Results from the present study show that sera from patients with aPL exhibit an enhanced capacity to activate the classical pathway of complement, defined by C1q and/or C4d deposition on heterologous platelets. In aPL patients with underlying SLE, deposition of C1q and especially C4d, exceeding twice the level observed with normal serum, was associated with an increased incidence of arterial thrombosis. Despite enhanced classical pathway complement activation by sera from patients with primary aPL, a similar association with arterial thrombosis was not observed. A significant association, however, was noted between the presence of aPL IgG and positive complement fixation on heterologous platelets in all patients, although the correlation between the magnitude of aPL levels and the extent of complement deposition was poor, likely reflecting heterogeneity in the complement fixing ability of the autoantibodies present.
At the time of inclusion, none of the patients were receiving any form of heparin therapy. This is relevant because heparin can inhibit complement activation.28
However, some patients in both the SLE and APSCORE registries were receiving anticoagulation therapy with warfarin. In the APSCORE cohort, 4/47 aPL patients were receiving warfarin, compared with 59/96 patients with APS. In the SLE cohort, 3/91 aPL negative patients and 28/78 aPL positive patients were treated with warfarin. Although vitamin K antagonism is not expected to directly impact the complement system, thrombin generation may indirectly enhance complement activation.23
For this reason, in-situ complement activation on immobilized platelets was assayed in the presence of a direct thrombin inhibitor (PPACK).
The association between serum complement fixation and arterial thrombosis in patients with SLE and aPL, but not primary aPL, may suggest that other SLE-related factors are mechanistically important for arterial thrombosis in the setting of aPL-mediated complement activation. Alternatively, or in addition, the variance in C1q and C4d deposition observed with sera from patients with primary aPL was greater than that noted with sera from normal controls or patients with SLE. This may reflect preanalytical variables associated with serum preparation and storage that could affect complement activation29
and may have contributed to the apparent lack of association between complement positivity and thrombotic events in patients with primary aPL.
The following mechanism for the interaction between aPL, complement activation and arterial thrombosis may be plausible. Circulating aPL auto-antibodies bind to the platelet surface, creating an immune complex that, depending on antibody isotype, is recognized by complement (i.e., C1q). In-situ complement activation subsequently occurs on the platelet surface, as evidenced by C4d deposition. Platelet activation by complement,30
as observed in vitro
using the serotonin release assay, and the generation of complement-derived inflammatory mediators 31
may promote arterial thrombosis. In the setting of SLE, vascular injury may create an environment that predisposes to platelet accumulation and subsequent in-situ complement activation.
The complement system plays an important role in the pathogenesis of SLE. Complement deficiencies in the classical pathway (C1q, C4 and C2) predispose to the development of autoimmunity by a variety of mechanisms including impaired clearance of immune complexes and apoptotic cells, as well as aberrant tolerance induction and changes in cytokine regulation.26,27
During SLE flares, the complement system is activated, giving rise to partial deficiency or dysfunction due to consumption.27
Complement activation takes part in inflammatory reactions that give rise to tissue and organ damage.26
Because the extent of complement consumption in SLE patients likely impacts measurable complement deposition on platelets in the current assay, a limitation of our study is the inability to assay C1q levels and C4 levels in all serum samples due to limited sample volume. Thus, it was not possible to normalize complement deposition to serum complement levels. Indeed, C4d deposition was lowest in samples with lowest C4 levels. This may have increased false negative results.
Another significant limitation of the present study is the inability to correlate complement activation with lupus anticoagulant information. Determination of lupus anticoagulant status is indeed important for an APS series. Although these data were available for most samples in the APSCORE cohort (), platelet–poor plasma samples were unavailable for analysis in the SLE cohort, and thus, concurrent lupus anticoagulant measurements could not be made. It should be noted, however, that various APSCORE and patients with SLE were on warfarin therapy, which may interfere with lupus anticoagulant measurement. Thus, even if lupus anticoagulant data were available for all patients, the percentage of true positive patients would be overestimated.
The role of complement in APS is under intense investigation. Multiple pathogenic mechanisms have been suggested.32
Exposure of human endothelial cells to aPL has been shown to result in tissue factor production and upregulation of adhesion molecules.33
Although the cause of tissue injury in APS is likely to be multifactorial,32,34
a substantial body of evidence shows that complement activation is a requirement for pregnancy complications and thrombosis.12
Potentially, aPL-mediated complement fixation contributes to arterial thrombosis via production of inflammatory mediators: C3a responsible for leukocyte recruitment35
and C5a stimulating endothelial cell production of tissue factor.36
The presence of receptors for anaphylatoxins has also been described in human coronary plaques, suggesting that they may play a role in plaque formation/progression.37
In patients with SLE, cross-sectional and prospective cohort studies show a predictive role of aPL for future vascular events.38,39
However, patients with SLE are at significantly increased risk of premature atherosclerosis and/or thrombosis,40,41
regardless of aPL status, and a significant aPL profile does not appear to be predictive of thrombosis in individual patients with SLE.42
Moreover, a positive aPL level does not predict the location of thrombotic events (arterial, venous, pregnancy complications). Results from the present study suggest that the ability of sera with positive aPL levels to substantially activate complement on platelets may be associated with arterial thrombosis in patients with SLE, particularly when the complement fixation assay is made more stringent by using C4d generation as the endpoint. This may suggest that observed C1q deposition on heterologous platelets reflects the presence of complement fixing antibodies on platelets, whereas the in-situ generation of C4d is indicative of the activation of the classical complement system and its inflammatory sequelae. The current study did not have sufficient power to distinguish between specific arterial thrombotic events, such as stroke and myocardial infarction. The majority of patients with arterial thrombotic events (22 of 32) had a history of cerebrovascular accidents, and a history of myocardial infarction was reported for two patients. Other reported arterial thrombotic events included digital, splenic and adrenal infarcts.
In summary, the present study provides further insight into potential pathophysiologic mechanisms associated with thrombotic complications in SLE patients with aPL. Prospective studies are warranted to determine whether direct measurement of C1q and C4d deposition on circulating platelets, or enhanced complement fixation on heterologous and/or homologous platelets in situ, may be useful in predicting arterial thrombosis risk in patients with SLE. This information could contribute to more targeted therapeutic interventions in patients having SLE with aPL.