Isolation and Characterization of an Antifactor VIII Antibody from a Patient with Acquired Hemophilia A and a Severe Hemorrhagic Syndrome.
Authors: Bukys Michael A. , Simioni Paolo , Smith Kerri , Spiezia Luca , Kalafatis Michael 
Published: April 2020

Patients with hemophilia A usually develop inhibitory antibodies to factor VIII (fVIII) that are directed against epitopes in the A2 and C2 domains of the cofactor and result in increased morbidity and mortality because of an extended hemorrhagic syndrome. However, in some cases individuals spontaneously develop autoantibodies to fVIII resulting in acquired hemophilia A. While the etiology and mechanism of the disorder are still unknown, the study and determination of the properties of the autoantibodies is necessary to provide insights into their mechanism of generation. A 78-year-old woman was admitted to the Internal Medicine Department of the University-Hospital of Padua for severe anaemia and syncope. She developed an acute hemorrhagic syndrome characterized by the development of extended subcutaneous and mild intramuscular haematomas on both legs. The patient’s family history was negative for hemorrhagic manifestations. Total body computed tomography (CT) scan confirmed the presence of small intramuscular haematomas. Preliminary laboratory analyses revealed a significant prolongation of the aPTT (activated partial thromboplastin time) to 60 sec and a mild prolongation of the PT (prothrombin time) to 15 sec. Mixing experiments with normal plasma failed to correct the patient’s aPTT. Plasma fVIII levels were ~6%. Assay for fVIII inhibitor revealed a level of 6 Bethesda Units (BU). The patient was treated with high dose of steroids and recombinant fVIII (3,000 Units daily). Subsequently, she developed swelling to the neck in the sub maxillary region and symptoms consistent with an acute obstruction to the upper airways requiring oral-tracheal intubations and ventilation. These findings prompted the use of recombinant FVIIa to prevent progression of the hemorrhagic lesion. Three sessions of plasmapheresis were performed, which were followed by the administration of high dose immunoglobulin and the administration of immunosuppressive treatment. After 45 days, treatment was continued with steroids only. The patient recovered completely and no new bleeding episodes developed. The total immunoglobulin fraction was isolated from the patient’s plasma and found to induce a prolongation of the clotting time in an aPTT assay using purified reagents. The immunoglobulin fraction was also found to inhibit intrinsic tenase activity in an assay using purified reagents and a chromogenic substrate that detects factor X activation. In contrast, the total immunoglobulin fraction purified from normal pooled plasma didn’t prolong the aPTT nor inhibited intrinsic tenase activity. Immunoprecipitation experiments with the total immunoglobulins fraction purified from the patient’s plasma revealed that the antibody recognizes epitopes on the light chain of the cofactor. Immunobloting experiments performed with the same material demonstrated that the antibody recognizes fVIII heavy and light chains. Following activation by thrombin it was found that the antibody recognizes the Mr 73,000 light chain and the A2 domain of the cofactor. These data demonstrate that the immunoglobulin fraction isolated from the patient’s plasma has more than one epitope on the cofactor. Our findings provide the demonstration of a strong anti-fVIII acquired autoantibody with low titer which is not related to the presence of antiphospholipid antibodies and is responsible for a severe hemorrhagic syndrome.