Research On The Mechanism Of Coagulation,inflammatory Molecules Expression And Thrombosis Formation Induced By Anti-?2GP?/?2GP? Complex

Antiphosphopid antibodies(aPL)/anti-?2-glycoprotein I(anti-?2GP?)complex plays an important role in the pathogenesis of antiphospholipid syndrome(APS),which could induce thrombus formation by triggering the expression of pro-inflammatory cytokines and thrombosis-related factors.Previous studies showed that TLR4 and ANX2 synergistically mediate the anti-?2GP?/?2GP? complex-induced expression of TF by THP-1 cells.Based on the previous studies,this paper validated the mechanism of anti-?2GP?/?2GP? complex in inflammation and thrombosis by THP-1 cells in vitro;and established experimental APS(EAPS)mouse model in TLR4 intact(C3H/HeN)and TLR4 deficiency(C3H/HeJ)mice.The analysis TLR4 function in aPL/anti-?2GP?-induced arteriovenous thrombosis in mice provided experimental basis for APS immune intervention and prevention of thrombosis.TLR4 and ANX2 synergistically mediate the anti-?2GP?/?2GP? complex-induced TF expression by THP-1 cells.This study investigated the roles of TLR4 and its adaptor molecules MD-2 and MyD88,and nuclear factor kappa B(NF-?B)in the anti-?32GP?/?2GP?-induced activation of THP-1 cells,and analyzed their influence on proinflammatory expression.The results showed that anti-?2GP?(10?g/mL)/?32GP?(100?g/mL)complex treatment can promote interleukin-6(IL-6),interleukin-8(IL-8)and tumor necrosis by THP-1 cells.Expression of factor ?(TNF-?);whereas the effects were blocked by the pre-treatment of TLR4 inhibitor TAK-242(5 ?M)or NF-kB inhibitor PDTC(20 ?M)was not elevated.The results suggest that TLR4/MD-2/MYD88 and NF-?B signaling pathways are involved in the expression of IL-6,IL-8 and TNF-? induced by anti-?2GP?/?2GP? complex,which may be related to the development of antiphospholipid syndrome(APS).This study analyzed the differences in TF mRNA and protein expression between peritoneal macrophages isolated from C3H/HeN and C3H/HeJ mice in vitro.The results showed that gene and protein levels of TF,TNF-?,IL1? and IL6 were significantly increased in the C3H/HeN mice group after anti-?2GP?/?2GP? complex or LPS stimulation(p<0.05).For FeC13-induced thrombus model in EAPS mice,C3H/HeN mice developed larger thrombus in the carotid artery and more red blood cell accumulation in the femoral vein,suggesting the effects of anti-?2GP?/?2GP? complex on thrombus formation and pro-inflammatory state were mediated by TLR4.Compared with C3H/HeJ EAPS model mice,macrophages activity and carotid TF expression in C3H/HeN EAPS model mice weresignificantly increased,as well as the expression of TF,TNF-? and IL1? protein in macrophages and endothelial cells isolated from those mice.After intraperitoneal injection of anti-?2GP? or IgG-APS in C3H/HeN mice,the expression of VCAM-1,ICAM-1 and E-selectin genes and proteins in the thoracic aortic adhesion molecules was increased,compared with C3H/HeJ mice treated by the same stimuli.The results indicated that anti-?2GP?/?2GP? complex was involved in the formation of adhesion-promoting state,namely the elevated expression of VCAM-1,ICAM-1 and E-selectin in vascular endothelial cells of thoracic aortic isolated from EAPS mice,which depends on TLR4 function.Our findings suggested a role played by TLR4 in APS-related thrombosis and provided new view angle for the prevention and treatment of the disease.