Immune response to hemostatic proteins

Principal investigators
Jan Voorberg

Hemophilia is an X-linked bleeding disorder that is caused by a deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B). Coagulation factor replacement therapy of hemophilia may be complicated by the formation of inhibitory or neutralizing antibodies (inhibitors). This side-effect occurs in approximately 25% of the patients with severe hemophilia A, and in about 5% of the patients with hemophilia B. Inhibitor development renders patients unresponsive to coagulation factor replacement therapy. The eradication of inhibitory antibodies in patients with haemophilia A can be accomplished by frequent administration of high or intermediate doses of factor VIII (FVIII), so-called immune tolerance induction (ITI). We have monitored the distribution of IgG subclasses of anti-FVIII antibodies during ITI. FVIII-specific antibodies of subclass IgG1 were detected in all inhibitor patients tested, anti-FVIII IgG4 in 16, IgG2 in 10 and IgG3 in one of 20 patients analyzed. Levels of anti-FVIII IgG1 and IgG4 correlated well with inhibitor titres as measured by Bethesda assay. In low-titre inhibitor patients, anti-FVIII antibodies consisted primarily of subclass IgG1 whereas, anti-FVIII antibodies of subclass IgG4 were more prominent in patients with high titre inhibitors who needed prolonged treatment or who failed ITI. Overall, our findings document the distribution and dynamics of anti-FVIII IgG subclasses during ITI. Future studies will address whether monitoring of the relative contribution of anti-FVIII subclasses IgG1 and IgG4 may be useful for the identification of patients who are at risk of failing ITI.

Thrombotic thrombocytopenic purpura (TTP) is a micro-angiopathy that is related to an acquired or congenital deficiency of the von Willebrand Factor (VWF) cleaving protease ADAMTS13. In the absence of ADAMTS13, ultra large VWF (UL-VWF) polymers, originating from endothelial cell specific organelles, designated Weibel-Palade bodies, accumulate in the circulation. These UL-VWF polymers mediate the formation of platelet-rich thrombi in the microcirculation that give rise to hemolytic anemia and thrombocytopenia. In plasma of the majority of patients with acquired TTP, antibodies directed towards ADAMTS13 are present. Epitope mapping studies revealed that most antibodies interact with the spacer domain of ADAMTS13. Recently, we have shown using an anti-idiotypic antibody specifically recognizing the VH1-69 variable heavy chain gene segment that anti-ADAMTS13 antibodies present in plasma of patients with acquired TTP frequently incorporate this gene segment. Based on these findings we propose that the frequent usage of the VH1-69 gene segments underlies the restricted epitope specificity of anti-ADAMTS13 antibodies that develop in patients with acquired TTP.

Patients suffering from the antiphospholipid syndrome (APS) have antibodies with reactivity against several domains of beta2-glycoprotein I (beta2GPI). Domain I of beta2GPI proved to contain a major binding site for thrombosis-related antiphospholipid antibodies. We investigated the heterogeneity of the antibody response against beta2GPI by isolating a panel of human monoclonal antibodies from two APS patients. Two patients were selected based on a history of recurrent thrombosis and the presence of anti-beta2GPI IgG antibodies in plasma. B-cells were isolated from these patients and cDNA coding for the variable heavy chain repertoire was cloned into pHEN1-VLrep. Single chain Fv fragments displayed on phages were selected for reactivity towards beta2-glycoprotein I and domain I. Unique patient-derived monoclonal antibodies were isolated which are now being characterized for LAC activity and other beta2GPI-dependent molecular interactions. Our current data raise the possibility that pathogenic anti-domain I antibodies contribute to the thrombotic complications observed in patients with APS.

Key publications
Van Helden PM, van den Berg HM, Gouw SC, Kaijen PH, Zuurveld MG, Mauser-Bunschoten EP, Aalberse RC, Vidarsson G, Voorberg J. IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A. Br J Haematol 2008; 142:644-52.
Pos W, Luken BM, Hovinga JA, Turenhout EA, Scheiflinger F, Dong JF et al. VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura. J Thromb Haemost 2009; 7:421-8.
De Laat B, Mertens K, de Groot, PG. Mechanisms of disease: antiphospholipid antibodies – from clinical association to pathologic mechanism. Nat Clin Pract Rheumatol 2008; 4:192-9.

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