Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly ␤ 2 GPI, and activate endothelial cells (ECs) in a ␤ 2 GPI-dependent manner after binding of ␤ 2 GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-␤ 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-B has been proposed. In the present study, we confirm a critical role for TLR4 in anti-␤ 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-␤ 2 GPI Abs. These results provide new evidence for novel proteinprotein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-␤ 2 GPI-associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome. (Blood. 2012; 119(3):884-893) IntroductionAntiphospholipid syndrome (APS) is characterized by thrombosis and recurrent fetal loss in patients with circulating antiphospholipid Abs (APLAs) and is the most important cause of acquired thrombophilia. [1][2][3] Prospective studies have demonstrated that patients with APS experience significant morbidity and mortality despite recommendations for indefinite anticoagulation. 4 The term "antiphospholipid" is actually a misnomer, because the majority of APLAs are directed against phospholipid-binding proteins, of which ␤ 2 -glycoprotein I (␤ 2 GPI) is the most common. 5,6 The clinical importance of anti-␤ 2 GPI Abs has been demonstrated in several previous reports, 7 and recent studies have shown that affinity-purified human anti-␤ 2 GPI Abs induce thrombosis in mice. 8 Despite the clinical importance of APS, however, its pathogenesis has not been well defined. 1,3,9 One mechanism by which APLAs/anti-␤ 2 GPI Abs may promote thrombosis is through ␤ 2 GPI-dependent activation of endothelial cells (ECs). [10][11][12] ECs play a critical role in the maintenance of blood fluidity through expression of anticoagulant proteins on their luminal surface and the elaboration of antithrombotic substances. 13 However, EC activation leads to loss of these anticoagulant properties and transformation to a pro-adhesive, procoagulant phenotype. 13 APLAs/anti-␤ 2 GPI Abs induce EC activation in vitro and in vivo, as determined by their ability to increase the expression of adhesion molecules (E-selectin, ICAM-1, VCAM-1), and tissue factor (TF) and to enhance the expression, synthesis, and/or secretion of pro-inflammatory cytokines and chemokines. 3,[10][11][12] These effects may account for the ab...

The antiphospholipid syndrome (APS) is characterized by venous and/or arterial thrombosis, or recurrent fetal loss, in the presence of antiphospholipid antibodies (APL). The pathogenesis of APS is multifaceted and involves numerous mechanisms including activation of endothelial cells, monocytes, and/or platelets; inhibition of natural anticoagulant pathways such as protein C, tissue factor inhibitor, and annexin A5; activation of the complement system; and impairment of the fibrinolytic system. Fibrinolysis-the process by which fibrin thrombi are remodeled and degraded -involves the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) or urokinase-type plasminogen activator, and is tightly regulated. Although the role of altered fibrinolysis in patients with APS is relatively understudied, several reports suggest that deficient fibrinolytic activity may contribute to the pathogenesis of disease in these patients. This article discusses the function of the fibrinolytic system and reviews studies that have reported alterations in fibrinolytic pathways that may contribute to thrombosis in patients with APL. Some of these mechanisms include elevations in plasminogen activator inhibitor-1 levels, inhibitory antibodies against tPA or other components of the fibrinolytic system, antibodies against annexin A2, and finally, antibodies to β 2 -glycoprotein-I (β 2 GPI) that block the ability of β 2 GPI to stimulate tPAmediated plasminogen activation.

IntroductionThe antiphospholipid syndrome (APS) is characterized by arterial or venous thrombosis and/or recurrent fetal loss in the presence of antiphospholipid antibodies (APLAs). [1][2][3] It is now widely accepted that the majority of pathologic antibodies in patients with this disorder are actually directed against phospholipid-binding proteins, the most common of which is ␤ 2 -glycoprotein I (␤ 2 GPI).The pathogenesis of APS-associated thrombosis is multifactorial, and a number of mechanisms have been proposed. 4 These include inhibition of protein C activation and activity, 5,6 inhibition of annexin V assembly on exposed phospholipid surfaces, 7 and prevention of appropriate interactions of antithrombin with glycosaminoglycans, 8 among others. 4,9 Studies from our laboratory and others suggest that ␤ 2 GPI-dependent activation of vascular cells by APLA/anti-␤ 2 GPI antibodies plays a central role in disease pathogenesis 10,11 and may initiate the cascade of events that leads to thrombus development. For example, ␤ 2 GPI binds to endothelial cell annexin A2, and subsequent cross-linking of annexin A2-bound ␤ 2 GPI initiates endothelial cell activation through a pathway that may involve Toll-like receptor 4 (TLR-4) [11][12][13] and leads to activation of NF-B. 14 A similar pathway may be functional in monocytes, activation of which also contributes to the development of thrombosis in patients with APLA. 15 In addition, APLA may promote platelet activation in the presence of subthreshold concentrations of agonists, 16 although whether this is a receptor-mediated process, and if so, its relationship to platelet ␤ 2 GPI binding sites, such as GP1b 17 and apoER2, 18 requires further study.In endothelial cells, activation of NF-B stimulates an inflammatory and procoagulant response 19 and plays a critical role in the ability of APLA to promote thrombosis. 20 Thus, modulation of NF-B activity may provide an opportunity to reverse the pathologic vascular response in APS. The Krüppel-like factors (KLFs), 21 particularly KLF2 and KLF4, inhibit inflammatory cytokinemediated responses in endothelial cells, 22,23 at least in part through inhibition of NF-B activity. 23 However, expression of KLF2 itself may be inhibited by inflammatory cytokines and/or vascular injury, [23][24][25] although the expression of KLF4 appears to be increased under these conditions. 26 In considering the importance of NF-B in endothelial cell activation mediated by APLA/anti-␤ 2 GPI antibodies 14 and the potentially opposing effects of KLF2 and KLF4, we hypothesized that changes in expression of these transcription factors might influence the endothelial cell response to APLA/anti-␤ 2 GPI antibodies. Here, we report that, unlike responses to inflammatory cytokines, the expression of both KLF2 and KLF4 is decreased in response to APLA/anti-␤ 2 GPI antibodies. Moreover, restoring the expression of these KLFs blocks endothelial cell activation in response to APLA/anti-␤ 2 GPI antibodies. These activities result from inhibition of NF-B transcrip...

Persistent infection with oncogenic human papillomaviruses (HPVs) is the most important factor in the induction of uterine cervical cancer, a leading cause of cancer mortality in women worldwide. Upon cell transformation, continual expression of the viral oncogenes is required to maintain the transformed phenotype. The viral E6 protein forms a ternary complex with the cellular E6-AP protein and p53 protein which promotes the rapid degradation of p53. Recent studies have revealed that lignans from the creosote bush (3'-O-methyl-nordihydroguaiaretic acid) can repress the viral promoter responsible for E6 gene expression. Work reported here shows that the lignan can subvert viral oncogene function resulting in stabilized p53 protein within treated HPV-containing tumor cells. The stabilized p53 is transcriptionally active as demonstrated by a luciferase reporter vector and induction of genes for Bax and PUMA proteins. Apoptosis is detected by annexin V binding to treated cells as analyzed by flow cytometry. Programmed cell death is confirmed by the induction of active caspases and TUNEL assay. Initiator caspase-9 is activated first, followed later by the effector caspase-3 enzyme. The stabilization and induced apoptosis are not observed within treated HPV-negative cervical tumor cells. Quantitative real time RT-PCR analysis of endogenous E6 gene transcription from the integrated HPV 16 promoter shows at least a fivefold repression of expression as compared to untreated cells. These results indicate that the loss of E6 protein in treated cells could be, in part, responsible for the stabilization of p53 within the lignan treated cells.

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4315 Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy loss in the presence of antiphospholipid antibodies (APLA). These antibodies are directed primarily against phospholipid-bound β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells, enhancing the expression of adhesion molecules and tissue factor, and the secretion of proinflammatory cytokines. Krüppel-like factors (KLF) regulate endothelial cell inflammatory responses. KLF2 and KLF4 mediate anti-atherosclerotic and anti-inflammatory effects in endothelial cells, and we have hypothesized that alterations in the expression or activity of KLF2 or KLF4 may modulate the endothelial cell response to APLA. In preliminary studies, we have observed that endothelial cell activation induced by APLA/anti-β2GPI antibodies inhibits the expression of KLF2 and KLF4, and as demonstrated by our laboratory and others, is accompanied by activation of NF-kB. However, forced expression of KLF2 or KLF4 by plasmid-mediated transfection of endothelial cells inhibits neither the phosphorylation of ser536 of the p65 subunit of NF-kB, nor the nuclear translocation of p65 in response to APLA/anti-β2GPI antibodies. Despite the lack of effect on forced KLF2 or KLF4 expression in endothelial cells on p65 phosphorylation, expression of either of these factors inhibits NF-κB transcriptional activity with corresponding inhibition of cellular activation as measured by inhibition of cell-surface E-selectin expression as well as E-selectin promoter activity. Inhibition of NF-kB transcriptional activity by KLF2 and KLF4 appears to be due to recruitment of the CBP/p300 cofactor away from NF-kB by KLF2 or KLF4, since augmenting the cellular pool of CBP/p300 by transfection restores NF-κB activity and endothelial cell activation responses. Similarly, treatment of APLA-activated endothelial cells with CBP/p300 siRNA inhibits NF-kB transcriptional activity regardless of the levels of KLF2 or KLF4. These data suggest that APLA inhibit KLF expression and that these changes promote the acquisition of a prothrombotic endothelial cell phenotype. CBP/p300 may serve as a molecular switch that determines the relative antithrombotic activities of KLFs versus the prothrombotic, inflammatory responses induced by NF-kB in APLA/anti-β2GPI antibody activated endothelial cells. Disclosures: No relevant conflicts of interest to declare.

151 Antiphospholipid syndrome (APS) is a multisystem autoimmune disorder characterized by an increased risk of arterial and/or venous thrombosis and recurrent fetal loss in the presence of elevated levels of antiphospholipid antibodies (APLA). Most pathologic APLA actually recognize phospholipid-bound proteins, the most common of which is beta-2 glycoprotein I (β2GPI). Previous work from our laboratory and others have demonstrated that anti-β2GPI antibodies activate endothelial cells in the presence of β2GPI through cross-linking or clustering of endothelial cell surface Annexin A2, to which β2GPI binds with high affinity. Endothelial cells activated by β2GPI/anti-β2GPI antibodies increase their expression of cell surface adhesion molecules including ICAM-1, VCAM-1, and E-selectin. Since annexin A2 is not a transmembrane protein, APLA-induced endothelial cell activation may occur through a TLR4-MyD88-dependent pathway leading to NFκB activation. However, the nature of the interactions between annexin A2, β2GPI and TLR4 have not been well defined and whether additional proteins may contribute to the formation of a signaling complex remains unknown. To address this issue, we used a cell surface ELISA for E-selectin to measure endothelial cell activation in response to β2GPI/anti-β2GPI antibodies. We observed that inhibition of TLR4 expression by siRNA caused a 90% inhibition of E-selectin expression following exposure of cells to β2GPI/anti-β2GPI antibodies, while TLR2 siRNA had no effect. siRNA-induced inhibition of apolipoprotein E receptor 2 (ApoER2) expression also did not affect the activation of endothelial cells by β2GPI/anti-β2GPI antibodies. To determine whether annexin A2 binds directly to TLR4, we determined whether TLR4 could be affinity purified from APLA-induced endothelial cell extracts using immobilized annexin A2. These studies led to the isolation of not only TLR4, but two additional proteins, calreticulin and nucleolin. The interaction between Annexin A2 and TLR4 was also demonstrated using Annexin A2 coupled to Affigel-HZ. To explore the role of calreticulin and nucleolin in APLA-induced endothelial cell activation, we measured APLA/anti-β2GPI antibody-induced E-selectin expression by endothelial cells after siRNA mediated inhibition of calreticulin and nucleolin expression, observing that siRNAs against either of these proteins significantly inhibited endothelial cell activation. Additional studies confirmed that siRNA-mediated knockdown of Annexin A2, TLR4, calreticulin and/or nucleolin inhibited the cell surface expression of not only E-selectin, but ICAM-I and VCAM-I. Interestingly, endothelial cell activation caused by APLA/anti-β2GPI antibodies was induced increased expression of mRNA encoding annexin A2, TLR4, MD2 (a TLR4 co-receptor involved in dimerization), MyD88, and S100A10 (a component of the endothelial cell annexin A2-S100A10 heterotetramer) (12, 3, 12, 5 and 6-fold, respectively). Taken together, these studies suggest that a complex consisting of annexin A2, TLR4, calreticulin and nucleolin may mediate endothelial cell activation by APLA/anti-β2GPI antibodies. We hypothesize these proteins may form a multi-component signaling complex, perhaps in lipid rafts, that culminates in the activation of NF-κB. Disclosures: No relevant conflicts of interest to declare.

3272 Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLA). The majority of APLA are directed against phospholipid binding proteins, particularly β2GPI. Anti-ß2GPI antibodies activate endothelial cells and monocytes in a β2GPI-dependent manner through a pathway that involves NF-κB and leads to increased expression of adhesion molecules, tissue factor and proinflammatory cytokines. Krüppel-like factors (KLFs) regulate endothelial cell and monocyte responses to inflammatory stimuli; increased expression of these transcription factors inhibits proinflammatory and procoagulant gene expression, and maintains vascular homeostasis. We recently reported that anti-ß2GPI antibodies decrease the expression of KLF2 and KLF4 in endothelial cells (Allen et al, Blood 2011), promoting endothelial cell activation. Subsequent studies demonstrate that these antibodies decrease expression of KLF2 in monocytes as well. Statins have been proposed as a potential alternative to anticoagulation for APS patients, and stimulate the expression of KLFs. We hypothesized that the ability of statins to block endothelial cell activation in response to anti-β2GPI antibodies was mediated by KLFs. Treatment of endothelial cells and monocytes with 100 nM fluvastatin, lovastatin, or simvastatin upregulated KLF2 and KLF4 mRNA, even in the presence of anti-ß2GPI antibodies. In parallel, statin treatment inhibited the anti-β2GPI antibody-mediated induction of E-selectin, VCAM-1, and TF mRNA in endothelial cells, and ICAM-1 and TF mRNA in human monocytes. To assess the dependence of these effects on KLF expression, endothelial cells were pretreated with KLF2 or KLF4 siRNA prior to treatment with statins. siRNA-mediated inhibition of KLF expression completely blocked the ability of statins to prevent anti-β2GPI antibody-induced endothelial cell activation, as measured by adhesion molecule and TF mRNA levels and expression of E-selectin on the endothelial cell surface. Taken together, these data demonstrate that KLFs are critical modulators of the effects of statins on endothelial cells, and that increased expression of KLFs may represent a mechanism by which these drugs inhibit the activation of endothelial cells and monocytes by APLA/anti-β2GPI antibodies. Disclosures: No relevant conflicts of interest to declare.