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Classical complement pathway activation in immune thrombocytopenia purpura: inhibition by a novel C1s inhibitor

2015; Wiley; Volume: 173; Issue: 6 Linguagem: Inglês

10.1111/bjh.13648

1365-2141

Ellinor I.B. Peerschke, Sandip Panicker, James B. Bussel,

Complement system in diseases

Immune thrombocytopenia purpura (ITP) is an autoimmune disorder, the pathophysiology of which is characterized by immune-mediated platelet destruction and decreased platelet production (Johnsen, 2012). Platelet destruction in ITP occurs by a variety of immune-mediated mechanisms, including humoral and cell-mediated immunity. Increasing evidence suggests a role for complement activation in ITP (Peerschke et al, 2009; Najaoui et al, 2011). Although early work by Frank et al (1975) demonstrated that an intact classical complement pathway was required for damage of antibody-sensitized mammalian cell membranes and the development of thrombocytopenia in a guinea pig model, the role for classical pathway (CP) complement activation in human ITP has not been definitively established. The lectin pathway has also been reported to be activated by antibodies and autoantibodies, generating the same activated complement proteins downstream of C1 as the CP (Malhotra et al, 1995). In the present study, we performed an in vitro trial of a novel CP complement inhibitor (TNT003), a murine monoclonal antibody directed against C1s, to evaluate the role of CP activation in ITP and provide in vitro proof of principle that CP inhibition prevents complement activation in ITP patient plasma. TNT003 is a novel C1s inhibitor that has been shown to inhibit cold agglutinin-mediated complement deposition on the surface of red blood cells in vitro (Shi et al, 2014). Patients (n = 55) consisted of males (n = 21), age 39 ± 24 years (mean ± SD) (range 8–87 years) and females (n = 34), age 48 ± 23 years (range 17–86 years), with a median ITP duration of 106 months and 99·5 months, respectively. At the time of blood collection, patients were undergoing treatment with a variety of modalities including rituximab, intravenous immunoglobulin, eltrombopag, romiplostin, veltuzumab, ciclosporin, danazol, azathioprine, prednisone, dexamethasone and mycophenolate mofetil, either alone or in combination. Fifteen patients had undergone splenectomy. Results of CP activation were compared to platelet count, obtained as part of the patient's clinical laboratory assessment, and the presence of antiplatelet antibodies (IgG, IgM, IgA) directed against major platelet membrane glycoprotein antigens, IIb/IIIa, Ia/IIa, and Ib/IX, using the Lifecodes Pak12 assay (Immucor GTI Diagnostics, Inc., Waukesha, WI, USA). This study was approved by the Institutional Review Boards of Weill Cornell Medical School and Memorial Sloan Kettering Cancer Center. Classical pathway activation was evaluated using a previously described assay (Peerschke et al, 2009). Given that complement activation occurs spontaneously on activated or immobilized platelets (Peerschke et al, 2010), CP activation by patient plasma was expressed as a ratio relative to pooled normal control plasma, in order to detect enhanced complement activation. Enhanced complement activation was defined as a ratio of ≥1·5, representing values greater than 3 SD above the reference interval (97·5% confidence limit). Increased complement activation was noted in 26/55 patients with ITP (c. 47%). Elevated C1q deposition was found in 42% of patients (23/55 patient plasma samples). Enhanced C4d deposition was demonstrated in c. 13% of patients (7/55 plasma samples) and was associated with a statistically significant inverse correlation (P = 0·042) with platelet count (Fig 1). In six of seven patients with heightened C4d deposition, the circulating whole blood platelet count was below 100 × 109/l, including five patients with platelet counts below 50 × 109/l. Increased C4d deposition was associated with the presence of autoantibodies directed against major platelet antigens in all five patients with platelet counts below 50 × 109/l. In these patients (5/5), antibodies directed against GPIIb-IIIa were identified. Three patients additionally demonstrated antibodies against GPIa/IIa. A single patient also exhibited detectable autoantibodies against GPIb/IX. These findings are consistent with previous reports summarized by McMillan (2009), who described anti-platelet antibodies in approximately 58% of patients, with reactivity to GPIIb-IIIa being the most common. Classical pathway activation was inhibited by TNT003, as demonstrated by reduced C4d deposition, and markedly reduced downstream C3b and C5b-9 deposition from patient plasma (n = 55) (Table 1). More complete complement inhibition was achieved by chelation of divalent cations with 10 mmol/l EDTA, confirming the participation also of the alternative pathway in complement activation on platelets (Peerschke et al, 2009). TNT003 appears to impact CP activation predominantly downstream of C1 binding, and supports the notion that CP activation plays a major role in terminal complement pathway activation in ITP plasma. Indeed, in vitro platelet lysis has been described following normal platelet exposure to autoantibodies from ITP patient sera (McMillan & Martin, 1981). In addition to direct cellular damage via C5b-9 lytic complexes, autoantibody-mediated complement deposition promotes platelet clearance by the reticuloendothelial system (Johnsen, 2012). Interestingly, ITP patients with a high degree of platelet-associated complement deposition/fixation have been reported to benefit significantly from splenectomy (Bell, 2002). In the present study, increased plasma CP activation was noted in 10 of 15 patients who had undergone splenectomy (P = 0·032, Fisher's Exact Test). These preliminary in vitro findings may suggest that inhibition of CP activation could be a potential alternative to splenectomy for selected patients with ITP. Taken together, the results of the present study provide direct evidence of increased CP activation in ITP and proof of principle that CP inhibition may effectively target this process. CP blockade using C1 esterase inhibitor (C1 INH) has been used clinically, predominantly in patients with hereditary angioedema. Although C1 INH therapy is well tolerated in humans, it exerts effects beyond regulation of the classical complement pathway, including modulation of the lectin pathway and kinin, coagulation and fibrinolytic systems. Targeted inhibition of C1s, therefore, may represent more specific inhibition of CP activation. Indeed, TNT003, when tested in vitro against C1 INH, was found to be >3 orders of magnitude more potent for inhibiting antibody-dependent complement activation in haemolysis-based assays (data not shown). Evaluation of CP complement inhibition in ITP and the potential risk for infection by pyogenic bacteria with chronic use awaits clinical trails. This work was supported in part by TrueNorth Therapeutics, Inc., South San Francisco, CA. The authors are grateful to Nenita Francisco and Kajal Kothadia for expert technical assistance. EP, SP and JB designed the study; EP performed the research; SP and JB contributed essential reagents; EP, SP and JB analysed the data; EP, SP and JB wrote the paper. Sandip Panicker is an employee of TrueNorth Therapeutics, Inc.