Studies On The Clinical Features,Immunologic Characteristics And FⅧ Binding Epitope In Bullous Pemphigoid Complicating With Acquired FⅧ Inhibitor

Research background: Acquired factorâ…§(Fâ…§) inhibitor is an uncommon hemorrhagic disorder, which caused by the development of antibodies directed against the coagulation Fâ…§. The incidence of acquired Fâ…§inhibitor has been estimated to be 0.2～1.0 case per 1 million persons per year. Acquired Fâ…§inhibitor is more frequent in the elderly and affects both sexes equally. Patients with Fâ…§antibodies hemorrhage into the skin,muscles,gastrointestinal and urological system,prolonged postpartum bleeding and excessive bleeding following trauma or surgery, whereas hemarthroses are unusual. The hemorrhage in acquired Fâ…§inhibitor is serious or life threatening, patients with anti-Fâ…§antibodies become resistant to substitution therapy, the mortality rate is as high as 16%～22%. The disease is commonly associated with other underlying medical conditions such as pregnancy and postpartum status,immunological disorders et al. However, in approximately 50% of cases, Fâ…§autoantibodies occure in patients lacking relevant concomitant diseases. Acquired Fâ…§inhibitor is rarely found to be associated with bullous pemphigoid. In the literature, only 8 cases with acquired Fâ…§inhibitor have been described and limited in clinical analysis, however, there is nothing literature similar to our investigation in China. Most Fâ…§inhibitors were IgG, IgA or IgM were rarely to be reported. The IgG subclass of anti-Fâ…§antibody have been characterized as being predominantly polyclonal, a prevalence of the IgG₄ subclass (although IgG₄ constitutes only 4% of serum IgG) often combined with at least one other heavy chain subclass (primarily IgG₁). Previous studies have shown that the major inhibitor epitopes were located in the A2,A3,C2 Fâ…§domains, majority bound to the 44kD and or 72KD thrombin cleaved fragment. We observed and analyzed the clinical features and laboratory examination and identified the diagnosis of a patient, who suffered from bullous pemphigoid with acquired Fâ…§inhibitor. Treated the patient with plasmapheresis therapies, the therapeutic efficacy was estimated by observing the change of symptoms,Fâ…§:C activity and Fâ…§inhibitor titre; By Fâ…§:C neutralizing test in vitro and "temporary" animal hemophilia model was set up, we not only proved Fâ…§inhibitor in patient's plasma was able to inhibit Fâ…§:C activity obviously, but also strengthened the clinical diagnosis; we used immuno-nephelometric methods, combined with the immunoblotting assay, and the distribution table of IgG subclass was obtained; By solid-phase binding assay of Fâ…§and Fâ…§inhibitor, combined with western blot, the binding epitope at which Fâ…§inhibitor binded with Fâ…§was recognized, thus we elucidated the immune characteristics of Fâ…§inhibitor and molecular mechanism for Fâ…§inhibitor pathogeny partly; and provided new data for the clinical features recognizing, laboratory monitoring and reasonable treatment. Partâ… The clinical features and diagnosis of an acquired Fâ…§inhibitor in a patient with bullous pemphigoidObjective: To analyze the clinical and laboratory features of an acquired Fâ…§inhibitor in a patient with bullous pemphigoid, determine the diagnosis of acquired Fâ…§inhibitor, and to improve the understanding of acquired Fâ…§inhibitor. Methods:â‘ Collected and analyzed the patient's case history in detail;â‘¡Laboratory tests about hemorrhagic disorder such as activated partial thromboplastin time (APTT),Prothrombin time (PT),Thromboplastin generation test(TGT) were performed; The plasma Fâ…§:C activity was determined based on One-stage assay; the titre of Fâ…§inhibitor was evaluated by Bethesda Unit(BU); ) The therapeutic efficacy of immunosuppressant combined with plasmapheresis was estimated by observe the change of clinical phenotype,Fâ…§:C activity and Fâ…§inhibitor titre. Results:â‘ Patient's clinical manifestation was consistent with the phenotype of Fâ…§deficiency, who presented with multiple episodes of bleeding after she was diagnosed with bullous pemphigoid. There was no family or prior bleeding history, so we concluded that the bleeding was acquired;â‘¡APTT was 80.5s (normal: 25～35s) and the abnormal APTT could not be corrected by normal plasma; PT was 12.0s (normal, 11～14s); TGT showed Fâ…§deficiency, and the Fâ…§activity was < 1.5%, Fâ…§inhibitor titer in patient's plasma was 147.8 BU;â‘¢Patient's hematoma slowly resolved and skin ecchymoses disappeared, the Fâ…§:C activity went up to 13.2% and Fâ…§inhibitor titer dropped to 28 BU/ml after treated with plasmapheresis and cyclophosphamide. Conclusion: We determined the clinical and laboratory diagnosis of the patient suffered from bullous pemphigoid with acquired Fâ…§inhibitor; APTT and PT were the preliminary screening tests of hemorrhagic disease, further APTT correct test,TGT,the detection of Fâ…§:C activity and Fâ…§inhibitor titre can determine the diagnosis; Immunosuppressant and plasmapheresis are effective in treating acquired Fâ…§inhibitor.Partâ…¡Inhibitory effect of Fâ…§inhibitor on Fâ…§:C in normal pooled plasma and rabbit modleObjective: To purify the IgG from patient plasma or normal pooled plasma; To certify the effects of Fâ…§inhibitor on Fâ…§:C activity in normal pooled plasma or rabbits in vivo. Methods: IgG purification was finished by protein A-agrose column chromatograph and identified by SDS-polyacrylamide gel ectrophoresis (SDS-PAGE); Purified patient IgG was added into normal pooled plasma with different dose (Oμg, 6.25μg, 12.5μg, 25μg, 50μg, 75μg), then APTT was assayed for discovering Fâ…§inhibitor effect on Fâ…§in vitro, equal amounts of normal human IgG was added into normal pooled plasma as control; Pateint's plasma was infused into rabbits by intravenous injection and blood sample were collected at different time-point (including pre-infusion 30′, post-infusion 30′,60′,90′,120′separately). Then rabbit plasma APTT values were evaluated, equal volume of normal plasma was infused into rabbits as control. Results:â‘ we got the purified IgG, the molecular weight of purified IgG are 55KD (heavy chain) and 28KD light chain);â‘¡The patient's IgG was able to prolong normal human plasma APTT significantly with a dose-dependent manner, while normal IgG had no effect on normal human plasma APTT;â‘¢Animal experiments showed that the patient's plasma markedly prolonged rabbits plasma APTT in a time-dependent fashion; while normal human plasma did not interfere with rabbits APTT. Conclusion: The Fâ…§inhibitor in the patient with bullous pemphigoid is IgG; The Fâ…§inhibitor has significant inhibitory effect on Fâ…§:C in vivo or vitro, which strengthened the clinical diagnosis of acquired Fâ…§inhibitor.Partâ…¢IgG subclass assay and Fâ…§binding epitope identification of acquired Fâ…§inhibitorObjective: To determine the IgG subclass concentration of patient; To identify Fâ…§inhibitor IgG subclass and Fâ…§binding epitope, and to elucidate the molecular mechanism for Fâ…§inhibitor pathogeny. Methods:â‘ The determination of IgG subclass concentration by immuno-nephelometric method;â‘¡Western blot analysis was used, combined with optical density(OD)scanning for protein band, we determined the relative concentration of patient's IgG subclass;â‘¢The Fâ…§of thrombin cleaved was run on 8% SDS-PAGE, then separated Fâ…§fragments were electrophoretically transferred on PVDF membranes, the membranes were incubated with patient's plasma overnight (solid-phase binding assay of Fâ…§and Fâ…§inhibitor), combined with Western blot, the IgG subclass and the binding epitope of Fâ…§Inhibitor was identified; The membranes were incubated with normal human plasma as control. Results:â‘ Immuno-nephelometric method displayed the concentration of IgG₄ in patient's plasma or purified IgG was significantly higher than that in normal plasma or purified IgG, the patient's IgG₁ subclass (both plasma and IgG) was also somewhat higher than that in normal. However, there are not obviously difference in IgG₂ and IgG₃ between patient and normal (both plasma and IgG);â‘¡Western blot analysis combined with OD scanning displayed the patient's inhibitors were IgG₄ predominant, followed by IgG₁;â‘¢Solid-phase binding method followed by Western blot showed that reactivity of the 44KD Fâ…§fragment with monoclonal anti-IgG₄ and IgG₁, but lacked the reactivity with monoclonal anti-IgG₂ and IgG₃ when incubated PVDF membrane with patient plasma; However, there is not evidence for the binding when incubated PVDF membrane with normal plasma(IgG₁,IgG₂,IgG₃ or IgG₄). Conclusion: We definited the IgG subclass of Fâ…§inhibitor in Chinese patient with bullous pemphigoid, which was IgG₄ predominant and IgG₁ was secondary; the binding epitope for Fâ…§inhibitor was identified as Fâ…§44KD fragment, the immunologic characteristics and molecular mechanism for acquired Fâ…§inhibitor pathogeny were elucidated partly.in our case.