Study On The Mechanism Of Rifampicin-induced Immune Thrombocytopenia

ObjectiveDrug-induced immune thrombocytopenia(DITP)is a major adverse drug effect mediated by drug-dependent antibodies(D-d Abs).It can cause severe thrombocytopenia and life-threatening bleeding,which can be actuated by a wide range of medications including rifampicin.In this study,we investigated the mechanisms of thrombocytopenia induced by rifampicin during treatment of tuberculosis,and assessed effects of IVIG and corticosteroid on their alone or combined in the treatment of these diseases.These may provide some guidance to clinicians when they treat DITP patients.Methods1.In vitro experiments:(1)The presence of rifampicin-dependent antiplatelet antibodies(rd-Ab)in RITP patient's serum was determined by flow cytometry analysis.(2)Monoclonal antibody immobilization of platelet antigen(MAIPA)and Immunoprecipitation assay was used to detect the binding of Rifampicin-dependant patient antibody to glycoprotein complex GPllb/llla on platelet membrane.(3)To detect the reactions of rifampicin-dependant antibodies in patient serum with platelet membrane glycoproteins and the localization of rd-antibodies on GPllb/llla complex,flow cytometry and MAIPA assay was preformed.(4)Platelet aggregation assay was carried out to determined the Inhibited effect of rd-antibody on platelet aggregation2.In vivo experiments:NOD/SCID mouse model of RITP was used to To Investigate the Protective effects of intravenous immunoglobulin(IVIG),methylprednisome and combination of both against platelet clearance mediated by rd-antibodiesResults1.The presence of rifampicin-dependent anti-platelet antibody in patient's serum of RITP was assessed using flow cytometry.We found that rd-Ab in the patient's serum is an Ig G antibody.Binding of the rd-antibody to normal platelets was detected at concentrations of rifampicin as low as 6 mg/L,which was much lower than a standard dose of 600 mg in the pharmacological levels of human plasma.2.The rd-Ab was characterized by using flow cytometry,MAIPA,and immunoprecipitation assay.We found that the rd-Ab targets glycoprotein GP IIb-IIIa.Our results have further demonstrated that the binding of rd-Ab to platelets was blocked by the anti-GPIIb/IIIa m Ab(AP2),indicating that the rd-Ab is not only specific for GPIIb/IIIa but also identical or close to the binding site of AP2,calcium-dependent epitopes on the GPIIb/IIIa complex.3.Functional study has shown that platelet aggregation was partially inhibited by rd-Ab,indicating that rd-Ab not only caused thrombocytopenia,but also platelet dysfunction.4.In mouse model of RITP,rapid human platelet clearance was observed with the rd-Ab,which was partially prevented by the treatments of IVIG and corticosteroids alone.However,the platelet clearance mediated by rd-Ab was more rapidly inhibited with the treatment of IVIG than corticosteroid that is timeand dose-dependent.The combination of IVIG and corticosteroids has shown better results than IVIG or corticosteroids alone.Conclusions1.Our results have demonstrated that the rd-Ab is not only specific for GPIIb/IIIa but also identical or close to the binding site of AP2,calcium-dependent epitopes on the GPIIb/IIIa complex,which platelet aggregation was partially inhibited by rd-Ab.2.In NOD/SCID mouse model of RITP,rapid human platelet clearance was observed with the rd-Ab,which was partially prevented by the treatments of intravenous immunoglobulin(IVIG)and corticosteroids alone.However,the platelet clearance mediated by rd-Ab was more rapidly inhibited with the treatment of IVIG than corticosteroid.The combination of IVIG and corticosteroids has shown better results than IVIG or corticosteroids alone.