This pilot study investigated neutrophil- and platelet-derived soluble markers as well as galectin-3 in patients with r-axSpA. We found significantly higher levels of HNL in both plasma and serum, as well as significantly elevated concentrations of plasma MPO and serum sCD40L, in r-axSpA patients compared with controls. In contrast, no differences were detected in neither plasma nor serum regarding sP-selectin and galectin-3. Across both studied groups, serum concentrations of granule-associated proteins exceeded those in plasma (except for galectin-3), consistent with clotting-induced release [16, 17].

Neutrophils are increasingly recognised as important players across the spondyloarthritis (SpA) spectrum [18]. We found that HNL, a sensitive marker of neutrophil degranulation [6], was significantly elevated in both plasma and serum, consistent with an earlier study demonstrating increased serum HNL in r-axSpA [19]. Our detection of increased plasma levels suggests circulating neutrophil activation in r-axSpA, with clotting-amplifying release in serum. Additionally, plasma MPO levels were also significantly elevated, consistent with previous findings of increased MPO (both mRNA and protein) in r-axSpA [5, 20]. Neutrophils release their granules in a hierarchical order, with the specific granules (containing HNL) being mobilised already under modest stimulation whilst the azurophilic granules (containing MPO) require a stronger activation for mobilisation to the plasma membrane [21]. Therefore, increased levels of both plasma HNL and MPO shows a sustained neutrophil activation in r-axSpA patients.

Previous research has linked platelet count and platelet-to-lymphocyte ratio to disease severity [4], and transcriptomic analyses have identified altered expression of immune-related genes, including CD40L [22]. In this study, serum sCD40L was significantly increased in r-axSpA patients despite normal platelet counts and treatment with NSAIDs, well-known inhibitors of platelet activation, supporting a pro-inflammatory role of CD40-CD40L signalling in autoimmunity [7]. However, a previous study reported conflicting results regarding sCD40L in r-axSpA [10]. In contrast, platelet-derived sP-selectin was not elevated, consistent with one earlier report [10] but in disagreement with another study linking increased sP-selectin mRNA expression to a poorer prognosis [23]. The reasons for this discrepancy remain speculative. As previously outlined, it may be influenced by the fact that 69% of patients in this pilot study received treatment with NSAIDs. It could also relate to the fact that mRNA expression is not invariably proportional to the amount of translated protein, or to differential regulation of platelet α-granule proteins. Mechanistically, both proteins are inserted into the plasma membrane post degranulation and then enzymatically cleaved (likely by different enzymes) from the platelet surface to produce soluble ectodomains [24] adding to the complexity when interpreting results. In the current study, serum sCD40L levels were elevated in r-axSpA indicating augmented CD40L expression or a pre-activated (primed) platelet phenotype, reinforcing the notion of platelet involvement in r-axSpA pathogenesis.

Galectin-3, produced by neutrophils and multiple other cell types including monocytes and epithelial cells, regulates inflammation and tissue remodelling [12]. Although elevated serum galectin-3 has previously been reported in r-axSpA [11], we observed no group differences. This may relate to methodological differences, as Cao et al. [11] used a chemiluminescent microparticle immunoassay rather than an ELISA. Interestingly, plasma galectin-3 levels were significantly, albeit modestly, higher than serum levels in patients with r-axSpA, whereas no such difference was observed in controls. This modest increase is not readily explained by clotting-induced release and may instead reflect enhanced plasma stability of galectin-3, although this remains speculative at present.

A strength of this study is the combined analysis of plasma and serum, allowing a more nuanced understanding of proteins released under anticoagulated versus clotting-induced conditions. The inclusion of multiple markers provided a broader perspective on cellular contributions to r-axSpA pathogenesis compared with studies restricted to single analytes. To minimise potential confounders, we focused on a homogeneous group of HLA-B27-positive male patients with established disease and without DMARD treatment. However, most patients were receiving NSAIDs at the time of sampling, which may influence platelet activation and therefore represents a potential confounding factor that should be considered when interpreting the platelet-related findings. Accordingly, this design and the small sample size limit generalisability, and the findings should therefore be interpreted with caution and viewed as exploratory and hypothesis-generating, warranting validation in larger, more heterogeneous r-axSpA populations. Moreover, as two of the included analytes are neutrophil-derived, the lack of data on absolute neutrophil counts represents a limitation that should be addressed in future studies. Nevertheless, HNL and MPO primarily reflect neutrophil activation rather than absolute neutrophil counts and may therefore provide complementary information on neutrophil involvement in r-axSpA. Despite the limited cohort, a positive correlation between plasma HNL and markers of systemic inflammation (CRP and ESR) was observed, suggesting that elevated plasma HNL may reflect inflammatory activity. None of the other elevated markers showed significant correlations with CRP, ESR, or other clinical parameters.

In conclusion, the results of this pilot study support the involvement of neutrophils and platelets in r-axSpA, with HNL, MPO, and sCD40L showing disease-associated increases. Future studies incorporating longitudinal sampling will be essential to evaluate the clinical utility of these markers for diagnosis and disease monitoring.