) suggested that ADAMTS-13 deficiency was present also in several patients with thrombocytopenia caused by a variety of conditions other than TTP. However, in a large study on 68 patients with thrombocytopenia not caused by TTP or HUS, ADAMTS-13 activity was in none of these cases <10% even though it was mildly or moderately decreased in some patients (5). Therefore, we conclude that a severe deficiency of ADAMTS-13 activity (<3-5% of normal plasma) is a specific feature of TTP and must be distinguished from moderately (10-25%) and mildly decreased (25-50%) values. Another question addresses the sensitivity of ADAMTS-13 deficiency for the diagnosis of TTP. Furlan et al. (10) found 86% (26 of 30) of patients with TTP to be lacking ADAMTS-13 activity, and Tsai and Lian (36) reported a severe deficiency in 100% (37/37 patients). A prospective study by Veyradier et al. (41) found a severe protease deficiency in 71% (47/66) of the cases diagnosed as TTP. In a large inception cohort of 142 consecutive patients with TMA, Vesely et al. (40) found severely deficient ADAMTS-13 activity in only 33% (16/48) of the patients with idiopathic TTP. Thus, even though a severe deficiency of ADAMTS-13 activity is specific for a TMA that is generally diagnosed as TTP not all patients with TTP have severe ADAMTS- 13 deficiency. In our opinion, the absence of severe ADAMTS-13 deficiency should not rule out the diagnosis of TTP, which is based on clinical findings. It may be speculated that other mechanisms regulating the size of vwf multimers play a role in the pathogenesis of TTP. One interesting hypothesis concerns defective binding of ADAMTS-13 to the endothelium without interfering with the active site of the protease (24). This could, for instance, be due to anti-glycoprotein IV (CD36) antibodies, found in patients with TTP (34). Such autoantibodies might lead to impaired anchoring of ADAMTS-13 to the endothelium that is not detectable by current assays of ADAMTS-13 activity in plasma (Fig. 4). Finally, other still unknown pathogenetic mechanisms besides ULvWF multimers may lead to a clinical picture indistinguishable from TTP. Our patient initially presented with a severe ADAMTS-13 deficiency of <3% (Fig. 2). A weak inhibitor was detected. After 4 and 10 days of plasma exchange therapy the ADAMTS-13 activity had increased to about 30% and 100%, respectively. At the time of the clinical relapse, the protease activity interestingly was 15%, which is outside the range of severe deficiency (<3-5%) typically seen in many patients with classical TTP. No inhibitor was detectable during the second episode.the relapse, despite the absence of severe ADAMTS-13 deficiency, supports the hypothesis that additional Therapeutic strategies in TMAs If untreated, TTP generally follows a rapidly progressive fatal course in most patients. The mortality rate of >90% (1) was reduced to 8-25% by the introduction of therapeutic plasma exchange in clinical routine (15, 29). Plasma exchange should be started immediately when TTP is diagnosed or suspected. Plasma removal eliminates the acquired autoantibody and plasma infusion replaces the missing ADAMTS-13. It was shown by the Canadian Apheresis Study Group (28) that plasma exchange with fresh frozen plasma (FFP) was more effective than plasma infusion alone. However, this may reflect a dose phenomenon, as the patients treated with plasma exchange received threefold as much plasma as those with plasma infusion alone. It has been suggested that cryosupernatant plasma may be more efficient than FFP. However, equal outcomes were reported by the North American TTP Group (42). Mori et al. (26) showed a better response to plasma exchange therapy in those patients with severe ADAMTS-13 deficiency (10/12 patients with severe deficiency survived whereas 4/6 without severe deficiency died).this may suggest that plasma exchange with FFP replacement is not the optimal treatment in patients without severe autoantibody-mediated ADAMTS-13 deficiency. However, in the absence of knowledge of the pathomechanism in these patients, standard treatment with plasma exchange remains mandatory until improved therapeutic options based on the underlying pathogenesis become available. The strategy of performing plasma exchange in all patients with clinically diagnosed TTP is supported by data reported by Vesely et al. (40):Among the 48 patients with idiopathic TTP, treatment response, TTP-associated deaths and total mortality were not significantly different among the 16 patients with and the 32

5 107/16 without severe acquired ADAMTS-13 deficiency. Because many patients in all ADAMTS- 13 activity categories apparently respond to plasma exchange treatment, it does not seem justified at present to withhold plasma exchange in those without severe ADAMTS-13 deficiency. Plasma exchange is generally performed daily until platelet concentration and lactase dehydrogenase (LDH) activity are normalized. Usually, the neurological symptoms tend to improve first, followed by LDH and the platelets (35). After normalization of these parameters the frequency of plasma exchange is tapered. Typically, a response is seen during the first week and usually recovery is complete after 3 weeks, but large variations exist (40). For unknown reasons not all patients respond to plasma exchange therapy. In these cases, plasma exchange can be intensified to twice daily and/or additional FFP infusion between apheresis sessions may be applied. High doses of glucocorticosteroids are generally added to the treatment. This seems justified with regard to the frequent presence of autoantibodies inhibiting ADAMTS-13 activity. Vincristine is also often used in refractory TTP. Even though it acts as immunosuppressive agent, it is also known to have a direct effect on platelets by interfering with the polymerization of microtubules of the platelet membrane. Splenectomy was performed empirically in several patients with plasma refractory or relapsing TTP. As shown in the first patient described with autoantibody-mediated ADAMTS-13 deficiency and a relapsing course of TTP (12), splenectomy may lead to long-lasting remission by eliminating an important source of B-lymphocytes producing the inhibiting autoantibodies. Rituximab, the chimeric monoclonal anti-cd 20 antibody, currently is under investigation (17, 44). In the absence of life-threatening bleeding, platelet transfusions are contraindicated since they may lead to expanding microvascular thrombosis (19). Relapse of acquired TTP is common with an estimated recurrence rate of 36% after 10 years (32). Usually, patients relapse within the first year, but recurrences may occur as late as 10 years after the initial bout of TTP. Splenectomy might be beneficial in these cases as discussed above. The treatment of hereditary TTP differs from that of acquired TTP. Patients with severe constitutional ADAMTS-13 deficiency often manifest a first bout of TTP in early childhood but sometimes only in adulthood (9). They respond dramatically to FFP infusion alone (39). Regular infusions of FFP in order to substitute the deficient protease are an efficient prophylactic measure to avoid otherwise frequent relapses (3, 18). Children with diarrhea-associated HUS are generally not treated with plasma exchange and usually recover from acute episodes (33). Adults with HUS after E. coli O157 : H7 infection, however, have high mortality rates. One study suggested that plasma exchange therapy might be effective for them as well (7). Patients with ticlopidine-associated TMA had lower mortality rates when treated with plasma exchange therapy (4). Neoplasia-associated TMA and haematopoietic stem cell transplantation-associated TMA are sometimes treated by plasma exchange even though the outcome is usually fatal (40). References 1. Amorosi EL, Ultmann JE. Thrombotic thrombocytopenic purpura: report of 16 cases and review of the literature. Medicine (Baltimore) 1966; 45: Antoine G, Zimmermann K, Plaimauer B et al. ADAMTS13 gene defects in two brothers with constitutional thrombotic thrombocytopenic purpura and normalization of von Willebrand factor-cleaving protease activity by recombinant human ADAMTS13. Br J Haematol 2003; 120: Barbot J, Costa E, Guerra M et al. 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