Naturally occurring free thiols within β2-glycoprotein I in vivo: nitrosylation, redox modification by endothelial cells, and regulation of oxidative stress–induced cell injury

Abstract

␤2-Glycoprotein I (␤2GPI) is an evolutionary conserved, abundant circulating protein. Although its function remains uncertain, accumulated evidence points toward interactions with endothelial cells and components of the coagulation system, suggesting a regulatory role in vascular biology. Our group has shown that thioredoxin 1 (TRX-1) generates free thiols in ␤2GPI, a process that may have a regulatory role in platelet adhesion. This report extends these studies and shows for the first time evidence of ␤2GPI with free thiols in vivo in both multiple human and murine serum samples. To explore how the vascular surface may modulate the redox status of ␤2GPI, unstimulated human endothelial cells and EAhy926 cells are shown to be capable of amplifying the effect of free thiol generation within ␤2GPI. Multiple oxidoreductase enzymes, such as endoplasmic reticulum protein 46 (ERp 46) and TRX-1 reductase, in addition to protein disulfide isomerase are secreted on the surface of endothelial cells. Furthermore, one or more of these generated free thiols within ␤2GPI are also shown to be nitrosylated. Finally, the functional significance of these findings is explored, by showing that free thiol-containing ␤2GPI has a powerful effect in protecting endothelial cells and EAhy926 cells from oxidative stress-induced cell death. IntroductionThioredoxin 1 (TRX-1) is a ubiquitous, 12-kDa oxidoreductase enzyme whose multiple intracellular functions include reducing protein disulfides, scavenging oxygen free radicals, and redox regulation of signaling pathways involved in cell growth, apoptosis, and inflammation. 1 TRX-1 requires nitrosylation of the unpaired cysteine (Cys69) thiol to mediate key intracellular functions. 2 S-nitrosylation of thiols has been implicated as being important in several multisystemic physiologic and pathologic processes pertaining to hemostasis and vascular disease. 3 In addition to being a key intracellular molecule, TRX-1 is an enzyme also found in circulating plasma. It is released from cells in response to oxidative stress 4,5 and is found at higher levels in the circulation in a variety of acute and chronic inflammatory or metabolic conditions associated with an increase in systemic oxidative stress load. [6][7][8] Studies have shown that exogenous administration of TRX-1 exerts a protective role in animal models of high oxidative stress such as smoking and rheumatoid arthritis, 9,10 both of which represent risk factors for cardiovascular morbidity in humans. 11 Oxidative stress is known to be associated with vascular pathology in terms of promoting atherosclerotic plaque development, rupture and atherothrombosis. 12 However, the extracellular mechanism through which elevated TRX-1 may mediate a protective effect against oxidative stress-induced vascular injury has not been fully delineated.␤ 2 -Glycoprotein I (␤2GPI) is an evolutionary conserved 50-kDa plasma protein. The function of ␤2GPI remains uncertain, although it is thought to play a role in apoptotic cell clearance and coagulation through m...

Vascular thiol isomerases

“…28,29 Thiol isomerases on endothelium expose a free thiol (Cys326) on the surface of b 2 GPI and b 2 GPI reduced by endothelial thiol isomerases protects endothelium from cell death induced by oxidative stress. 30 …”

The Pathophysiology of Antiphospholipid Syndrome

“…Because heparin inhibits complement activation [78], mice treated with heparin after the injection of aPL do not develop fetal loss, unlike mice treated with the direct thrombin inhibitor, hirudin [79]. Some authors suggest that heparin prevents fetal loss through its effect on the complement pathway rather than an anticoagulant effect [80]. Although the current recommendations for APS patients are initial low molecular weight heparin (LMWH), LMWH has not been tested in patients with APS, and data from mice models are unclear [79].…”

“…In addition, approximately 35% of patients with SLE have LA [80] and ß2-GPI [81] antibodies respectively. In a mice model of SLE, the spontaneous development of domain 1 antibodies and analogous APS supported a relationship between the two syndromes and their pathogenesis [82].…”

Antigen and substrate withdrawal in the management of autoimmune thrombotic disorders

“…53 Approximately 80% of circulating plasma ␤ 2 GPI is maintained in the reduced form by platelet and endothelial cell oxidoreductases, 54,55 which may protect cells from hydrogen peroxide-induced apoptosis. 56 Free cysteines in reduced ␤ 2 GPI, particularly Cys 288 and Cys 326 , are susceptible to oxidation and nitrosylation as is the Cys 32 -Cys 60 disulfide bond in domain 1, which spans the primary antigenic region. 56 Oxidation increases the affinity of polyclonal rabbit and murine monoclonal antibodies for ␤ 2 GPI, 53 but its effect on patient-derived antibodies requires additional study.…”

“…56 Free cysteines in reduced ␤ 2 GPI, particularly Cys 288 and Cys 326 , are susceptible to oxidation and nitrosylation as is the Cys 32 -Cys 60 disulfide bond in domain 1, which spans the primary antigenic region. 56 Oxidation increases the affinity of polyclonal rabbit and murine monoclonal antibodies for ␤ 2 GPI, 53 but its effect on patient-derived antibodies requires additional study. …”