Factor VIII inhibitors in mild haemophilia A

Source: OAI

ABSTRACT The X-linked bleeding disorder haemophilia A is due to a deficiency or functional defect of coagulation factor VIII. The bleeding tendency can be corrected by administration of factor VIII concentrates. A serious complication of factor VIII replacement therapy is the development of anti-factor VIII antibodies (inhibitors) that neutralize factor VIII activity. In recent years, the epitope-specifities and the inhibitory mechanisms of factor VIII inhibitors have gained increasing interest. The generation of factor VIII knock-out mice has opened the possibility of studying the immunobiology of inhibitor formation in murine models of haemophilia A. In spite of these recent developments however, the immunological mechanisms underlying the anti-factor VIII immune response have remained poorly understood so far. Most of our current knowledge is based on studies on inhibitor formation in the severe form of haemophilia. However, inhibitors also occur in patients with mild haemophilia A, in particular after a period of extensive factor VIII replacement therapy. These patients differ from severe haemophiliacs in that they have low levels of circulating factor VIII activity (5-25% of normal). The presence of endogenous factor VIII may have major impact on the immune response to exogenous factor VIII during replacement therapy. The studies presented in this thesis were performed to obtain a better understanding of the immunobiology of inhibitor development in mild haemophilia A. In the introduction (chapter 1), recent studies on the immunobiology of factor VIII inhibitors in haemophilia A patients are summarized and discussed. We have characterized the anti-factor VIII antibodies in patients with mild haemophilia A employing phage display technology. In chapter 2, anti-C2 antibodies were isolated and characterized from the repertoire of a mild haemophilia A patient. Our results provide evidence for the presence of two classes of anti-C2 antibodies that recognize distinct antigenic sites in factor VIII. The characteristics of the anti-C2 antibodies were further analysed in chapter 3, and compared to the epitopes of previously described murine monoclonal antibodies. The first class of anti-C2 antibodies bind to the epitope defined by monoclonal antibody ESH4. The second class of antibodies bind to the epitope defined by monoclonal antibody CLB-CAg 117. Antibodies belonging to this second class of antibodies were also isolated from a different patient with mild haemophilia A (chapter 4). The VH gene segment usage of the antibodies directed at the epitope defined by CLB-CAg 117 is less restricted compared to the first class of anti-C2 antibodies. Based on the long CDR3 region, we argue that this second class of antibodies originates from a pool of polyreactive human antibodies. In chapter 5, we describe the inhibitor development of a patient with mild haemophilia A caused by an Arg593 to Cys mutation. We have isolated and characterized anti-A2 antibodies using phage display and we have performed epitope-mapping studies of anti-factor VIII antibodies in plasma using immunoprecipitation analysis. The data presented in chapter 5 provide a possible explanation for anamnestic responses observed in patients with a history of inhibitor development. We propose that activation of a quiescent pool of memory B cells underlies the rise in inhibitor titer observed in haemophilia A patients with a history of inhibitor development. Chapter 6 describes the epitope specificities of anti-factor VIII antibodies in another patient from our cohort of mild haemophilia A patients with the Arg593 to Cys mutation. Results from this chapter and previous studies show that high responder patients with the Arg593 to Cys substitution develop inhibitory antibodies predominantly directed at the A2 domain of factor VIII. This suggests that inhibitor formation proceeds via a common mechanism in these patients. The role of HLA class II alleles in inhibitor formation was investigated by HLA genotyping of 42 patients with the Arg593 to Cys mutation. Our data suggest a weak association between inhibitor development and HLA class II alleles in mild haemophilia A patients with the Arg593 to Cys mutation. In Chapter 7, we present the characteristics of a mouse transgenic for human factor VIII with the Arg593 to Cys mutation (hufVIII-R593C mouse). The anti-factor VIII immune response was analysed in transgenic hufVIII-R593C mice crossed with factor VIII-deficient mice (exon 16 knock out, or E-16 KO mice). Serial intravenous injections of human factor VIII do not evoke an immune response in hufVIII-R593C/E-16 KO mice. The introduction of the human factor VIII–R593C transgene renders the mice tolerant to human factor VIII. However, when hufVIII-R593C/E-16 KO mice were subcutaneously injected with factor VIII in the presence of an adjuvant, loss of tolerance to factor VIII was observed. The results of chapter 7 demonstrate that hufVIII-R593C/E-16 KO mice provide a valuable model for studies directed at the mechanisms underlying inhibitor development in haemophilia A. Finally, chapter 8 provides a general overview that discusses the implications of our findings.

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    New England Journal of Medicine 07/2001; 344(23):1773-9. DOI:10.1056/NEJM200106073442307 · 55.87 Impact Factor
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    ABSTRACT: Twenty six patients with mild or moderate haemophilia A and inhibitors are described. The inhibitor was detected at a median age of 33 years, after a median of 5.5 bleeding episodes. This usually following intensive replacement therapy. The median presenting inhibitor titre was antihuman 11.6 BU/ml, antiporcine 1.45 BU/ml. Plasma basal factor VIII level declined from a median of 0.08 IU/ml to 0.01 IU/ml following the inhibitor development. This caused spontaneous bleeding in 22 and a bleeding pattern similar to acquired haemophilia in 17. Bleeding was often severe and caused two deaths. The inhibitor disappeared spontaneously, or following immune tolerance induction, in 16 cases after a median of 9 months (range 0.5-46), with a return to the original baseline VIIIC level and bleeding pattern accompanied inhibitor loss. The inhibitor persisted in the remainder of the cases over a median period of 99 months (range 17-433 months) of follow-up. Inhibitors are an uncommon complication of mild haemophilia which frequently persist and may be associated with severe, life-threatening, haemorrhage. Forty-one percent of treated haemophilic family members had a history of factor VIII inhibitors, suggesting a familial predisposition to develop inhibitors in these kindreds. Sixteen patients from 11 families were genotyped. Seven different missense mutations affecting the light chain were detected and two in the A2 domain. Five patients from three families had a mutation causing a substitution of Trp2229 by Cys in the C2 domain which appears to predispose to inhibitor formation since 7 of the 18 affected individuals have a history of inhibitor development.
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    ABSTRACT: The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.
    Journal of Biological Chemistry 07/1995; 270(24):14505-9. DOI:10.1074/jbc.270.24.14505 · 4.57 Impact Factor
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