In the present study we have demonstrated that aPL inhibit the activation of eNOS and that the resulting decline in NO production underlies the promotion of leukocyte–endothelial cell adhesion and thrombus formation by aPL (summary in Figure ). Considering the primary role of eNOS-derived NO in both the anti-adhesive and antithrombotic characteristics of healthy endothelium (10
), eNOS antagonism by aPL is likely to be a critical initiating process in the pathogenesis of the vascular manifestations of APS.
aPL promote leukocyte adhesion and thrombosis by antagonizing eNOS via β2GPI, apoER2, and the phosphatase PP2A.
We found that aPL antagonize eNOS activity in cultured endothelial cells derived from multiple species, including humans. Studies of carotid vascular conductance in mice also showed that aPL attenuate eNOS activation, demonstrating the physiologic importance of this effect in vivo. These observations are consistent with the finding in APS patients that endothelium-dependent, NO-dependent flow-mediated dilation of the brachial artery is impaired, whereas endothelium-independent vasodilation is normal (48
). In addition, in APS patients there is a negative correlation between flow-mediated dilation and circulating levels of the adhesion molecules VCAM-1 and ICAM-1 (48
). Consistent with this inverse relationship between the capacity for NO production and the degree of vascular inflammation in APS patients, our work in both cultured endothelial cells and in vivo in mice now reveals a causal link between aPL-induced eNOS antagonism and increased leukocyte–endothelial cell adhesion.
To define the underlying mechanisms, we examined the role of β2GPI. Using both loss-of-function and gain-of-function strategies, we determined that β2GPI mediates eNOS antagonism by aPL. Furthermore, monoclonal Ab to domain I of β2GPI, which is the domain primarily targeted by pathogenic aPL that recognize β2GPI (7
), and not Ab to domain II, caused eNOS antagonism and the resulting increase in adhesion (Figure , i). Moreover, purified dimeric β2GPI, but not monomeric β2GPI, inhibited eNOS activation. Thus, we have demonstrated that the recognition of domain I of β2GPI and its dimerization are the upstream events in aPL antagonism of eNOS and its consequences. We further discovered that the downstream process leading to eNOS inhibition is attenuated eNOS S1179 phosphorylation caused by the activation of the phosphatase PP2A (Figure , iii).
There is considerable evidence that complement activation plays a role in the ultimate manifestations of APS. The inhibition of the complement cascade by the C3 convertase inhibitor complement receptor 1–related gene protein y–Ig (Crry-Ig) or the administration of anti-C5 monoclonal antibody reverses aPL-mediated thrombosis in mice, and C3- and C5-null mice are resistant to the effects of aPL (51
). Complement activation also participates in aPL-induced fetal loss during pregnancy (28
). Our finding that aPL-mediated eNOS inhibition does not require complement is consistent with the recent proposal that APS pathogenesis entails initial direct effects of aPL on endothelium, and possibly also on platelets, which are then amplified by the ensuing activation of complement via mediators such as C3a and C5a (51
). Our elucidation of the proximal mechanisms responsible for aPL effects on endothelium now makes it possible to test potential cause-effect linkage between endothelial aPL action and complement activation.
To date, the basis by which pathogenic aPL recognition of β2GPI on the cell surface elicits a transmembrane signal to modify intracellular events in endothelium has been poorly understood (52
). In biochemical analyses, it has been demonstrated that β2GPI binds directly to LDLR family members including apoER2, VLDLR, LRP, and megalin (14
), and apoER2 and VLDLR are expressed in endothelium (55
). Using RAP in cultured endothelial cells, we first showed that an LDLR family protein is required for aPL-mediated eNOS inhibition. Then in in vivo studies, we identified that receptor to be apoER2. We provided further evidence of the requirement for apoER2 in studies of aPL-induced endothelial cell–monocyte adhesion entailing knockdown of the receptor in endothelium by RNAi. Moreover, by using a recombinant soluble form of LDL-binding domain 1 of ApoER2 (sBD1) that inhibits dimeric β2GPI binding via domain V to apoER2 (38
), we showed that interaction between β2GPI and the receptor mediates aPL actions in endothelium (Figure , ii). We then expanded on these numerous in vitro and ex vivo findings in studies of leukocyte–endothelial cell adhesion and thrombus formation in wild-type, eNOS–/–
, and ApoER2–/–
mice. Although the results for aPL-induced thrombosis in eNOS+/+
mice should be interpreted conservatively because of the differences in thrombosis under control conditions, the cumulative findings indicate that apoER2 and eNOS are important linchpins in aPL vascular actions in vivo. Thus, the basis for transmembrane signaling induced by aPL binding to extracellular β2GPI in endothelium has now been identified, and the molecular events by which apoER2 initiates intracellular processes in endothelium including PP2A activation can now be investigated.
Although direct actions of aPL on platelets are likely contributory to APS-related thrombosis, there is a major role for the activation of endothelial cell adhesion in aPL-induced thrombus formation (9
). This has been most clearly indicated by studies demonstrating that ICAM1–/–
mice and mice administered anti–VCAM-1 monoclonal antibody are fully protected from aPL-induced thrombus formation (16
). However, the presence of aPL and aPL actions on endothelium are not sufficient, because most of the time patients with circulating aPL do not have thromboses. A “two-hit hypothesis” has been proposed in which aPL (the first hit) induces a threshold cellular perturbation in endothelium, or possibly in platelets, and another condition (the second hit) is required to trigger actual clot formation (60
). In most prior studies of APS-related thrombosis in rodents, as well as in our experiments, thrombus formation must be induced (16
). The second hit may involve a proinflammatory stimulus, since rats administered aPL with anti-β2GPI activity have spontaneous thromboses if they also receive LPS (61
). These observations have led to the suggestion that infectious agents may play a dual role in APS pathogenesis by initially triggering the generation of cross-reactive anti-β2GPI antibodies (first hit) due to considerable amino acid sequence homology between β2GPI and a number of bacterial and viral components, and then by inducing an inflammatory response that serves as the second hit (62
). With the molecular underpinning of the first hit now in hand, the nature of possible second-hit conditions can be better interrogated.
The proximal processes by which aPL cause changes in endothelial cell behavior have been elusive (52
). We now provide multiple lines of evidence demonstrating that aPL-induced increases in leukocyte–endothelial cell adhesion and thrombus formation are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI, apoER2, and PP2A. The identification of these molecules and the elucidation of how they interplay to invoke the endothelial actions of aPL and their sequelae now provide the framework for the development of new strategies to combat APS. With the new knowledge gained, it is now possible that the lifelong requirement for anticoagulation in the APS patient will be replaced by a mechanism-based therapy offering both far fewer complications and greater efficacy against this often devastating condition.