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Studies On The Clinical Feature,IgG Subclass And FXIII Binding Epitope In Acquired FXIII Deficiency Due To Autoantibodies Against FXIII

Posted on:2008-10-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y Y LuoFull Text:PDF
GTID:1104360215998924Subject:Internal Medicine
Abstract/Summary:PDF Full Text Request
Research background: Acquired coagulation factorⅩⅢ(FⅩⅢ)deficiency due to an inhibitor is an extremely rare blood disorder. Only34 patients with acquired FⅩⅢinhibitors have been reported by far.None has yet been reported in China. Acquired FⅩⅢinhibitor is morefrequent in the elderly and affects both sexes equally. FⅩⅢinhibitorsneutralize activities of FⅩⅢand block the cross-linking of fibrin, whichresults in instability of the patients' fibrin clot, and severe spontaneousrecurrent bleeding. The bleeding happens in various sites, including skin,subcutaneous tissue, muscles, visceral bleeding, prolonged bleeding aftersurgery, or even intracranial hemorrhage. The hemorrhage caused byacquired FⅩⅢinhibitor may be severe and difficult to treat. Themortality rate is as high as 50%. The cause of emergence of the inhibitorsis not dear. The studies suggest that development of FⅩⅢinhibitors maybe has some relationship with disorders in immune tolerance mechanism.Most cases of FⅩⅢinhibitors have been associated with someautoimmune diseases, especially with systemic lupus erythematosus, orprolonged drug ingestions including isoniazid, penicillin, phenytoin, andpractolol. Incidence of inhibitors in patients with inherited FⅩⅢdeficiency accepted replacement therapy with FⅩⅢconcentrate appearsto approximately 1%. Few FⅩⅢinhibitors develop in persons in good health. In most cases reported, the FⅩⅢinhibitors have been shown to bepolyclonal or monoclonal IgG, with a prevalence of the IgG1 subclass.FⅩⅢinhibitors interfere with one or more of the biochemical reactionsinvolved in the physiological pathway pertaining to fibrin stabilization.According to their modes of action, three major types can bedistinguished: the typeⅠinhibitor prevents activation of FⅩⅢ; the typeⅡinhibitor interferes with the transamidating function of FⅩⅢ; the typeⅢinhibitor is directed against the fibrin subunit itself, blocking thereactivity of the cross-linking site for access to FⅩⅢ. The A subunit ofFⅩⅢis functional subunit, and folds into five domains which areactivation peptide (including thrombin binding site), beta sandwich, thecatalytic core region (including Ca2+ binding site, active site), barrel 1 andbarrel 2. There is no data about FⅩⅢinhibitor epitopes located in thefunctional domains of FⅩⅢA subunit. We diagnosed the first case ofFⅩⅢinhibitor in monoclonal gammopathy of unknown significance andthe third FⅩⅢinhibitor in systemic lupus erythematosus, and studied theclinical features, the immune characteristics, and molecular mechanismfor FⅩⅢinhibitor pathogeny of the both. PartⅠClinical features, diagnosis and treatment ofacquired coagulation factorⅩⅢdeficiency arising fromFⅩⅢinhibitorObjective: To analyze the clinical and laboratory features ofacquired FⅩⅢdeficiency due to FⅩⅢinhibitor, determine the diagnosisof acquired FⅩⅢinhibitor, to enhance the understanding of the clinicaland laboratory features of the bleeding caused by acquired FⅩⅢinhibitor,and to improve the diagnosis and remission rate of the disease. Methods:①Collected and analyzed the patient's case history in details;②Laboratory tests about hemorrhagic disorder such as activated partialthromboplastin time(APTT), prothrombin time(PT), thrombin time(TT),fibrinogen(FIB), D-dimer(D-D), platelet count and function wereperformed; FⅩⅢinhibitor was established by clot solubility assay andclot solubility assay mixing studies; The plasma FⅩⅢtiter wasdetermined based on western blot combined with optical density(OD)scanning for protein band;③The therapeutic efficacy ofimmunosuppressant combined with fresh frozen plasma was estimated byobserve the change of clinical phenotype. Results:①The clinicalmanifestation of the two patients were consistent with the phenotype ofFⅩⅢdeficiency. Before presenting with multiple episodes of bleeding,the patient of the first case was diagnosed with monoclonal gammopathy of unknown significance while the patient of the second case wasdiagnosed with systemic lupus erythematosus. There was no family orprior bleeding history, so we concluded that the bleeding was acquired;②APTT, PT, fibrinogen, platelet count and function were within normallimit. Clots from both of the patients' plasma dissolved in 5M urea by 24hours. Clots prepared from normal plasma were stable for 48 hours. Theabnormal solubility of patients' plasma clot could not be corrected bynormal plasma, which established the presence of FⅩⅢinhibitor. TheFⅩⅢtiter in the patient of the first case was 2.9mg/L(normal: 31mg/L),while the FⅩⅢin the patient of the second case was too few to bedetermined;③The bleeding of the two patients was remarkablyresolved. Keeping receiving prednisone and cyclophosphamide, thepatient of the second case didn't experience episodes of bleeding anymore. Conclusion: We determined the diagnosis of acquired FⅩⅢinhibitors in the two patients suffered from monoclonal gammopathy ofunknown significance and systemic lupus erythematosus, respectively.Clot solubility assay is the preliminary screening test of such hemorrhagicdisease. Further clot solubility assay mixing studies and the detection ofFⅩⅢtiter can determine the diagnosis; Immunosuppressant and freshfrozen plasma are effective in treating acquired FⅩⅢinhibitor. PartⅡInhibitory effect of FⅩⅢinhibitor on FⅩⅢin normalpooled plasma and on fibrin cross-linksObjective: To purify the IgG from patient plasma or normalpooled plasma; to certify the effects of FⅩⅢinhibitor on FⅩⅢactivityand on fibrin cross-linking of clots from normal pooled plasma; todisscuss effects of FⅩⅢinhibitors on the rigidity of fibrin clot from amicroscopic angle. Methods:①IgG and light chain purification werefinished by protein A-agrose column chromatograph and identified bySDS-polyacrylamide gel ectrophoresis (SDS-PAGE) andnative-polyacrylamide gel ectrophoresis (native-PAGE);②Purifiedpatient IgG or light chain or normal IgG was added into normal pooledplasma with different dose (0 mg/ml,1.38 mg/ml,2.76mg/ml), thenclot solubility assay was performed to discover FⅩⅢinhibitor effect onFⅩⅢin vitro;③Fibrin cross-links of clots from two patients' plasmaand normal plasma were identified by SDS-PAGE; Purified patient IgGor normal IgG was added into normal pooled plasma with a final titer of2.76mg/ml, then fibrin clot was obtained and analyzed by SDS-PAGE,too;④Ultrastructure of fibrin clots from the two patients' plasma ornormal plasma was discovered using scanning electron. Results:①wegot the purified IgG of two patients and the light chain of the patient of thefirst case;②IgG of the two patients were able to increasing the solubility of normal plasma clots in 5M urea significantly with a dose-dependentmanner, while normal IgG and the light chain of the patient of the first casehad no effect on normal human plasma clots;③Both an polymers andγ-γdimers of the fibrin clot of the first case were modest, and there wereγmonomers, too. The fibrin clot of the second case had neither a chain norγchain cross-linking. The IgG of the patient of the first case inhibited thea chain andγchain cross-linking of normal clots partially, while the IgGof the patient of the second case completely inhibited both a chain andγchain cross-linking;④Analyzed by scanning electron, Fibrincross-linked from normal plasma consisted of tight and thick fibers, withalmost no fibrin "mesh"; whereas Fibrin cross-linked from the patient'splasma of the second case consisted of looser and thinner fibers, whichformed a larger "mesh". The structure of the patient's clot of the first casewas between normal and the patient of the second case. Conclusion: TheIgG in the two patients have significant inhibitory effect on FⅩⅢin vitroand result in that the ultrastructure of the two patients' clots showedmesh-like appearance different in size with thin and loose fibers, whichindicates the deficiency of FⅩⅢor the loss of FⅩⅢactivity. Stabilizingeffect of FⅩⅢon fibrin clot is weakened, which leads to the hemorrhagicsymptoms clinically. PartⅢIgG subclass and FⅩⅢbinding epitope identification foracquired FⅩⅢinhibitorObjective: To identify FⅩⅢinhibitor IgG subclass and FⅩⅢbinding epitope, and to elucidate the molecular mechanism for FⅩⅢinhibitor pathogeny. Methods:①The IgG subclass of the two FⅩⅢinhibitors were identified by solid-phase binding method followed byWestern blot;②Inhibitory effect of the two patients' IgG on activation ofFⅩⅢby thrombin was certified by SDS-PAGE;③The peptides of thefunctional domains of FⅩⅢA subunit were synthesized. Binding of thetwo acquired FⅩⅢinhibitors to the functional domains of FⅩⅢwasidentified using dot blot. Results:①Solid-phase binding methodfollowed by Western blot showed, when PVDF membrane incubated withthe patient's plasma of the second case, there was reactivity of the Asubunit of FⅩⅢwith monoclonal anti-IgG1 and IgG4, but lacked thereactivity with monoclonal anti-IgG2 or IgG3; when PVDF membraneincubated with patient's plasma of the first case, there was reactivity ofthe A subunit of FⅩⅢwith monoclonal anti-IgG1, but lacked thereactivity with monoclonal anti-IgG2, IgG3 or IgG4;②SDS-PAGEanalysis displayed electrophoresis of FⅩⅢwith thrombin showed oneband for the A' and B subunit different with two bands for the A and Bsubunit of purified FⅩⅢ; Incubated with the patient's IgG of the second case and thrombin, FⅩⅢhad one band for the A' and B subunit, whileincubated with the patient's IgG of the first case and thrombin, FⅩⅢhadtwo bands for the A and B subunit;③PeptideCa2+ containing Ca2+ bindingsite (NH2-YKFQEGQEEE-CONH2) and peptideactive containing activesite (NH2-GWQAVDSTPO-CONH2) from FⅩⅢA subunit weresynthesized; Dot blot analysis with HRP-rabbit anti-human IgG showedthat when PVDF membrane incubated with the patient's plasma of thesecond case, there was reactivity of FⅩⅢ, PeptideCa2+ and peptideactive;when PVDF membrane incubated with patient's plasma of the first case,there was reactivity of FⅩⅢ, and no activity of peptideCa2+ or peptideactive;However, there is no evidence for the binding when incubated PVDFmembrane with normal plasma. Conclusion:①We defined the IgGsubclass of FⅩⅢinhibitor in the patient of the first case with monoclonalgammopathy of unknown significance, which was IgG1, and the IgGsubclass of FⅩⅢinhibitor in the patient of the second case with systemiclupus erythematosus, which was IgG1 and IgG4;②The binding epitopefor FⅩⅢinhibitor in the patient of the first case was identified as FⅩⅢthrombin binding site and activation of FⅩⅢby thrombin was inhibited,which results in that stabilizing effect of FⅩⅢon fibrin clot is weakened;While the binding epitope for FⅩⅢinhibitor in the patient of the secondcase was identified as FⅩⅢCa2+ binding site and active site, andactivity of FⅩⅢand fibrin cross-links reaction activated by Ca2+ were inhibited. The exact binding epitopes of FⅩⅢinhibitors on functionaldomains of FⅩⅢwere determined. The molecular mechanism foracquired FⅩⅢinhibitor pathogeny was elucidated in our cases.
Keywords/Search Tags:FXIII inhibitor, acquired FXIII deficiency, IgG subclass, fibrin clot, ultrastructure, FXIII binding epitope
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