In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to β2 glycoprotein I (β2GPI) induce endothelial cell-leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of β2GPI and β2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2 -/-mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte-endothelial cell adhesion and thrombus formation were absent in eNOS -/-and in ApoER2 -/-mice. Thus, aPL-induced leukocyte-endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.

Subarachnoid hemorrhage (SAH) is a devastating neurological injury associated with significant patient morbidity and death. Since the first demonstration of cerebral vasospasm nearly 60 years ago, the preponderance of research has focused on strategies to limit arterial narrowing and delayed cerebral ischemia following SAH. However, recent clinical and preclinical data indicate a functional dissociation between cerebral vasospasm and neurological outcome, signaling the need for a paradigm shift in the study of brain injury following SAH. Early brain injury may contribute to poor outcome and early death following SAH. However, elucidation of the complex cellular mechanisms underlying early brain injury remains a major challenge. The advent of modern neuroproteomics has rapidly advanced scientific discovery by allowing proteome-wide screening in an objective, nonbiased manner, providing novel mechanisms of brain physiology and injury. In the context of neurosurgery, proteomic analysis of patient-derived CSF will permit the identification of biomarkers and/or novel drug targets that may not be intuitively linked with any particular disease. In the present report, the authors discuss the utility of neuroproteomics with a focus on the roles for this technology in understanding SAH. The authors also provide data from our laboratory that identifies high-mobility group box protein-1 as a potential biomarker of neurological outcome following SAH in humans.

3039 Poster Board II-1015 The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL), with pathogenic aPL directed primarily against the cell surface protein β2 glycoprotein I (β2GPI), increased risk for thrombosis and cardiovascular events, and pregnancy morbidity. The endothelium is a critical direct target of aPL, which upregulate adhesion molecule expression and procoagulant activity. However, the molecular basis for aPL actions on endothelium is unknown. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) has potent antiadhesive and antithrombotic properties. In this study we determined if aPL-induced alterations in endothelial cell phenotype are mediated by aPL actions on eNOS. Normal human IgG (NHIgG) and polyclonal aPL were obtained from healthy adults and APS patients, respectively. We found that aPL prevented acetylcholine (Ach)- or VEGF-mediated attenuation of monocyte adhesion to cultured endothelial cells and this was reversed by an NO donor, indicating a role for eNOS antagonism. In contrast, NHIgG did not affect adhesion. Whereas NHIgG did not alter eNOS activation, stimulation of eNOS by VEGF and other agonists was fully antagonized by aPL. In mice, NO-dependent, Ach-induced increases in carotid vascular conductance were unaffected by NHIgG but impaired by aPL, indicating that these processes are operative in vivo. Additional studies in culture showed that aPL attenuates eNOS activation by inhibiting Ser1179 phosphorylation, deprivation of β2GPI prevented aPL inhibition of eNOS, and monoclonal antibody against β2GPI mimicked the effects of aPL. Furthermore, receptor-associated protein (RAP) antagonism of LDL receptors, which are known to bind dimerized β2GPI, prevented aPL inhibition of eNOS in culture, and mice null for the LDL receptor apolipoprotein receptor 2 (apoER2) were protected from aPL inhibition of eNOS in vivo. Moreover, aPL-stimulated increases in leukocyte-endothelial adhesion were absent in both eNOS-/- and apoER2-/- mice. Thus, aPL-induced increases in leukocyte-endothelial adhesion are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI and apoER2. Novel therapies for APS can now be developed targeting these mechanisms. Disclosures Thomas: NIcox Research Institute: Research Funding. Thorpe:Peregrine Pharm.: Consultancy, sponsored research agreement.

Intracerebral hemorrhage (ICH) induces neurovascular injury following the rupture of a blood vessel within the brain via poorly defined mechanisms. The aim of the present study was to determine whether glio‐vascular communication may restrict hemorrhagic vascular injury. Hemin concentration‐ and time‐dependently increased oxidative cytotoxicity in mouse bEND.3 microvessel cells. Cell death was preceded by a NFκB‐mediated increase in inflammatory gene expression, including up‐regulation of inducible nitric oxide synthase (iNOS) expression and activity. The inhibition of iNOS or the addition of a peroxynitrite decomposition catalyst reduced cell death. Interestingly, co‐treatment with astrocyte conditioned media (ACM) reversed hemin‐induced NF ?ÛB activation, nitrotyrosine formation, and apoptotic cell death via the release of the endogenous antioxidant, reduced glutathione (GSH). Prior treatment of astrocytes with the glutathione‐depleting agent, DL‐buthionine (S,R)‐sulfoximine (BSO) or direct addition of diethyl maleate, a thiol depleting agent, to ACM reversed the observed protection. In contrast, the addition of exogenous GSH or the GSH precursor, N‐acetylcysteine, were protective in bEND.3 cells. These data identify a previously unrecognized vasculoprotective role for astrocyte‐derived GSH and indicate therapeutic targeting of GSH may reduce neurovascular injury following ICH.