Antiphospholipid syndrome pathogenesis in 2023: an update of new mechanisms or just a reconsideration of the old ones?

Abstract

Antibodies against phospholipid (aPL)-binding proteins, in particular, beta 2 glycoprotein I (β2GPI), are diagnostic/classification and pathogenic antibodies in antiphospholipid syndrome (APS). β2GPI-aPL recognize their target on endothelium and trigger a pro-thrombotic phenotype which is amplified by circulating monocytes, platelets and neutrophils. Complement activation is required as supported by the lack of aPL-mediated effects in animal models when the complement cascade is blocked. The final result is a localized clot. A strong generalized inflammatory response is associated with catastrophic APS, the clinical variant characterized by systemic thrombotic microangiopathy. A two-hit hypothesis was suggested to explain why persistent aPL are associated with acute events only when a second hit allows antibody/complement binding by modulating β2GPI tissue presentation. β2GPI/β2GPI-aPL are also responsible for obstetric APS, being the molecule physiologically present in placental/decidual tissues. Additional mechanisms mediated by aPL with different characteristics have been reported, but their diagnostic/prognostic value is still a matter of research.

Introduction

Antiphospholipid syndrome (APS) is classified as the association between recurrent arterial/venous thrombotic events and/or pregnancy morbidity and the persistent presence of autoantibodies detectable by at least one of the three classification laboratory assays: the anticardiolipin (aCL), anti-β2glycoprotein I (GPI) or the lupus anticoagulant (LA) [1].

From a clinical point of view, APS is characterized by isolated vascular and obstetric events or by the combination of both types of manifestations. A less frequent but more aggressive variant has been also reported: the ‘catastrophic APS’ (CAPS) [2].

The syndrome can be associated with another underlying systemic autoimmune disease—more frequently systemic lupus erythematosus (SLE)—at the variance with the isolated or primary APS (PAPS).

All the above-mentioned variants share common pathogenic mechanisms only in part and will be discussed separately.

The common denominator

The common denominator of the different APS clinical variants is represented by the presence of the so-called antiphospholipid antibodies (aPL) detectable by aCL, anti-β2GPI and LA assays. These autoantibodies are directed against PL-binding proteins and there is evidence that they are pathogenic in addition to be diagnostic/classification markers. Although different aPL types have been described, the key pathogenic autoantibodies are those directed against β2GPI [2–4].

Beta2GPI is an abundant plasma protein highly conserved across the animal kingdom. This finding and the demonstration of its involvement in several biological pathways suggest that the protein plays important functions. However, the whole biological role of β2GPI remains to be fully elucidated [5].

The preserved sequence/structure among animal species and the induction of antibodies against murine (self) β2GPI by mice initially immunized with human (exogenous) β2GPI and adjuvant is consistent with the possibility that molecular mimicry may be responsible for the loss of tolerance against self-β2GPI in subjects predisposed to autoimmunity [6]. The autoimmune nature of APS is further supported by the presence of an IFN signature comparable to that found in other systemic autoimmune diseases characterized by the production of autoantibodies [7–9]. Moreover, the recent demonstration of the presence of β2GPI on the neutrophil NETs may support the possibility that the naive molecule can undergo post-transcriptional modifications as several other molecules do in the NET environment [10, 11]. Additional post-transcriptional modifications of the molecule (e.g. redox switch, nitrosylation, carbamylation) may eventually be responsible for triggering an autoimmune response directed to the self-molecule [12–14].

Beta2GPI is a relatively large molecule composed of five domains (i.e. sushi domains), and a conformational sequence located in the domain (D)1 close to the link with D2 has been shown to be the immunodominant epitope recognized by most of the anti-β2GPI antibodies [15]. Anti-D1β2GPI antibodies display the highest diagnostic and prognostic power and have been shown to be pathogenic in both in vivo and in vitro experimental models [3, 16].

Vascular APS

Vascular APS is an autoimmune thrombotic vasculopathy characterized by recurrent clot formation in both arteries and veins in the absence of other known causes. Histopathological analysis of thrombotic tissues does not show clear signs of local inflammation supporting the view that APS cannot be associated with inflammatory vasculitis [17, 18]. The demonstration that circulating levels of inflammatory mediators (e.g. pro-inflammatory cytokines) are in the normal range or slightly increased tends to exclude their systemic critical role in vascular APS [19, 20].

As previously stated, β2GPI-dependent aPL are widely accepted as pathogenic autoantibodies. This finding is supported by epidemiological associations with the clinical vascular manifestations and by animal models showing the enlargement of blood clots triggered by mechanical or chemical stimuli in hamsters and mice [3, 21, 22]. Another model characterized by spontaneous thrombus formation was developed in rats that received a sub-liminal inflammatory stimulus (i.e. LPS) prior to the administration of aPL [22]. This model mirrors the common situation observed in APS patients who, despite the long-term persistence of circulating aPL (first hit) develop thrombosis only after a second hit, such as an infectious episode [23]. We have shown that LPS administered to mice promotes β2GPI deposition on vascular endothelial cells followed by the binding of antibodies and complement activation [22, 24] (Fig. 1).

Although the biological functions of β2GPI are still unclear, it is well known that β2GPI may bind to membranes of different cell types via the engagement of several receptors. In vitro models showed that anti-β2GPI antibodies may recognize the target molecule complexed with these receptors and induce their clustering, resulting in cell signalling and modulation of biological function [3]. These findings are not reported in animal models. Table 1 reports the list of cell types expressing the different receptors for β2GPI and their signalling ability.

Beta2GPI can adopt different conformations (i.e. J-elongated, S-twisted and O-circular) and redox states: (i) oxidized, in which all the disulfide bonds are formed, and (ii) reduced, in which one or more disulfide bonds are broken. There is evidence that the oxidized molecule displays the J-form exposing the N-terminal domain (in D1) which is mainly recognized by pathogenic aPL [12, 29–31]. This conformation favors the engagement of the so-called PL-binding site in the D5 with negatively charged structures (e.g. CL-coated plates) or with the cell membrane receptors [12, 29, 32]. Consequently, the J-form may represent the proper conformation adopted by the molecule to expose the immunodominant epitope and the high-density tissue concentration of β2GPI bound to the cell receptors favors the deposition of a relatively high number of antibodies overcoming the limitation of their low avidity [33]. The finding that aPL recognize β2GPI bound to tissue or to a negatively charged structure better than the free molecule in solution is consistent with this view [29, 30, 32].

The typical vascular APS is characterized by thrombotic events localized at restricted sites at the arterial or venous tree, suggesting the critical role of the endothelium in the initial phase of thrombus formation. The detection of β2GPI on endothelial cells both in experimental models and in human tissues is consistent with the hypothesis that endothelium behaves as the aPL target [3, 24, 34–38]. The anti-β2GPI mediated activation (perturbation) of the endothelium is responsible for the induction of a pro-adhesive (e.g. by expressing adhesion molecules) and pro-coagulant [e.g. by expressing tissue factor (TF)] endothelium phenotype [35–40]. These events may represent the localized pro-coagulant/pro-adhesive ‘initial loop’ followed by the downstream recruitment/activation of other cells (e.g. monocytes, platelets and neutrophils) usually involved in the coagulation cascade. All these cells contribute to the ‘amplification loop’ and reinforce the initial trigger leading eventually to clot formation. Both in vitro and in vivo models have shown that circulating monocytes and neutrophils are activated by aPL contributing to the thrombophilic state [39–41]. These cells are further activated following their interaction with the pro-adhesive/pro-inflammatory perturbed endothelium and contribute to the hypercoagulable environment expressing, for example, TF or in the case of neutrophils, through NETosis [42–46]. NETs consist of lattices of extracellular DNA, which display pro-coagulant effects through the activation of the intrinsic pathway, thrombin generation, and by impairing thrombomodulin-dependent protein C activation [41–43].

The finding of increased arachidonic acid metabolites in APS patients suggestive of platelet involvement [47] has been further supported by clinical studies and experimental models [25, 39, 40, 47–49]. Antiphospholipid antibodies do not react with resting platelets but recognize β2GPI present on the cell membrane of activated platelets once it is bound to their receptors (see Table 1) [25, 38, 49]. Mild thrombocytopenia is frequently found in APS patients but it is still unknown whether aPL themselves play a role in platelet decrease [50]. As previously reported for monocytes and neutrophils, there is also evidence from in vitro models that β2GPI-dependent antibodies may activate platelets triggering aggregation and inducing TF cell membrane expression [51].

Additional mechanisms may contribute to the ‘amplification loop’ and eventually to the hypercoagulable state. For example, aPL may affect fibrinolysis, the natural anti-coagulant pathways, and are associated with the presence of circulating macro- and micro-vesicles conveying, for example, miRNAs able to affect the coagulation itself [52–56].

In a recent study, we showed that β2GPI can bind to clots obtained in vitro or collected from patients after vascular surgery, suggesting additional physiological roles for β2GPI and, more importantly, the ability of the bound molecule to favour further deposition of aPL—if present—and complement activation [57].

Obstetric APS

The persistent presence of aPL is associated with pregnancy morbidities which also represent classification criteria for the syndrome [1]. Why some patients display only vascular or obstetric manifestations even in the presence of comparable high aPL levels raises the issue of whether or not the two types of clinical scenarios are supported by unrelated pathogenic mechanisms [58].

Beta2GPI and β2GPI-dependent aPL are the main players in obstetric and vascular APS. This finding is supported by epidemiological studies as well as by in vitro and in vivo experimental models [59, 60].

Anti-phospholipid antibodies in ‘pure obstetric’ APS are detectable with the same aPL assays used in the vascular variant. The majority of these antibodies are also reacting with β2GPI D1, suggesting that different epitope specificities cannot explain the association with one or with the other clinical variant [59]. Interestingly, some studies showed that IgG from pure obstetric APS may inhibit trophoblast invasion in vitro and affect monocyte gene expression differently from IgG isolated from vascular APS [61, 62]. Altogether these findings support the hypothesis that, despite similar antibody specificity, aPL in obstetric APS may display different biological effects in comparison with those found in vascular APS. The presence of one or the other aPL type may eventually cause different clinical manifestations.

There is sound evidence that β2GPI is present on trophoblast and decidua even in physiological conditions playing a key role in placentation [59, 63]. The tissue binding of the molecule via D5 [60] reproduces the high-density antigenic distribution and the presentation of the molecule with the elongated J-conformation that facilitates the autoantibody binding as discussed above (fourth paragraph of Vascular APS section). In other words, the molecule is present at high density on the cell membranes in close contact with the maternal blood containing aPL, offering the best conditions for antigen/antibody reaction. This condition differs from that observed on the vessel walls, where β2GPI can be demonstrated only after endothelial perturbation/damage and explains why the ‘second hit’ is apparently not required for the development of obstetric APS [24, 34]. In fact, passively infused aPL fail to be thrombogenic in naive animal and can only trigger clot formation after a sub-liminal pro-inflammatory stimulus (e.g. LPS) [22]. In contrast, aPL passively infused into pregnant mice can induce fetal resorptions and growth retardation (the animal equivalent of human miscarriages) without additional factors [64–67]. The reason for this difference is not clear, but both the high amount of β2GPI bound to placental tissues and the physiological changes in pregnancy (e.g. hormonal environment and placental vascular changes linked to pregnancy) may be a plausible explanation [58].

The β2GPI distribution at the fetal-maternal interface might also explain why low titres of aPL are associated with pregnancy complications whereas only medium or high aPL titres are strong risk factors for vascular manifestations and are included in clinical scores for the APS risk profile [68, 69]. In other words, the large amount of β2GPI at the placental level can offer the proper antigenic density to allow the binding of aPL even at low titres. This is not the case for the vessel walls, where an increase of β2GPI and higher aPL titre are required for endothelial perturbation after antibody binding/complement activation.

While blood clotting is the main pathogenic mechanism in the vascular variant of the syndrome, there is growing evidence that this is not apparently true for obstetric APS [58, 70]. Extensive thrombotic occlusions defined as an area of villous ischemic necrosis are relatively frequent in placentas from APS patients but are not specific because comparable lesions can also be reported in placentas from aPL-negative women with miscarriages. Abnormalities in uterine vessels’ vascular remodeling and/or local hypercoagulability without clear occlusions have also been reported [71, 72]. The findings from in vitro and in vivo models are consistent with the histopathological data from APS placentas and support the view that defective placentation rather than placental thrombosis represents the main pathogenic mechanism. Table 2 summarizes the pathogenic mechanism and pathways mediated by β2GPI-dependent aPL, which recognize the target molecule on trophoblasts and decidual cells. Needless to say, the same autoantibodies—via the interaction with β2GPI on the outer membranes—may also affect local endothelium or other circulating cells of the coagulation cascade (e.g. platelets, neutrophils, etc.) contributing to placenta damage by additional mechanisms [58, 70].

As mentioned above, the passive administration of β2GPI-dependent aPL IgG in pregnant naive animals can trigger fetal resorptions and growth retardation mimicking the human obstetric APS. In one model a large amount of infused human IgG (10 mg/mouse) was associated with overt placental inflammation [67, 74]. Comparable effects on the pregnancy outcomes without inflammation were reported in other models using substantially lower amounts of infused aPL (50 µg/mouse) [64–66]. The known signalling pathways triggered by aPL in the trophoblast and decidua cells (see Table 2) are consistent with localized placental inflammation. It is reasonable to believe that the strong inflammatory signature found in one model can be emphasized by the large quantity of the infused antibodies while it is not seen in the other model using low amounts of antibodies. This can also explain why a stronger complement activation and neutrophil recruitment at the placental level were reported in the first model (reviewed in [58]).

Catastrophic APS

Catastrophic APS is defined as a life-threatening disorder characterized by: (i) the clinical evidence of multiple organ involvement (commonly, three or more organs) developing over a very short period of time; (ii) histopathological evidence of multiple small vessel occlusions; and (iii) the presence of aPL, usually in high titres (reviewed in [75]).

Unlike the vascular variant, CAPS is an aPL-mediated systemic thrombotic microangiopathy characterized by increased levels of circulating pro-inflammatory mediators [75]. The clinical picture of CAPS is characterized by thrombotic complications affecting several anatomical compartments, more commonly intra-abdominal organs and less frequently lungs and skin. The systemic presentation and the hyper-inflammatory phenotype raise the issue of whether the pathogenesis is supported by mechanisms different from those of the classic vascular APS [75].

Despite the big clinical difference, there is evidence that β2GPI and the β2GPI-dependent aPL are also the main players in CAPS. Similarly, the two-hit hypothesis seems to apply to CAPS as well. In fact, infections (present in almost half of the cases), surgical procedures, malignancies, uncontrolled anticoagulation or its withdrawal, pregnancy complications, drugs, and disease activity in SLE have all been reported to behave as triggers (second hit) of CAPS in subjects carrying persistent positive aPL (first hit) [75].

Although studies on the pathophysiological mechanisms of CAPS are limited due to the difficulty in collecting biological samples, thrombotic microangiopathy is the main pathological finding. As mentioned above, and differently from the classic vascular APS, the hallmark is represented by increased circulating levels of pro-inflammatory cytokines and ferritin. All these mediators contribute to activate different cell types involved in the systemic hypercoagulable state (e.g. endothelium, monocytes, platelets and neutrophils). This cascade of events may take place simultaneously in several tissues explaining the systemic manifestations. In particular, platelet activation and thrombocytopenia in >60% of the patients are consistent with the view that CAPS display close similarities with other systemic thrombotic microangiopathies [76].

APS is a complement-mediated disease

Anecdotal reports published in the early nineties and more recent data from APS patients have shown that complement activation is a common denominator in all clinical APS variants [77–84]. The clinical findings have been supported by the demonstration that animals with inherited complement deficiencies or treated with molecules blocking complement activation were protected in APS experimental models (reviewed in [77]).

Although complement can be activated through the classical, alternative and lectin pathways, the pathway/second mainly involved in complement-mediated clinical manifestations of APS and the exact mechanism responsible for their activation is still open to discussion (reviewed in [77]).

Complement activation in vascular, obstetric and catastrophic APS will be discussed separately due to differences in the pathogenesis of the APS variants.

Complement in vascular APS

Anecdotical reports have revealed a decrease in the levels of C3, C4 and CH50 activity and an increase in the circulating levels of the activation products (e.g. C3a, C4a but not C5a, Bb, C4d) (reviewed in [77, 82, 85–87]) in the absence of defects in the regulatory proteins. Complement activation occurs in APS independently of specific vascular manifestations and is unrelated to the time of the vascular event. Comparable results were also reported in asymptomatic aPL carriers [49]. These data are in contrast with the findings of another study that reported an association between persistent complement activation and a more severe clinical APS phenotype [87]. Moreover, the same group found an increased prevalence of germline variants in genes controlling components of the alternative pathway implicated in complement-mediated atypical hemolytic uremic syndrome [88]. The authors suggested that patients bearing these genetic mutations are at higher risk of developing CAPS or recurrent thrombosis.

Direct evidence supporting the important role played by the complement system in thrombus formation has been obtained from animal models of APS. The infusion of a C5-neutralizing antibody or the use of C3- and C5-deficient mice showed that inhibition of complement activation prevents thrombus enlargement in mice [85, 86] and spontaneous clot formation in rats [22] supporting the crucial contribution of complement to aPL-induced thrombosis (reviewed in [77]). The finding that a complement-fixing anti-D1 β2GPI human monoclonal IgG induces clotting in vivo while the CH2-deleted variant of this antibody unable to fix C1q fails to trigger clot formation speaks in favor of the pathogenic relevance of complement activation through the classical pathway [89]. Complement activation through the classical pathway ending with the deposition of C5b-9 on target cells was also documented by another in vitro study [87, 88]. However, this does not exclude a potential activation of the lectin and alternative pathways that may contribute to amplify the complement cascade. We have recently shown that MBL binds to β2GPI in the absence of antibodies triggering activation of the lectin pathway and in turn promoting thrombin generation [90]. Increased levels of Bb, an activation product of the alternative pathway, have been reported in a large cohort of APS patients and found to be associated with the presence of LA and dual/triple aPL positivity [82]. Failure of aPL to induce thrombosis in C6-deficient rats and mice is a clear indication that clot formation in the animal models is dependent on the action of the terminal complex C5b-9 [22]. The co-localization of C9 and IgG on the endothelium of the mesenteric micro-vessels of rats infused with aPL, along with this observation, support the conclusion that complement activation proceeds till the assembly of the membrane attack complex at the sites of thrombosis [22]. Previous studies have shown that C5b-9, in a sublytic or cytolytically inactive form exerts a procoagulant effect on endothelial cells inducing the expression of tissue factor involved in the activation of the extrinsic pathway of coagulation [91, 92].

The involvement of complement has been further supported by the demonstration of deposits of complement components in the affected tissues not only in animal models [22, 31, 89] but also in human samples, although a limited number of patients were investigated in most studies [34, 93]. The finding of C1q and C5b-9 deposits supports the activation of the classical pathway till the assembly of the terminal complex [34].

Complement in obstetric APS

Complement involvement in obstetric APS was suggested by anecdotical reports in the early days of APS research [78]. After the initial reports of mild hypocomplementemia and increased activation products in APS pregnant women, a recent multicentre study confirmed the occurrence of low C3/C4 levels and their association with negative pregnancy outcomes [94]. Increased levels of Bb have been observed in aPL-positive patients with adverse pregnancy outcomes suggesting the contribution of the alternative pathway to pregnancy complications [95].

Deposits of both early and late complement components and their activation products have been observed in the placentas of aPL-positive women in two studies [77, 96]. There was, however, some disagreement with regard to the components involved, the tissue localization and the association with histological signs of inflammation or therapy [77, 96], possibly due to differences in the areas of the placenta examined from the way the samples were collected. The deposits of C1q in the placenta specimens reported in one of these studies are again in favor of the involvement of the classical pathway [77].

The most compelling evidence supporting the pathogenic role of complement in the adverse pregnancy outcome of APS patients was obtained from the experimental models of aPL-mediated fetal loss. Large amounts of aPL infused in pregnant mice were found to induce fetal resorptions and growth retardation that were not observed in C5-, C4- and B-deficient animals (reviewed in [58, 67, 74]). As for the model of vascular APS, human anti-D1 β2GPI monoclonal IgG that activates complement via the classical pathway [89] was found to cause fetal loss in pregnant mice whereas the CH2-deleted variant was ineffective and possibly protective.

C5a has been shown to play a major role in APL-induced fetal loss stimulating PMN to release TNF that induces placental inflammation [74].

Complement in CAPS

The beneficial effect of the administration of eculizumab in refractory CAPS cases has been the initial indirect demonstration supporting the role of complement activation in the catastrophic variant [97–100]. This finding has been confirmed by the ‘CAPS registry’, which showed low levels of C3 and C4 in >50% of the cases even without a clear association with clinical manifestations and aPL serology [101]. Recently, another group reported complement activation evaluated by a modified Ham assay in most CAPS sera [87, 88]. More intriguingly, the deposition of IgG and complement components (C3d, C4d, C5b9) in subendocardial capillaries and arterioles was described in a case report of CAPS [93].

Complement gene variants associated with aHUS, the prototypical complement-mediated thrombotic microangiopathy, have been also reported in CAPS and less frequently in vascular APS [87, 88]. Despite the absence of CAPS animal models, all these findings support the involvement of complement in CAPS and the rationale for using complement-blocking drugs.

Complement activation, other inflammatory mediators of innate immunity and NET formation, which are all expressions of ‘intravascular inflammation’, contribute to local clotting and placental damage. However, they are not associated with an overt inflammatory process in the vessel wall characterized by infiltration of inflammatory cells as seen in classical vasculitides. This finding is consistent with the definition of APS as a vasculopathy.

Complement and coagulation

The animal models clearly show that blocking complement (or complement deficiency) protects the animal from thrombosis even when the ‘2 hits’ are present. This suggests that complement is pivotal for clotting, likely thanks to the well-known cross-talk between the coagulation and complement cascades [102, 103]. The best examples of this cross-talk are represented by the expression of TF on cells activated by β2GPI-dependent aPL in the presence of complement and the binding of MBL to β2GPI, which promotes complement-dependent thrombin generation [3, 77, 90].

Open questions

Protective aPL

As discussed above (third paragraph of obstetric APS session), aPL with similar antigen specificity and detectable by the same assays can be associated with different clinical variants of APS for reasons that are not yet clear. The other side of the coin is represented by medium/high titre of aPL with serological characteristics comparable to those found in APS patients and persistently present for a long time in asymptomatic carriers. Recent data suggest that the epitope specificity of these anti-β2GPI-dependent aPL is mainly directed against D5 rather than D1 as in symptomatic patients [104, 105]. It has been speculated that antibodies against D5 are not pathogenic. The results obtained from the APS rat model revealed that IgG from monospecific anti-D5 positive sera fail to trigger complement activation and to promote blood clotting, suggesting that they are not pathogenic [31]. These antibodies react with an epitope close to the PL-binding site of the molecule and inhibit the binding of β2GPI to anionic structures/cell receptors behaving as a sort of ‘protective’ antibodies [31]. These findings were confirmed by in vitro experiments with human β2GPI on CL-coated plates and are consistent with the critical role of D5 in β2GPI binding supporting the putative anti-thrombotic effect of anti-D5 monospecific aPL [31]. The prevalence of these ‘protective’ aPL would explain why the carriers do not display any clinical manifestation.

The inhibition of β2GPI binding and the pathogenic effect of β2GPI-dependent aPL by a synthetic peptide mimicking the PL-binding site of D5 (TIFI) further supports the relevance of D5 in β2GPI binding and allowing the exposure of the immunodominant epitope in D1 [66, 106].

aPL against other PL-binding proteins

There is growing evidence that antibodies against prothrombin (PT), and in particular those specific for the phosphatidylserine (PS)-PT complex, may offer additional prognostic information in vascular APS, although the pathogenic effect of these antibodies remains a controversial issue [107–109]. Three papers reported a thrombogenic effect of anti-PT (in one study anti-PS/PT) antibodies in animal models similar to those used for β2GPI-dependent aPL [110–112], and a direct effect on the endothelium was suggested in two of these studies [110, 111]. However, the demonstration of the diagnostic power of anti-PT antibodies is still debated and this justifies why the test is not yet included in the new classification criteria [1]. For this reason, the possible role of anti-PT antibodies in APS pathogenesis has not been discussed in detail and the reader is referred to the specific papers [107, 108].

aPL against post-transcriptional modified β2GPI

Different post-translation modifications have been suggested to explain the loss of tolerance towards β2GPI consistently with the hypothesis that autoimmune responses may take place after post-translational modifications of the target self-antigens. For example, post-translational oxidative modification of β2GPI can produce new antigens able to elicit autoreactive T cells [113].

Carbamylation of β2GPI is another post-translational mechanism able to modify the protein and to trigger the production of specific autoantibodies. One study showed that IgG specifically reacting with carbamylated β2GPI but not with the wild-type molecule can be found in one-third of APS patients [14].

It is useful to speculate that these post-translational mechanisms may all take place in the NET environment where β2GPI was demonstrated to be present [10].

Co-factor and complement-independent aPL

Beta2-GPI-dependent aPL able to activate complement are widely accepted as the major players in APS pathogenesis. However, there is evidence that additional, not necessarily alternative, types of antibodies may be present in the patients.

For example, monoclonal anti-β2GPI IgG may increase the formation of platelet-rich thrombi in animals without the involvement of complement or the Fcγ cell receptor, as the equimolar amounts of F(ab)₂ molecule display similar clot formation [114].

Moreover, aPL reacting with endosomal lysophosphatidic acid presented by the CD1d-like endothelial protein C receptor were found to induce thrombosis and endosomal inflammatory signalling in experimental models. These aPL recognize a single cell surface lipid–protein receptor complex and perpetuate a self-amplifying autoimmune signalling loop engaging both the innate immune complement and coagulation pathways. While this finding further supports the close cross-talk between complement and coagulation cascades in APS, it is also paving the way for new pathogenic and possibly diagnostic autoantibodies [115, 116].

The involvement of neutrophils in the amplification loop triggered by aPL is behind the rationale of studies focused on the detection of antibodies against NETs. Anti-NET IgM and less frequently IgG have been reported in a large series of APS patients. These antibodies potentially activate complement and were related to different NET-associated antigens [117, 118].

Although definite clinical correlations cannot be drawn from these studies, the newly described antibodies may represent another mechanism sustaining the amplification loop triggered by aPL.

Conclusion

Our knowledge of APS pathogenesis has been evolving rapidly since the original description of the disease.

In the vascular APS, we have sound evidence for pathogenic mechanisms mediated by well-defined β2GPI-dependent aPL and additional humoral/cellular players that support the cascade of events ending in clot formation. Recently, antibodies apparently unrelated to β2GPI-dependent aPL have been described, raising the possibility of ancillary pathogenic pathways. This finding may stimulate new areas of research aimed at associating a given biomarker with a peculiar clinical manifestation at variance with what we have obtained with the formal diagnostic/classification aPL up to now. However, further clinical and experimental studies on these antibodies are warranted to support their direct role in vascular events independently of β2GPI-dependent antibodies.

Regarding the obstetric variant, we now have robust evidence that pregnancy morbidity is not mainly related to a vascular thrombotic disorder but that β2GPI-dependent aPL affect trophoblast and decidual cells directly and are responsible for defective placentation. In addition, there is evidence that cells/mediators of innate immunity play a pathogenic role in obstetric APS as well.

Experts in the field of APS are now encouraged to reconsider the pathogenetic mechanisms described in the past in the light of new knowledge. In particular, the use of the new drugs blocking complement activation and administered orally is promising, but still to be validated regarding the potential side effects (e.g. increase risk of infections which may represent harmful second hits in aPL positive patients).

Data availability

No new data were generated in support of this article.

Funding

This work was supported by Italian Ministry of Health (Ricerca Corrente 2022 to P.L.M.).

Disclosure statement: The authors have declared no conflicts of interest.

References