ABSTRACT
Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly ß(2)GPI, and activate endothelial cells (ECs) in a ß(2)GPI-dependent manner after binding of ß(2)GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-ß(2)GPI Ab-mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88-dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-ß(2)GPI Ab-mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-ß(2)GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-ß(2)GPI-associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome.
Subject(s)
Annexin A2/metabolism , Antibodies, Antiphospholipid/pharmacology , Endothelium, Vascular/metabolism , Signal Transduction , beta 2-Glycoprotein I/immunology , Annexin A2/genetics , Blotting, Western , Calbindin 2 , Cells, Cultured , Endothelium, Vascular/cytology , Humans , Immunoenzyme Techniques , Immunoprecipitation , Luciferases/metabolism , Membrane Microdomains , Phospholipids/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Real-Time Polymerase Chain Reaction , S100 Calcium Binding Protein G/genetics , S100 Calcium Binding Protein G/metabolism , Thrombosis , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Umbilical Veins/cytology , Umbilical Veins/metabolism , NucleolinABSTRACT
OBJECTIVE: Regulation of the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. The purpose of this study was to investigate the effects of beta(2)-glycoprotein I (beta(2)GPI), an abundant plasma glycoprotein, on tPA-mediated plasminogen activation. METHODS: The effect of beta(2)GPI on tPA-mediated activation of plasminogen was assessed using amidolytic assays, a fibrin gel, and plasma clots. Binding of beta(2)GPI to tPA and plasminogen was determined in parallel. The effects of IgG fractions and anti-beta(2)GPI antibodies from patients with antiphospholipid syndrome (APS) on tPA-mediated plasminogen activation were also measured. RESULTS: Beta(2)-glycoprotein I stimulated tPA-dependent plasminogen activation in the fluid phase and within a fibrin gel. The beta(2)GPI region responsible for stimulating tPA activity was shown to be at least partly contained within beta(2)GPI domain V. In addition, beta(2)GPI bound tPA with high affinity (K(d) approximately 20 nM), stimulated tPA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (K(cat)/K(m)) of tPA-mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of beta(2)GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following beta(2)GPI repletion. Stimulation of tPA-mediated plasminogen activity by beta(2)GPI was inhibited by monoclonal anti-beta(2)GPI antibodies as well as by anti-beta(2)GPI antibodies from patients with APS. CONCLUSION: These findings suggest that beta(2)GPI may be an endogenous regulator of fibrinolysis. Impairment of beta(2)GPI-stimulated fibrinolysis by anti-beta(2)GPI antibodies may contribute to the development of thrombosis in patients with APS.
