Pathogenesis of Thrombosis in Patients with Antiphospholipid Syndrome
Chary López-Pedrera, Francisco Velasco, and Maria J. Cuadrado
Introduction
Antiphospholipid syndrome (APS) is an acquired autoimmune disorder of unknown etiology. The syndrome is defined by the association of arterial or venous thrombosis and/or pregnancy morbidity, in the presence of antiphospholipid anti- bodies (aPL), anticardiolipin antibodies (aCL), and lupus anticoagulant (LA) [1].
aPLs are a heterogeneous family of autoantibodies with diverse cross-reactivities whose origin and role have not been fully elucidated. It is now recognized that many of the autoantibodies associated with APS are directed against phospholipid binding plasma proteins, such as β2-glycoprotein I (β2-GPI) and prothrombin, or phospholipid–protein complexes [2], expressed on or bound to the surface of vas- cular endothelial cells, platelets, or monocytes [3].
β2-GPI, a plasma protein bearing the major antigenic epitope for aPL, interacts with negatively charged phospholipids involved in the coagulation process, and has both procoagulant and anticoagulant properties. β2-GPI suppresses the thrombo- modulin–protein C system [4], factor XII activation, factor X activation, and pro- thrombinase activity. Antibodies against β2-GPI may modify the properties of β2-GPI and favor a prothrombotic state. However, individuals with β2-GPI deficiency do not have a thrombotic tendency [5]; thus, aPL-associated thrombosis cannot be explained merely by β2-GPI insufficiency.
Prothrombin, another plasma protein, is the second major target of aPL and the zymogen of the serine protease thrombin [6]. Thrombin is one of the most potent enzymes, and it catalyzes several reactions which may be important in blood coagu- lation. In this way, recent studies have demonstrated that anti-prothrombin anti- bodies with LA activity can inhibit coagulation reactions in a phospholipid and prothrombin dependent manner, by enhancing the intrinsic inhibitory effect of pro- thrombin itself [7]. Therefore, aPL may also modify prothrombin properties, ulti- mately leading to a thrombotic state.
Thrombosis is the key lesion of the APS [8]. Several non-exclusive mechanisms have been proposed to explain the involvement of aPL in the pathogenesis of thrombosis in APS, including the induction of tissue factor (TF) expression by endothelial cells and monocytes [9, 10]. This chapter focuses on the contribution of
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TF to the pathogenesis of thrombosis in patients with APS and the intracellular mechanisms involved in TF expression.
TF Pathway and Thrombosis in APS
TF is a specific transmembrane single chain glycoprotein composed of 263 amino acids (47 kDa), that requires interaction with specific membrane phospholipids (PL) to become functionally active [11–13]. TF serves as both high-affinity receptor and enzyme activator for plasma FVII or FVIIa in initiating a localized procoagulant activity (PCA) on the anionic PL cell surface. TF is widely accepted to be the major initiator of in vivo coagulation [14]. It is also believed that TF has a key role in fibrin deposition in immunologic disorders, as well as in disseminated intra-vascular coagulation and clot formation in gram-negative bacterial sepsis, cancer, and inflammatory bowel disease [12, 13, 15]. TF is expressed on the surface of many cell types but, in the resting state, is normally absent from cells in contact with blood.
However, TF can be induced, in vitro, to appear on endothelial cells and monocytes in a transcriptionally regulated manner by several physiologic or non-physiologic stimuli [16–18].
In APS patients, our recent in vivo studies have shown that patients with primary APS have increased expression of TF on the monocyte surface, along with increased mRNA-TF, TF antigen, and activity levels in peripheral blood mononuclear cells, where the source of TF is the monocyte [19–21]. Moreover, TF expression was found increased in APS patients with thrombosis when compared with those without and with healthy controls. TF expression in these patients was found to be further increased in those positive for IgG aCL, but not in those positive for IgM aCL of LA. In addition, TF expression in APS did not appear to be related to plasma levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), two inflammatory mediators that influence TF production, suggesting that inflamma- tory changes do not determine TF production in the steady state.
Recent evidence has suggested a role for the TF pathway in the pathogenesis of aPL-related thrombosis. Experimental data have shown that procoagulant activity in cultured EC and monocytes is induced by plasma from patients with APS and by purified aPL [22]. Furthermore, it has been demonstrated that antibodies against β2- GPI induce the expression and activity of TF in vitro [10]. In addition, β2-GPI expression on monocytes is significantly increased in patients with APS and corre- late with TF expression, thus contributing to the maintenance of a persistent pro- thrombotic state [23].
Signal Transduction Mechanisms Associated with the Increased Expression of TF in Response to aPL
The mechanism(s) by which aPL induce TF expression is unknown. A potential role or Fcγ receptors (FcγR) in the pathogenesis of APS was suggested [24], but Pierangeli et al [25] showed in a murine model that these effects are not dependent on binding of antibody to FcγR. Rand and coworkers have proposed a thrombo- genic mechanisms [26, 27] in which the high affinity of the aPL for anionic PL
bound proteins on the cell surface may in itself explain the prothrombotic effects including TF expression. They suggest that annexin V has a physiologic role in inhibiting coagulation, by forming clusters that bind with high affinity to anionic PL on cell surfaces exposed to flowing blood, and thus fully shielding these surfaces from the assembly of the TF–FVIIa and other coagulation complexes. More recently, annexin II, a phospholipid binding protein, has been identified as an EC or monocyte surface molecule and might be involved in aPL mediated cellular signal- ing, playing a critical role in the upregulation of monocyte TF [28].
At a molecular level, it has been suggested that aPL may interact with specific cell surface receptors (proteins and/or lipids), inducing signals that have consequences downstream, that ultimately will result in upregulation of cell surface proteins (i.e., TF). In an in vitro recent study, Pierangeli et al [29] have shown that aPL induces activation of the nuclear factor kappa B (NFκB) in EC. In turn, NFκB activation leads to upregulation of gene transcription of adhesion molecules on EC and to ini- tiation of various signal transduction pathways. In an in vivo recent study, our group has studied the activation of NFκB and the expression of its inhibitor IκBα in monocytes from patients with primary APS and their association with monocyte TF expression.
Our study, performed on purified monocytes from APS patients, showed both increased proteolysis of the cytoplasmic inhibitory protein IκBα and nuclear translocation and activation of the NFκB proteins p65 and p50, compared to con- trols. Moreover, NFκB activation was accompanied by an increased expression of TF in these patients. Furthermore, the analysis of the p38 and pERK MAP kinase activity, which have been demonstrated to be involved in the aPL induced upregula- tion of TF in endothelial cells [29], indicated that mean levels of phosphorylated forms of p38 and ERK1/2 MAP kinases were significantly higher in monocytes of APS patients compared to controls. In addition, Pearson’s relational statistic indi- cated significant inverse correlation between levels of pp38 MAP kinase and IκBα (P
= 0.018). Hence, increased levels of pp38 correlated with diminished levels of cytosolic IκBα expression. Thus, our data might suggest an implication of p38 MAP kinases in NFκB activation in monocytes of APS patients [30].
As an intermediate early gene in activated EC, TF is rapidly induced in response to pathophysiologically relevant stimuli such as cytokines and growth factors, including vascular endothelial growth factor (VEGF), in EC and monocytes [31].
VEGF is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolitic activity of EC. Although the mitogenic activity of VEGF is EC specific, recent reports indicate that VEGF is able to stimulate chemotaxis and TF production in monocytes [32]. Previous studies have demonstrated significantly higher levels of plasma VEGF in APS patients compared to control [33]. In a recent study by our group, and in order to elucidate the origin of this plasma VEGF, we measured VEGF expression in monocytes from APS patients by several methods, including real time polymerase chain reaction (RT-PCR), Western blot, immunocy- tochemistry, and flow cytometry. We found significantly higher levels of both mRNA and protein VEGF in monocytes from APS patients compared to controls, thus pointing to the monocytes as the possible source of this plasmatic VEGF. In addition, increased VEGF expression correlated positively with the levels of both mRNA and cell surface TF expression. VEGF stimulated activity in monocytes is mediated by the VEGF receptor Flt-1. Monocytes, in contrast to endothelium, express only this specific receptor, which functions as a mediator of monocyte
recruitment and procoagulant activity [34]. In our study, VEGF-Flt-1 expression in monocytes from APS was found elevated in parallel with VEGF. Moreover, a posi- tive correlation was found between the expression of this receptor and that of monocyte TF. Thus, increased VEGF activity could be responsible for TF overex- pression in monocytes of APS patients [35].
Recent studies have suggested that p38 and ERK1/2 MAP kinases are two essen- tial mediators of VEGF-induced endothelial TF [36]. Moreover, VEGF induced acti- vation in monocytes and EC has been proved to be mediated by p38 MAP kinase pathway. Our group recently showed that phosphorylated forms of p38 and ERK1/2 were significantly increased in APS patients compared to controls (described in pre- vious paragraphs). The increased ERK activity observed (which did not seem to be involved in NFkB activation) might play a role as a mediator of VEGF induced TF expression in monocytes of PAS patients, as suggested for EC [36]. Nevertheless, as p38 MAP kinase has also proved to be involved in both the upregulation of VEGF [37, 38] and the VEGF induced activation of TF expression, an additional and may be independent role for this kinase cannot be ruled out. Additional in vitro studies are required to evaluate this hypothesis.
TF and Therapeutic Intervention in APS
APS is associated with an increased risk of recurrent thrombosis [39], being throm- boprophylaxis similar to that used in the general population. However, the contri- bution of TF to a prothrombotic state in this syndrome provides a renewed focus on antithrombotic therapies in current use (i.e., oral anticoagulation) and supports the need for antithrombotic strategies that more specifically target the activity of the TF–VIIa complex or the antibody mediated TF expression.
Oral anticoagulation reduces the availability of functional FVII and minimizes thrombotic risk, even if TF is highly expressed. Retrospective studies have strongly suggested the efficacy of high-intensity oral anticoagulation for the prevention of recurrent thrombosis in APS, but there is some debate regarding the duration of therapy. High recurrence rates have been seen after the cessation of anticoagulant therapy as well as in patients who were not receiving anticoagulants, suggesting that patients with APS require long-term treatment [39]. In patients with APS, monocyte TF expression and procoagulant activity remain high for years after the last throm- botic episode despite receiving oral anticoagulants [19]. Thrombotic complications should be expected if the intensity of anticoagulation drops below target values.
Strategies aimed to prevent antibody mediated TF overproduction or agents that could lower TF expression by other mechanisms must be identified. In this regard, the newly discovered anti-inflammatory, anticoagulant, antiproliferative, and immunoregulatory effects of statins are of great interest. These compounds are being tried as an additional therapeutic tool in the treatment of thrombosis in APS patients. Meroni et al [40] have recently described the effects of statins on EC acti- vated in vitro by pro-inflammatory cytokines and/or by aPL (anti–β2-GPI antibod- ies). Fluvastatin and simvastatin were found to be able to inhibit the induction of a pro-adhesive and pro-inflammatory phenotype in a specific manner because the addition of mevalonate reversed the inhibition.
Statins effects on coagulation process have also been described. Statins have antithrombotic properties although the mechanisms leading to this effect are still
unclear. Undas et al [41] found that simvastatin treatment reduces blood clotting by decreasing rates of prothrombin activation, factor Va generation, fibrinogen cleav- age, factor XIII activation, and increased rate of factor Va inactivation.
Furthermore, impaired TF expression on cultured human macrophages and mono- cytes induced by statins has been demonstrated in vitro and has been attributed to the inhibition of the TF gene induction [42, 43]. If statins are able to reduce, in vivo, the TF expression in these patients, this could have an important impact in the future therapeutic approach of patients with aPL.
Conclusions
Increased TF expression may contribute to thrombosis in patients with APS. This effect might ultimately depend on antibody engagement of PL binding proteins on the monocyte and the EC surface, leading to signal transduction and altered cell activity. Understanding the intracellular mechanism(s) of aPL mediated TF activa- tion may help to establish new therapeutic approaches to revert the prothrombotic state observed in APS patients.
Acknowledgements
This work was supported by grants from the Junta de Andalucia (exp. 171/03) and the Fondo de Investigación Sanitaria (FIS 01/0715 and 03/1033) of Spain.
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