Mechanisms of autoantibody-induced monocyte tissue factor expression

Thrombosis Research (2004) 114, 391--396

intl.elsevierhealth.com/journals/thre

Mechanisms of autoantibody-induced monocyte tissue factor expression Alisa S. Wolberg a, Robert A.S. Roubey b,* a

Department of Pathology and Laboratory Medicine, USA Department of Medicine, Division of Rheumatology and Immunology, The University of North Carolina at Chapel Hill, CB #7280, Rm. 3330 Thurston Building, Chapel Hill, NC 27599-7280, USA

b

Received 25 May 2004; accepted 9 June 2004 Available online 19 July 2004

KEYWORDS Tissue factor; Tissue factor pathway inhibitor; Monocytes; Endothelial cells

ABSTRACT The expression of tissue factor (TF) activity to flowing blood is the trigger for physiological coagulation as well as many types of thrombosis. A growing body of evidence suggests that increased tissue factor activity is a significant contributor towards the hypercoagulability associated with the antiphospholipid syndrome (APS). The increase in tissue factor activity appears to be due to increased transcription and translation of nascent tissue factor molecules but is not due to deencryption of existing tissue factor molecules on cells. Autoantibodies and/or immune complexes circulating in APS patients appear to enhance the expression of tissue factor activity on monocytes and endothelial cells. Anti-h2-glycoprotein I (h2GPI) autoantibodies have been specifically implicated in the antibody-mediated enhancement of tissue factor activity. The presence of antibodies against tissue factor pathway inhibitor (TFPI) in certain APS patients suggests that negative regulation of tissue factor activity might also be impaired in these patients. Given a mechanism involving increased tissue factor activity in APS-associated thrombosis, agents specifically targeting tissue factor activity may be a novel and efficacious therapy that is safer than current approaches to the management of APS. A 2004 Elsevier Ltd. All rights reserved.

Tissue factor and coagulation Tissue factor (TF) is the physiological initiator of normal coagulation as well as clotting observed in thrombotic disease. TF is a high-affinity receptor for * Corresponding author. Tel.: +1-919-966-0578; fax: +1-919966-1739. E-mail address: [email protected] (R.A.S. Roubey).

coagulation factor VII(a) and functions as an essential cofactor for factor VIIa to efficiently cleave factors IX and X to their active forms (factors IXa and Xa, respectively). The factors Xa/Va complex then cleaves prothrombin to thrombin (see Fig. 1). Cell-bound TF is a 47-kDa transmembrane glycoprotein that is constitutively expressed on the surfaces of various cell types outside the vasculature but is not expressed on endothelial cells or peripheral

0049-3848/$ - see front matter A 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2004.06.012

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Figure 1 Regulation of the TF pathway. Functionally active TF expressed on a cell is a high-affinity receptor and cofactor for the enzyme, factor VIIa. Factor VIIa/TF cleaves factors X and IX to form factors Xa and IXa (factors IX and IXa not shown). Together with factor Va, factor Xa forms the prothrombinase complex and cleaves prothrombin (factor II) to yield thrombin (factor IIa). TFPI inhibits this pathway by binding to and inactivating factor Xa. The factor Xa/TFPI complex then inhibits factor VIIa/ TF, forming a quaternary complex.

blood cells (at least not in a functionally active form). A soluble form of TF also circulates in blood at approximately 60 pg/ml [1,2]. This circulating TF is likely in an inactive form, but can become procoagulant and is hypothesized to support thrombus propagation at sites of vascular injury [1]. Structurally, TF is a member of the cytokine receptor superfamily. Genetic deficiency of TF in mice is lethal at the embryonic stage [3].

Regulation of TF activity on cells in contact with blood Endothelial cells, blood monocytes, and other cells in contact with flowing blood do not constitutively express functional TF and do not have intracellular stores of TF [4]. However, in response to stimulation with certain agents, including lipopolysaccharide (LPS) [5], endothelial microparticles [6], chemokines [7], anti-platelet factor 4/heparin antibodies [8], homocysteine [9], P-selectin [10] and/or certain inflammatory cytokines, these cells express TF activity via transcription and synthesis of nascent TF molecules. Some cells may express a nonfunctional (encrypted) form of TF. These and other TF-expressing cells can regulate TF via a rapid mechanism (de-encryption) whereby the TF activity on a cell increases without a concomitant increase in TF antigen. De-encryption results from

A.S. Wolberg, R.A.S. Roubey the stimulation of TF-bearing cells with agents, such as calcium ionophore, cycloheximide, hydrogen peroxide or freeze/thaw cycles, and results in up to 11-fold increases in the measured TF activity of a cell [11--14]. De-encryption can be attributed to changes in morphology, membrane lipid expression (increased cell surface PS expression) and to changes in the TF molecule itself [13,15]. It has been shown that TF encryption is due in part to the dimerization of TF molecules on the cell surface. In this model, TF dimers are inactive and TF activity after stimulation is due to the rapid dissociation of dimers into active TF monomers [12].

Tissue factor pathway inhibitor Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type protease inhibitor that modulates the initiation of coagulation via factor Xa-dependent feedback inhibition of TF/VIIa. TFPI inhibits TF activity by forming a quaternary complex (TFPI, TF, VIIa, Xa) via an interaction that requires calcium ions and is enhanced by anionic phospholipid membrane (reviewed in Ref. [16]). TFPI can also inhibit VIIa/TF without factor Xa [17] and may inhibit factor Xa directly in a phospholipid-independent manner [18]. There are three intravascular pools of TFPI. About 10--50% of TFPI circulates in plasma at a concentration of 50--150 ng/ml, much of which is complexed to lipoproteins. Approximately 50--90% of TFPI is bound to vessel wall glycosaminoglycans and can be released into plasma by injection of heparin. A small amount of TFPI is stored in platelets and released upon platelet activation.

The TF pathway and thrombosis Increased TF activity has been implicated in a number of thrombotic conditions and hypercoagulable states. Increased expression of TF on vascular endothelial cells and monocytes has been reported in patients with cancer [19], gram-negative bacterial sepsis [20], atherosclerosis [21], and OKT3induced coagulopathy in renal transplant patients [22]. The physiological importance of TFPI is evidenced by the fact that ‘‘knockout’’ of the TFPI gene in mice is lethal at the embryonic stage [23]. Low levels of endogenous free TFPI have not been associated with thrombosis. A TFPI polymorphism

Mechanisms of autoantibody-induced monocyte tissue factor expression has been described [24]; however, the role of this polymorphism in thrombosis is controversial.

The TF pathway in APS Sera from certain patients with systemic lupus erythematosus enhance the procoagulant activity of cultured endothelial cells [25--28]. Most data support the hypothesis that the stimulating factor(s) are the patients’ autoantibodies, although the effects of the autoantibodies on cellular procoagulant activity may be enhanced by suboptimal concentrations of TNF-a [27,29]. There is growing evidence that increased TF activity on circulating blood monocytes is an important mechanism of hypercoagulability in antiphospholipid syndrome (APS) and that autoantibodies are directly responsible. In 1990, de Prost et al. [30] reported that monocyte procoagulant activity was increased in patients with systemic lupus erythematosus, about half of whom had lupus anticoagulants. Serum from these patients increased TF activity on normal monocytes, although the serum factor responsible did not appear to be immunoglobulins. In retrospect, the experiments with purified IgG were performed under serum-free conditions and the absence of h2-glycoprotein I (h2GPI) may have been a key factor. Subsequently, a number of groups have found that serum, plasma, purified total IgG, and anti-h2GPI antibodies from APS patients enhance TF expression and procoagulant activity on normal monocytes [31--37]. F(abV)2 antibody fragments retain these procoagulant effects, suggesting that Fc receptors are not required for the procoagulant activity of these antibodies [31,38]. Additionally, several of these studies demonstrated that monocytes isolated from APS patients exhibit increased expression of TF and TF mRNA [33,37,39,40]. Anti-h2GPI human monoclonal antibodies derived from peripheral B cells of APS patients enhance monocyte TF activity and levels of TF mRNA in a h2GPI-dependent fashion [33,35]. Using an anti-h2GPI monoclonal antibody [41], as well as affinity-purified anti-h2GPI autoantibodies from an APS patient, we have demonstrated that antih2GPI antibodies are at least one specificity involved in inducing monocyte TF. Time course experiments demonstrated that APS patient IgG increased both TF mRNA and activity, with peaks in expression at 2 and 6 h, respectively [38]. These same antibodies did not up-regulate TF activity by de-encryption, suggesting that de-encryption of existing TF on cells is not an APS thrombosis-related mechanism [42].

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We and others have also detected autoantibodies directed against TFPI in APS patients and found an association of these antibodies with arterial thrombosis and stroke [43,44]. Adams et al. [45] identified anti-TFPI activity in IgG from 5 of 33 APS patients examined. Another mechanism may be related to recent reports of cellular autoimmunity to h2GPI. Visvanathan and McNeil [46] reported that h2GPI-specific T cells are of the TH1 phenotype and produce interferon-g, a cytokine known to stimulate monocyte TF expression [47].

Pharmacological inhibition of TF as an APS therapy Given the relationship between increased TF activity, thrombosis, and APS, pharmacological agents that block monocyte TF activity may be a novel and attractive therapeutic approach in APS. Such targeted treatments would likely have less risk of bleeding complications than long-term anticoagulation with warfarin (the current treatment for prevention of recurrent thrombosis in APS). Several agents are known to decrease TF activity in vitro and in vivo. Dilazep, an antiplatelet agent, inhibits in vitro monocyte and endothelial cell TF expression induced by several stimuli including TNF-a, thrombin, and phorbol ester [48]. In vivo, dilazep decreases plasma levels of soluble TF, D-dimer, thrombin--antithrombin complexes, and fibrinogen in patients with a hypercoagulable state associated with malignancy [48]. Our data demonstrate that dilazep inhibited APS IgG-induced monocyte TF activity in a dose-dependent fashion. Because dilazep inhibits the uptake of adenosine by increasing the extracellular concentration of adenosine, and because adenosine inhibits TF expression [49,50], we have also examined the ability of theophylline, a nonspecific adenosine receptor antagonist, to block dilazep-mediated inhibition of TF expression. Theophylline partially blocked the inhibition of TF expression by dilazep, suggesting that dilazep mediates TF expression via its effect on adenosine transport. Future studies with specific adenosine receptor antagonists will better define dilazep’s mechanism of action. In addition to dilazep, several other drugs have been shown to inhibit increased expression of TF on monocytes and endothelial cells induced by LPS and other stimuli. The antiplatelet agent dipyridamole is an adenosine uptake inhibitor similar to dilazep and may have similar anti-TF properties. Pentoxifylline inhibits LPS-induced monocyte TF expression [51,52]. Several angiotensin-converting

394 enzyme inhibitors (captopril, imidapril, and fosinopril) significantly inhibit LPS-induced monocyte TF activity, antigen expression, and gene transcription [53,54]. Lastly, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG--CoA) reductase inhibitors (‘‘statins’’), including simvastatin and fluvastatin, reduce TF activity, antigen expression, and gene transcription [55--57]. In particular, fluvastatin has recently been shown to inhibit thrombosis in a murine model of APS [58]. Direct inhibitors of TF activity could also prove excellent therapeutics in the treatment and management of APS. Active site-inactivated factor VIIa (FVIIai) is a modified form of recombinant factor VIIa in which the enzymatic active site has been blocked by a synthetic inhibitor. FVIIai has exhibited potent antithrombotic effects in two separate animal models (rat and rabbit) of arterial thrombosis [59,60].

Future directions Growing evidence supports the hypothesis that thrombosis in the antiphospholipid syndrome is caused by the increased expression of TF activity on monocytes and vascular endothelial cells. Recent work has identified increased transcription and translation of nascent TF as the mechanism of this increased expression of TF activity. Experiments examining the autoantibody-mediated regulation of TF activity will provide important information as to the mechanism of this activity. Therapies that specifically target the regulation of TF activity may provide an improved approach to the management of the antiphospholipid syndrome.

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