E. A. M. Turenhout

The spacer domain of ADAMTS13 contains a major binding site for antibodies in patients with thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy often associated with a severely decreased activity of ADAMTS13. In plasma of the majority of patients with TTP, antibodies are present that inhibit the von Willebrand factor (VWF) processing activity of ADAMTS13. We describe a sensitive assay that monitors binding of recombinant ADAMTS13 to immobilized IgG derived from patient plasma. Analysis of fifteen patients with TTP and severely reduced ADAMTS13 activity revealed that in all patients antibodies directed to ADAMTS13 were present. Levels of anti-ADAMTS13 antibodies varied considerably among patients, specific antibody levels in plasma range from less than 100 ng/ml to over 1 microg/ml. Longitudinal analysis in three patients revealed that anti-ADAMTS13 antibody levels declined with different kinetics. For further characterization of anti-ADAMTS13 antibodies, we prepared a series of recombinant fragments corresponding to the various ADAMTS13 domains. All seven TTP plasma samples tested, showed reactivity of antibodies towards a fragment consisting of the disintegrin/TSR1/cysteine-rich/spacer domains. In one patient, we also observed reactivity towards the TSR2-8 repeats. No binding of antibodies to propeptide, metalloprotease and CUB domains was detected. To further delineate the binding site in the disintegrin/TSR1/cysteine-rich/spacer region, we prepared additional ADAMTS13 fragments. Antibodies directed towards the cysteine-rich/spacer fragment were found in all plasma samples analyzed. No antibodies reacting with the disintegrin/TSR1 domains were detected. A recombinant fragment comprising the spacer domain was recognized by all patients samples analyzed, suggesting that the 130-amino-acid spacer domain harbors a major binding site for anti-ADAMTS-13 antibodies.

Amino acid regions 572-579 and 657-666 of the spacer domain of ADAMTS13 provide a common antigenic core required for binding of antibodies in patients with acquired TTP.

Antibodies directed against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). We have previously localized a major antigenic determinant within the spacer domain of ADAMTS13. To identify the amino acid residues of the spacer domain that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of fifteen hybrids (designated A-O) in which 5-10 amino acids of the spacer domain were exchanged for the corresponding region of ADAMTS1. Plasma from six patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of amino acid residues 572-579 (hybrid C) and 657-666 (hybrid M) completely abolished the binding of antibodies from all six patients analyzed. Regions 580-587 (D), 602-620 (G, H), 629-638 (J), and 667-767 (N) contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). Antibodies derived from patient 1 required region 602-620 (G, H) for binding (CGHM-epitope). For antibodies of patient 3, residues 564-571 (B), 580-587 (D), and 629-638 (J) were required (BCDJM-epitope), whereas replacement of residues 602-610 (G) and 629-638 (J) greatly diminished binding of antibodies from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of patients, our results suggest that residues 572-579 (C) and 657-666 (M) comprise a common antigenic core region that is crucial for binding of anti-ADAMTS13 antibodies. Other regions that spatially surround this antigenic core further modulate binding of antibodies to the spacer domain.

VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Summary.  Background: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1‐69 heavy chain gene segment for the assembly of anti‐ADAMTS13 antibodies. Objective: We explored the ability of two VH1‐69 germline gene‐encoded antibodies to inhibit the von Willebrand factor (VWF)‐processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1‐69 encoded anti‐ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1‐69 encoded antibodies. Methods and Results: Binding of the two VH1‐69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS‐VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL‐VWF strings on the surface of endothelial cells. G8‐reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. Conclusions: These results suggest that VH1‐69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.

Factor VIII inhibitor in a patient with mild haemophilia A and an Asn618→Ser mutation responsive to immune tolerance induction and cyclophosphamide

Summary.  We describe a patient with mild haemophilia A (original value of factor VIII activity 0·30 U/ml) who developed an inhibitor (36·1 Bethesda U/ml) which cross‐reacted with his endogenous factor VIII. This caused a decline in basal factor VIII level (< 0·01 U/ml) and severe haemorrhagic events. Treatment to induce immune tolerance was started with factor VIII/von Willebrand factor (VWF) concentrates, but inhibitor levels increased progressively and the patient suffered serious bleeding. Cyclophosphamide was administered and, after 8 months treatment, factor VIII levels increased to 0·20 U/ml and the inhibitor could no longer be detected. Screening of his factor VIII gene revealed a missense mutation in exon 13 that predicts substitution of Asn618→Ser in the A2 domain of factor VIII. Immunoprecipitation analysis showed that the antibodies present in the patients plasma reacted with metabolically labelled A2 domain and, to a lesser extent, with factor VIII light chain. Inhibitory antibodies were completely neutralized by recombinant A2 domain, whereas no neutralization was observed after the addition of factor VIII light chain (A3–C1–C2) and C2 domain. More detailed analysis showed that the majority of inhibitory antibodies were directed against residues Arg484–Ile508, a previously identified binding site for factor VIII inhibitors. Our findings suggest that immune tolerance therapy and cyclophosphamide were successful in eradicating inhibitory antibodies against a common epitope on factor VIII.

Analysis of factor VIII inhibitors in a haemophilia A patient with an Arg593-->Cys mutation using phage display.

Summary. We characterized anti‐factor VIII antibodies in a mild haemophilia A patient with an Arg593→Cys mutation in the A2 domain, using V gene phage‐display technology. All isolated single‐chain variable‐domain antibody fragments were directed against residues Arg484‐Ile508, a binding site for factor VIII inhibitors in the A2 domain. After a further period of replacement therapy, a transient rise in inhibitor titre was observed. These antibodies were directed against the A2 domain. Activation of a pre‐existing pool of B cells, which express antibodies against residues Arg484‐Ile508, could explain the rapid anamnestic response.

Disappearance of factor VIII autoantibodies preceding autoimmune haemolytic anaemia.

We describe a previously healthy woman who at the age of 44 years developed a factor VIII inhibitor, that over the years increased to a maximum level of 3600 Bethesda units (BU) mL–1 in 1978. The epitope specificity of the factor VIII inhibitor was investigated and antibodies directed against the A2 and C2 domains of factor VIII were detected. The majority of these antibodies were of subclass IgG4. Over the years, the inhibitor titre gradually decreased and in 1989, the inhibitor could no longer be detected. Shortly after, the patient developed autoimmune haemolytic anaemia. A possible link between the disappearance of factor VIII inhibitors and the development of other autoantibodies may be explained by concomitant development of anti‐idiotypic antibodies that neutralize the activity of factor VIII inhibitors. We were unable to detect anti‐idiotypic antibodies, which could explain the decline in factor VIII inhibitor titre in this patient.