Involvement of the complement system in immune thrombocytopenia: review of the literature

Abstract Immune thrombocytopenia (ITP) is a thrombocytopenic condition induced by autoimmune mechanisms and includes secondary ITP with underlying diseases such as connective tissue diseases (CTD). In recent years, it has been elucidated that the subsets of the ITP are associated with complement abnormalities but much remains unclear. To perform a literature review and identify the characteristics of complement abnormalities in ITP. PUBMED was used to collect the literature published up to June 2022 related to ITP and complement abnormalities. Primary and secondary ITP (CTD-related) were examined. Out of the collected articles, 17 were extracted. Eight articles were related to primary ITP (pITP) and 9 to CTD-related ITP. Analysis of the literature revealed that the ITP severity was inversely correlated with serum C3, C4 levels in both ITP subgroups. In pITP, a wide range of complement abnormalities was reported, including abnormalities of initial proteins, complement regulatory proteins, or the end products. In CTD-related ITP, reported complement abnormalities were limited to the initial proteins. Activation of the early complement system, mainly through activation of C3 and its precursor protein C4, was reported for both ITPs. On the other hand, more extensive complement activation has been reported in pITP.


Introduction
Immune thrombocytopenia is narrowly defined as an immune-mediated decrease in platelets alone and can cause the autoimmune bleeding disorder known as primary immune thrombocytopenia (pITP).The incidence rate of pITP is 2.1-2.8/100,000personyears, and it mainly affects children aged <6 years, women aged 20-34 years, and the elderly [1,2].The disease is mainly caused by the phagocytosis and destruction of opsonized platelets in the reticuloendothelial system by autoantibodies against platelet membrane glycoproteins (GPIIb/IIIa (CD41/CD61), GPIb/V/IX (CD42b), etc.).Anti-platelet autoantibodies also impair platelet production by inducing megakaryocyte maturation defects and apoptosis in the bone marrow.
Thrombocytopenia may often be part of a larger spectrum of autoimmune diseases, especially in systemic lupus erythematosus (SLE) and related disorders, such as antiphospholipid syndrome (APS).A large proportion (24%) of persistent secondary immune thrombocytopenia (sITP) cases are of SLErelated thrombocytopenia [3] (Figure 1).
The prognosis of both primary and sITP have improved markedly in recent years with the availability of more treatment options.However, adults with pITP still have a 1.3-2.2-foldhigher mortality rate than the general population [4], and the prognosis of thrombocytopenia with underlying connective tissue diseases (CTD), such as SLE, is relatively poor [3].Therefore, there has been a great deal of research regarding novel therapeutic options, and rituximab (RTX) has been particularly effective in treating refractory cases [5], with the anti-BAFF antibody belimumab (BEL) showing therapeutic potential against refractory immune thrombocytopenia [6].In addition to these B-cell elimination/suppression therapies, treatments based on different mechanisms, such as thrombopoietin receptor agonists and Syk inhibitors, are being investigated to expand the treatment options [7].
The complement system comprises a group of proteins responsible for innate immunity, which are activated sequentially following specific triggers resulting in opsonization, local inflammation, and cell membrane destruction.The complement system is involved in a variety of systemic manifestations of some autoimmune diseases, such as SLE and APS.In particular, activation of the classical complement pathway by immune complexes, and subsequent amplification by the alternative pathway, leads to local inflammation and, ultimately, tissue damage through the production of membrane attack complex (MAC, C5b-9) (Figure 2).A recent phase I trial of sutimlimab, an antibody against C1s approved for cold agglutinin disease, showed favorable results in a limited number of pITP patients [8].Hypocomplementemia is a frequent complication of SLE, but has also been reported to be associated with hypocomplementemia in 1/3 of patients and is beginning to be considered as a possible mechanism of platelet destruction [9].Hypocomplementemia is observed in patients with severe thrombocytopenia, and the rate of progression from pITP to SLE is higher in such cases [10].Thrombocytopenia is a frequently observed complication of SLE and was reported in 16.8% of 2104 SLE cases reviewed in China [11].In that study, hypocomplementemia was identified in a multifactorial analysis as a factor correlated with thrombocytopenia, along with lupus nephritis and various other factors, but no specific decrease in complement protein or complement system abnormalities were mentioned [11].
Complement activation has also been noted in other cytopenias.Complement activation is thought to be a major pathogenetic mechanism in autoimmune hemolysis, such as paroxysmal nocturnal hemoglobinuria (PNH) and cold autoimmune hemolytic anemia, but especially in the former, for which anti-C5 antibodies have been used in clinical practice [12].It has been reported that CD55, which  suppresses C3 convertase, and CD59, which suppresses end-product production, are involved in erythrocyte and leukocyte depletion in SLE [13].Thus, complement activation is commonly involved in thrombocytopenia and may be an important therapeutic target even in pITP without systemic autoimmune pathology, such as connective tissue disease (CTD).
Here, we present a literature review on complement abnormalities associated with thrombocytopenia in pITP, SLE, and related diseases representative of immune thrombocytopenia, and discuss possible therapeutic targets.

Methods
This literature review was performed to identify complement abnormalities associated with thrombocytopenia in pITP, as well as SLE and related diseases (SLE and/or APS and CTD-ITP).The criteria of literature selection were defined as original articles written in English from 2002 to June 2022 that examined human adults.Of these, the Englishlanguage articles and publications period was defined by the following search formula, and the inclusion of original articles and human adults discussed-articles was confirmed during the literature screening phase described below by the researchers.In addition, to eliminate case reports, reports with fewer than 10 cases examined were excluded.PubMed was searched using the following terms: Two researchers from two different institutions that worked independently conducted primary and secondary screenings.One group performed a literature screening for pITP and the other for CTD-ITP.Therefore, a total of four researchers have done the literature screening.The methodology was based on the advice of a statistician.Analyses were performed manually and no automated tools were used.
The primary screening was performed based on the article title and abstract, and the secondary screening was performed by screening the full text of the articles.
The outcome data to be obtained from the article were defined as abnormalities in complement molecules associated with thrombocytopenia.This was assumed to include changes in serum complement levels, deposition of complement molecules on platelet membranes, and the presence of anti-complement antibodies.
If any of the articles that were not picked up in the negotiation process or were excluded in the screening process were considered important by one or more of the researchers, the article was reviewed again among all the researchers to reach a consensus on its adoption.
This study was not pre-registered as the literature review.

Results
The literature search identified 1,585 potentially eligible articles.After primary screening based on the titles and abstracts, 1,507 articles were determined to be ineligible.The full texts of the remaining 78 articles were reviewed, which resulted in the exclusion of a further 63 articles.Thus, a total of 16 articles were included in the final review.Seven of these sixteen articles were on pITP and nine were on CTD-ITP (Figure 3).One article on pITP presented the results of a phase I trial of anti-complement therapy and was added due to its importance, as agreed upon by all of the authors [14].The other studies were observational.A narrative-based review of the literature on the involvement of the complement system in pITP and CTD-ITP is presented below.

Primary ITP and the complement system
The articles included in the final review are listed in Table 1.Complement activation in pITP is relatively well documented.In a study of 108 adult patients with active pITP, Cheloff et al. [9] reported that the serum levels of C3, C4, and CH50 were significantly lower in pITP patients compared to healthy controls; of the patients, 32% exhibited a decrease in one of C3, C4 or CH50, while 10% had a decrease in all three.The severity of pITP was correlated with serum C4 and C3 levels.There was no correlation between the response to existing and the level of hypocomplementemia.On the other hand, another study showed no clear differences in serum C3 and C4 levels between pITP patients and healthy controls [15].However, consistent reports of abnormalities in the serum levels of proteins further upstream in the complement system, such as C1q, which is the initiator protein of the classical pathway of the complement system, were reported [15,16].Complement deposition on the platelet membrane is another common finding in patients with pITP and is considered to indicate complement-related platelet damage.Moreover, deposition of C1q and C4d, which appear in response to C1q and C4 activation, was reported [16][17][18].These complement molecules do not bind to platelet membranes in healthy individuals and are correlated with an increased immature platelet fraction (IPF) and severe thrombocytopenia in patients with pITP [17].Deposition of C3b, which occurs as a result of activation of the downstream molecule C3, was also observed, but in relatively small amounts.The deposition of these complement proteins on platelet membranes was correlated with the binding rate of anti-platelet autoantibodies to platelet membrane proteins [19], and the binding capacity of complement was reduced on platelets lacking the antibody binding sites GPIIb/IIIa and GPIb/IX.This complement deposition on platelets was observed in 58% of patients with pITP, and splenectomy has been reported to be an effective treatment in cases of advanced deposition [17].C5b-9, which is a complement complex comprising proteins from C5b to C9 and the end product of the complement system, disrupts the membrane structure when it appears on platelet membranes, and is often observed on the membranes of platelets from pITP patients.In addition, soluble C5b-9 is present in the serum of pITP patients in remission at the same level as in healthy individuals, but is highly concentrated in active pITP and inversely correlated with platelet count [15].
Activation of the alternative pathway and abnormalities in complement regulatory factors have also been reported.Sahip et al. measured factor H (CFH), factor B (CFB), and other parameters in serum samples from 92 pITP patients and compared them with 48 healthy controls; the levels of both CFH and CFB were significantly lower in pITP patients.Retests after treatment showed no change in CFB in pITP patients, while CFH was significantly elevated [20].They also reported frequent detection of anti-C1q antibodies in pITP.As in other autoimmune diseases, anti-C1q antibodies are assumed to be involved in the pathogenesis of pITP, not as neutralizing antibodies but rather as autoantibodies that activate the classical complement pathway [21,22].
Although therapeutic application of the complement system is still in its infancy, treatment with antibodies targeting an early molecule of the classical pathway has shown promising results.In an open-label phase I trial, Broome et al. [14] reported that the platelet counts in 12 patients with relapsing pITP increased from 25 Â 10 9 /L to 54 Â 10 9 /L at 24 h after starting treatment with sutimlimab, a monoclonal antibody that inhibits C1s.Five patients (42%) achieved sustained platelet count responses (!50 Â 10 9 /L in !50% of follow-up visits) and four achieved a complete response (platelet count !100 Â 10 9 /L).Five serious adverse events were recorded, of which one (migraine) was considered to be related to sutimlimab.

ITP related to SLE and/or APS (CTD-ILD) and the complement system
The final articles related to CTD-ILD and complement abnormalities are listed in Table 2.Although it has long been suggested that complement activation is associated with thrombocytopenia in SLE and APS, it is more difficult to isolate the effects of complement activation in thrombocytopenia in CTD-ITP because of the wide range of organs involved.A total of nine articles reported complement abnormalities specifically associated with thrombocytopenia in SLE and APS.
Several reports indicated an association of low serum complement protein levels with CTD-ITP.In a multivariate analysis, Ziakas et al. found that low serum levels of C3 and CH50 were correlated with thrombocytopenia, and low serum C3 level was a risk factor for poor prognosis (along with high disease activity) [23,24].Ho et al. reported that low serum levels of C3 and C4 were correlated specifically with recurrent thrombocytopenia in SLE [25].
Deposition of complement proteins on platelet membranes has been analyzed in patients with SLE [26][27][28][29][30].Such complement deposition has been analyzed in relation to thrombotic tendencies, as well as thrombocytopenia.Peerschke et al. reported that complement deposition on the platelet membrane was correlated with the presence of anti-phospholipid antibodies (aPL), as well as with platelet activation, arterial thrombosis [26,30], and APS [28].Complement deposition on the platelet membrane has primarily been examined in relation to vascular injury and thrombosis [18].
On the other hand, there have been few reports of molecules involved in the alternative pathway (other than C3), late components, or lectin pathway in CTD-ITP.Watanabe et al. [31] reported that the serum level of ficolin, an early component of the lectin pathway, was lower in CTD-ITP patients than controls, and thrombocytopenia was correlated with a low ficolin level, suggesting a role in the pathogenesis of thrombocytopenia.The serum ficolin level was not correlated with overall disease activity in SLE, but was significantly lower in patients with thrombocytopenia.

Discussion
Complement activation and abnormalities are widespread in both pITP and sITP, but mainly involve the early components of the classical pathway.Since it was difficult to integrate the data obtained in this study, the results from each article are listed separately (Tables 1 and 2).
Several articles also reported overexpression of the end product, C5b-9, in pITP (Figure 1).Activation of the classical pathway, which is initiated by immune complexes and their binding protein, C1q, is frequently observed in diseases involving pathogenic autoantibodies targeting molecules on the platelet membrane.Binding of complement proteins to the platelet membrane represents evidence of complement system activation on the platelet membrane, resulting in opsonization or membrane disruption that leads to thrombocytopenia.Abnormalities in complement regulatory factors, such as CHF and CHB, have also been reported, and may involve mechanisms that amplify complement system activity.One report described the involvement of the lectin pathway, in which the binding of L-ficolin, the initiator of the pathway, to N-acetylglucosamine on the platelet membrane led to membrane damage via activation of the complement system and the production of C5b-9.
Although the classical pathway is assumed to be primarily responsible for complement activation, activation of two other pathways may also occur, simultaneously or independently.Several articles on CTD-ITP reported that the presence of aPL is associated with complementrelated thrombocytopenia.The negatively charged phospholipids targeted by aPL are highly expressed on activated platelet membranes in particular, and are known sites of thrombus formation [32].Therefore, the thrombotic tendency is associated with thrombocytopenia in APS [33], and aPL-associated thrombocytopenia may present as pseudothrombocytopenia due to platelet aggregation [34,35].In addition, beta-2-glycoprotein I (b 2 GPI), a plasma protein that forms complexes with anionic phospholipids that are targets of aPL, shares a structural motif with complement regulatory factors, and blood levels of b 2 GPI were reported to be inversely correlated with the level of platelet membranebound C5b-9 [16].Note that complement activation occurs in a limited number of pITP patients, and as is the case in aPL-positive patients with pITP, the presence of aPL may at least partially explain the thrombocytopenia seen in ITP.Patients with pITP with APS/SLE-like features, such as aPL positivity and complement activation, may be at high risk of developing systemic autoimmune diseases.In fact, Yoo et al. reported that 7.7% of pITP patients developed SLE after 30 months of observation, with ANA positivity as one of the risk factors [12].
The conditions underlying autoimmune thrombocytopenia include not only CTD, but also malignant and infectious diseases such as lymphoproliferative disorders and hepatitis C [36], although we did not examine these diseases in the present study.Among infection-related ITPs, H. pylori ITPs are relatively frequent.In 50% of ITP cases with H.pylori infection, platelet counts improve with antimicrobialbased H.pylori elimination therapy [37].It has been reported that H.pylori infection activates complement [38] and induces C3 deposition and tissue damage, and platelet aggregation or destruction via local tissue damage, especially vascular endothelial cell damage, can be anticipated.However, the analysis of the pathogenesis of H.pylori-ITP that incorporates complement activation is scarce, and the proposed mechanisms are such as molecular mimicry, platelets aggregation via Fcc receptor activation, phagocytic perturbation and increased plasmacytoid dendritic cell (pDCs) response [36,37].
Recently, there have been reports of pITP development following mRNA vaccination against SARS-CoV-2, but this was also ruled out in our study [39].The pathophysiology of atypical hemolytic uremic syndrome (aHUS), a group of thrombotic microangiopathies (TMA) presenting as thrombocytopenia associated with complement disorder, has been analyzed [40].However, aHUS was also rule out in the present study as it shows secondary platelet destruction and complement activation primarily damages vascular endothelial cells.
In addition to aHUS, other autoimmune cytopenias that are prone to thrombocytopenia include PNH and cold agglutinin syndrome, the pathology of which involves complement abnormalities.Complement-related thrombocytopenia is relatively common, and some of these conditions may be improved by therapeutic interventions targeting the complement system.Furthermore, increasing numbers of diseases have recently been linked to complement abnormalities, thus highlighting the importance of the complement system as a therapeutic target and biomarker [41][42][43][44][45].
There are several limitations in this study.First, it must be said that the overall level of evidence in the articles obtained is comparatively low.The collected were mainly observational studies with no randomized-controlled studies, including one openlabel phase I trial.Studies with control groups were also scarce.Second, most of the studies have not conducted comprehensive analyses of the complement system, but only partial analyses.The analyses that highlight the involvement of the complement system as a whole in ITP, are needed.Third, Pubmed was the only database used for the literature search, and the possibility that other studies were obtained through searches by other search engines can not be ruled out.
Approximately 10% of patients with pITP become refractory to treatment [46,47];these patients have a poor quality of life and high mortality rate due not only to low platelet counts, but also to increased risks of bleeding and infection [48].CTD-ITP is also associated with a poor prognosis [23].Based on the results presented herein, it can be assumed that there are patients (mainly refractory cases) for whom complement therapy is effective, and that analysis of the complement system is important for stratifying patients.

Figure 2 .
Figure 2. The flow diagram for the database search of publications for the literature reviews.

Figure 3 .
Figure 3. ITP-associated complement abnormalities in the complement cascade diagram.The classical, lectin, and alternative pathways converge in the final common pathway when C3 conversion enzyme (C3 con) cleaves C3 into C3a and C3b during the course of these pathways, ultimately eliminating foreign substances by producing a membrane-damaging complex.In the activation pathway, complement-regulated proteins such as DAF, C4BP, MCP, and CFH, which are indicated by shading, regulate the progress of activation.Complement abnormalities associated with ITP are illustrated with the literature numbers collected.Upward arrows indicate increased complement protein or deposition on platelet membranes.Downward arrows indicate decreased complement protein.
schematic diagram of ITP is shown, which is divided into primary and secondary ITP, with secondary ITP underlying CTD (connective tissue disease), malignant disease, infection and drug complications.Primary ITP includes a group of conditions that are precursors to collagen diseases.Complementrelated ITP mainly includes part of CTD-related ITP and part of primary ITP. A

Table 1 .
Review of the literature on pITP and the complement abnormalities.

Table 2 .
Review of the literature on CTD-ITP and the complement abnormalities.