The association between aPL and thrombotic events is well established. Evidence from animal models of APS indicates that aPL may play a causal role in the vascular abnormalities in both the venous and arterial territories [8,9]. In an animal model of photochemically induced arterial thrombosis, monoclonal antibodies raised against human β2GPI promoted thrombus formation . Ramesh et al.  demonstrated that aPL inhibit the activation of nitric oxide and that the resulting decline in nitric oxide production underlies the promotion of leucocyte–endothelial cell (EC) adhesion and arterial thrombosis in mice. Injection of aPL in mice increased thrombus formation, carotid artery tissue factor (TF) activity, as well as peritoneal macrophage TF activity and expression . Furthermore, enhanced thrombus formation was observed in femoral vein of mice treated with aPL . Vega-Ostertarg et al.  found that mice injected with aPL have an enlargement in the thrombus size in the postcapillary venular endothelium in the cremaster muscle. Rapid endothelial deposition of fibrinogen and intravascular platelet–leucocyte aggregates were detected by intravascular microscopy on the mesenteric vessels of rats receiving an intraperitoneal injection of bacterial lipopolysaccharide followed by infusion of immunoglobulin G (IgG) purified from patients with APS .
Despite the persistent presence of aPL in circulation, thrombotic events in patients with aPL only occur occasionally, suggesting that the presence of aPL is necessary but not sufficient for clot formation in vivo. The ‘two-hit hypothesis’ has been proposed in which aPL (first hit) can only exert their prothrombotic influence in the presence of another thrombophilic condition (second hit). This ‘two-hit hypothesis’ was shown in an animal model of APS in which the injection of aPL in rats only resulted in increased thrombus formation when rats were pretreated with lipopolissacharide, but not when were injected with buffer .
Antiphospholipid antibodies and cell interactions
The major antigen structures recognised by aPL in patients with APS are phospholipid–binding proteins, β2GPI and prothrombin, expressed on the membranes of different cell types. The antibody forms a complex with the corresponding antigen, leading to the cell perturbation, the activation of cell signalling pathways, the transcription of procoagulant substances, adhesion molecules and subsequently thrombus formation.
Studies on the pathogenicity of aPL have been carried out mainly on the corresponding target molecules especially on the function of β2GPI and their modifications by aPL. However, to evaluate whether antiβ2GPI antibodies can block the function of β2GPI is difficult as true physiological role of β2GPI in coagulation cascade is not elucidated. Individual with complete β2GPI deficiency does not have any particular phenotype . Thus, the recent trend is to favour the hypothesis that the function of aPL on prothrombotic cells, via β2GPI, is more important than the function of β2GPI.
Membranes of activated platelets with negatively charged phospholipids are an important source of catalytic surface for blood coagulation. Activated factor X and thrombin are generated on activated platelets, and procoagulant microparticles shed by platelet activation. Platelets are prone to agglutinate and aggregate after exposed to aPL , and circulating activated platelets are found in patients with APS . β2GPI binds to membranes of activated platelets and inhibit the generation of activated factor X. Antiβ2GPI antibodies interfere with this inhibition . Thus, activated platelets may be a predominant immune target of antiβ2GPI antibodies and direct action of aPL in platelets contribute to APS-related thrombosis.
The endothelium is a predominant target of aPL. Pathogenic aPL binding to β2GPI cause the up-regulation of adhesion molecules , TF  and endothelin-1  causing a pro-inflammatory and prothrombotic EC phenotype. Prothrombin also binds to ECs, and this binding is enhanced by a human monoclonal IgG antiprothrombin antibody, IS6. IS6 up-regulates expression of TF and E-selectin on ECs .
Antiphospholipid antibodies exert also effect in the stimulation of the release of microparticles from ECs . Microparticle production is a hallmark of cell activation, but the role of microparticle in the pathophysiology of thrombosis has not been elucidated. Antiphospholipid antibodies bind to the negatively charged membrane of monocytes and induce TF up-regulation [23,24]. Monocytes are the source of most majority of circulating TF-bearing microparticles  and TF up-regulation is a major feature of monocyte activation in the APS .
Cell receptors for antiphosphospholipid antibody interactions
The cell activation mediated by aPL might require an interaction between phospholipid-binding plasma protein and a specific cell receptor(s). A number of potential receptors for the binding of β2GPI to cellular membranes have been identified including annexin A2, apolipoprotein E receptor 2 (ApoER2′), low-density-lipoprotein receptor (LDL-R) -related protein, megalin, Toll-like receptor (TLR) 2, TLR 4, the very-LDL-R and P-selectin glycoprotein (GP) ligand-1. β2GPI also directly binds to the platelet adhesive receptor GPIbα and to the platelet factor 4 (PF4) [27–32]. Most of these receptors are expressed on various cell types and whether those different receptors are involved in the pathophysiology of thrombosis is still matter of debate.
Annexin A2 is a receptor for tissue-type plasminogen and its ligand plasminogen. Annexin A2 is a membrane-bound protein found on the surface of ECs and monocytes, and on the brush-border membrane of placental syncytiotrophoblasts . Annexin A2 interacts with the β2GPI-antiβ2GPI antibody complex on the ECs and monocyte surfaces, mediating cell activation [27,28]. The involvement of annexin A2 in aPL-mediated pathogenic effects has been reported in vitro and in vivo models . However, it is unlikely that annexin A2 per se is actually involved in cellular activation because it lacks transmembrane domain. The activation of signalling responses requires the presence of another transmembrane adaptor protein(s) that associates with annexin A2 on the ECs surface . TLR-4 was identified as a potential putative adaptor protein for annexin A2 .
Several groups reported that TLR-2 and TLR-4 are involved in aPL-mediated cell activation [30,35,36]. TLR-4 signalling was shown in ECs after the incubation with aPL , but a direct interaction between TLR4 and β2GPI remains to be confirmed. Binding of β2GPI to TLR2 on endothelial surface has been reported .
Megalin/gp33 is an endocytic receptor that internalises multiple ligands including apolipoprotein E and B100. Megalin was shown to behave as a receptor of β2GPI and β2GPI-phospholipid complex . Pennings et al.  demonstrated that dimeric β2GPI can interact with LDL-R family members, including megalin.
Apolipoprotein E receptor 2 is a member of the LDL-R family expressed in many cell types. Studies on platelets suggested ApoER2′ as a receptor of β2GPI . The blockage of the platelet ApoER2′ using a receptor-associated protein abrogated the increased adhesion of platelets to collagen induced by β2GPI-anti-β2GPI antibody complex . Using a recombinant soluble form of LDL-binding domain 1 of ApoER2′, it was shown that the interaction between β2GPI and ApoER2′ mediated the aPL action in endothelium . The importance of ApoER2′ in the induction of prothrombotic state mediated by aPL was confirmed in vivo in a murine model of thrombosis and using ApoER2′ deficient mice . Injection of aPL caused a significant increase in thrombus formation, vascular TF activity and monocyte activation in the murine model of thrombosis, which were significantly reduced in the ApoER2′ deficient mice. Those data support the role of ApoER2′in thrombus formation in APS; however, the role of other potential receptors cannot be excluded as demonstrated by the partial protection from thrombogenic effects of aPL in ApoER2′-deficient mice.
β2 glycoprotein I directly binds to GPIbα subunit of the platelet adhesion receptor GPIb/IX/V in vitro [35,36]. The platelet GPIbα subunit has the von Willebrand factor as the most important ligand, but also serves to localise factor XI and thrombin on the platelet surface. Binding of β2GPI to GPIbα enables antiβ2GPI antibodies, directed against domain I, to activate platelets, resulting in thromboxane production and also to the activation of the phosphoinositol-3 kinase (PI3-kinase)/Akt pathway  contributing to the platelet adhesion and aggregation.
The involvement of Fcγ receptor on cellular activation has been investigated in vivo  and in vitro studies on platelets , monocytes  and ECs . Results suggest that this receptor is not strictly necessary for cellular activation.
The direct binding of β2GPI to PF4 derived from platelet granules has been reported . PF4 is a member of the C–X–C chemokine family secreted by activated platelets and has ability to bind to the platelets surfaces. PF4 contributes to the natural dimerisation of β2GPI, leading to the stabilisation of β2GPI binding onto the phospholipid cell surfaces which facilitates the antibody recognition. The β2GPI-PF4 complex is strongly recognised by serum of patients with APS . Moreover, platelets may be activated by β2GPI-antiβ2GPI antibody-PF4 or β2GPI-PF4 complexes. Almost every cell type can be a source of PF4 especially under some stimulation. Both, β2GPI and PF4 are abundant in plasma; thus, the preformed β2GPI-PF4 complexes may prime several pro-coagulants cells culminating in coagulation.
Those potential receptors proposed to be involved in the aPL-mediated cell activation have significantly increased in the last years, and additional studies are needed to clarify their biological and pathological roles.
Signalling pathways of cell activation
The signal transduction mechanisms involved in aPL-mediated cell activation have been the centre of interest for many researchers. How pathogenic aPL recognition of phospholipid-binding proteins on the cell surface elicits a transmembrane signal to modify intracellular events is not completely understood.
The adapter molecule myeloid differentiation protein (MyD)88-dependent signalling pathway and the nuclear factor kappa B (NFkB) have been involved in the ECs activation by aPL [44,45]. Incubation of ECs with antiβ2GPI antibodies resulted in a redistribution of NFkB from the cytoplasm to the nucleus, and this effect was accompanied by an increased expression of TF and leucocyte adhesion molecules . The p38 mitogen-activated protein kinase (MAPK) pathway is an important component of intracellular signalling cascades that initiate various inflammatory responses. It is recognised that the p38 MAPK pathway has a crucial role in mediating the effect of aPL in different cell types [24,47,48]. Activation of p38 MAPK increases activities of cytokines such as tumour necrosis factor (TNF) alpha, IL-1β and macrophage inflammatory cytokine 3β [24,36]. Monocytes stimulated by monoclonal antiβ2GPI antibodies from patients with APS induce phosphorylation of p38 MAPK, a locational shift of NFkB into the nucleus and up-regulation of TF expression. Such activation was not seen in the absence of β2GPI, indicating that the disturbance of monocyte by anti-β2GPI antibodies is started by interaction between the cell and the autoantibody-bound β2GPI [24,44]. The implication of p38 MAPK in cell activation has been also demonstrated in platelets  and ECs . Pretreatment of platelets with p38 MAPK-specific inhibitor, SB203580, completely abrogated aPL-mediated platelet aggregation. The induction of TF expression was also reported through the simultaneous activation of NFkB via the MAPK pathway and of the MEK-1/ERK pathway, but an inhibitor of the MEK-1/ERK pathway could not suppress the TF expression, implying the main role of p38 MAPK in those reactions .
Purified IgG from APS patients with venous thrombosis, without pregnancy morbidity, caused phosphorylation of NFkB and p38MPK and up-regulation of TF in monocytes. These effects were not seen with IgG fractions from patients with obstetric APS alone, suggesting that aPL from patients with different clinical aspects of APS may trigger different signalling responses . Figure 1 shows the procoagulant cell activation as one of the pathogenic mechanisms of thrombosis mediated by aPL.
Figure 1. Pathogenic mechanisms of cell activation mediated by antiphosphopholipid antibodies. Antiphospholipid antibodies interact with monocytes or endothelial cells through binding to phospholipid-binding protein (β2GPI or prothrombin) on cell surface. This interaction might require a specific cell receptor (s) and results in p38MAPK phosphorylation, nuclear translocation of NFkB and up-regulation of procoagulant substances and adhesion molecules, and subsequently thrombus formation. p38 MAPK, p38 mitogen-activated protein kinase; NFkB, nuclear factor kappa B; β2GPI, β2 glycoprotein I; PAI-1, plasminogen activator inhibitor-1; TNFα, tumour necrosis factor alpha; TF, tissue factor.
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Recently, two major findings in the antigenic structures recognised by aPL have been reported: first, the structural changes in β2GPI. β2GPI can exist in two different conformations, plasma β2GPI circulates in a circular (closed) conformation, whereas after interaction with antiβ2GPI antibodies undergoes a major conformational change into a fishhook-like (open) structure . Second, the finding that β2GPI can be reduced by thioredoxin 1 (TRX-1). β2GPI treated with TRX-1 generate free thiols within β2GPI, a process that may affect the function of β2GPI, and may have a regulatory role in platelet adhesion . Those novel biochemical findings into the structural changes that can occur within β2GPI and the consequences of these changes for the function of β2GPI might be relevant to our better understanding of the APS, but further studies are necessary to clarify their roles in the pathogenesis of APS.