This is the first study showing that calprotectin might be a potential predictor of microvascular manifestations in aPL-positive patients based on a large cohort. Serum calprotectin in aPL-positive patients was much higher than that in HCs (649.66 ± 240.79 ng/ml vs 484.62 ± 149.37 ng/ml, p < 0.001). Age of the first occurrence of aPLs positivity (OR 0.98, 95% CI 0.96–1), SLE (OR 2.08, 95% CI 1.15–3.75), calprotectin positivity (OR 1.83, 95% CI 1.02–3.26), hypertension (OR 2.73, 95% CI 1.36–5.45), hemolytic anemia (OR 2.66, 95% CI 1.13–6.23), and anti-β2GPI antibody (OR 2.06, 95% CI 1.11–3.83) are independent risk factors for microvascular manifestations in aPL-positive patients. Serum calprotectin had positive correlations with ESR (R = 0.155, p = 0.001), CRP (R = 0.119, p = 0.014), and a negative correlation with PLT (R = − 0.101, p = 0.031) at the time of blood sampling.

Extra-criteria manifestations, including microvascular manifestations, reflect a subtype of patients with aPLs positivity [4]. Those patients have a higher prevalence of arterial thrombosis, preeclampsia, relapse, and triple aPL positivity [5]. In the new APS classification criteria, microvascular manifestations were listed as clinical criteria as well, suggesting that microvascular manifestations play an important role in patients with APS [2]. However, there has been no generally accepted serum predictor for microvascular manifestations.

Calprotectin is a heterodimer of S100A8/S100A9 belonging to the family of Ca2+ binding proteins. It is mainly secreted by neutrophils and monocytes by necrosis, neutrophil extracellular trap formation, and Golgi-independent alternative pathway [20,21,22]. Macrophages, platelets, epithelial cells, keratinocytes, and cancer cells can also express calprotectin. Extracellular calprotectin is a steady marker of inflammation. It induces a proinflammatory response on endothelial cells, monocytes, neutrophils, and T cells mainly by Toll-like receptor 4 (TLR4) and receptor of advanced glycation end products (RAGE) [7]. Serum and plasma levels of calprotectin are elevated in many inflammatory diseases and thrombotic diseases [7]. Serum calprotectin is significantly higher in patients with SLE and has a positive correlation with disease activity [23]. In patients with COVID-19, calprotectin in circulation is correlated with thrombosis and poor prognosis [9, 24]. In patients with IBD, fecal calprotectin can be used to distinguish IBD from irritable bowel syndrome and monitor disease activity [25].

In our study, we found that serum calprotectin levels in aPL-positive patients were much higher than that in HCs regardless of SLE. There has been no study focusing on serum calprotectin levels in patients with APS before. Ruff et al. [11] found that the level of fecal calprotectin in patients with APS is elevated. Lood et al. [26] indicated that platelet calprotectin levels were increased in patients with SLE, particularly in patients with aPL positivity. However, the platelet level of calprotectin had no relationship with serum or plasma levels. These suggest that there might be an increment in both the expression and release of calprotectin in aPL-positive patients.

As an inflammation marker, calprotectin had positive correlations with ESR and CRP in our study. Patients with calprotectin positivity had a higher prevalence of microvascular manifestations. Endothelial cell dysfunction plays an important role in the pathogenesis of microvascular manifestations in patients with APS. Antibodies from patients with APS stimulate the mammalian target of rapamycin complex (mTORC) in endothelial cells, which is involved in the pathogenesis of microvascular manifestations [27]. Agostinis et al. [28] found that proinflammatory factors were needed for the binding between β2GPI and endothelial cells. These might explain the reason for the correlation between calprotectin and microvascular manifestations. Calprotectin could be an independent risk factor for microvascular manifestations, suggesting that patients in a hyperinflammation state are more likely to have endothelial cell dysfunction and develop microvascular manifestations.

Thrombocytopenia occurs in 20–50% of aPL-positive patients [29]. There are many proposed mechanisms of thrombocytopenia in patients with APS [30]. Anti-β2GPI–β2GPI complex could activate platelets by binding the glycoprotein Ibα (GPIbα) receptor, which might lead to thrombocytopenia [31]. In this study, we found that calprotectin was associated with thrombocytopenia at the time of blood sampling, but not thrombocytopenia history, which indicated that the process of calprotectin release was associated with the decrease of PLT. In vitro, calprotectin activated platelets with GPIbα as the receptor and the supporting role of CD36. Glycoprotein IIb/IIIa (GPIIb/IIIa) was activated, the expression of p-selectin was upregulated, and platelet-neutrophil aggregates were increased, but there was no platelet aggregation, which suggested a new mechanism of platelet activation [24]. Therefore, calprotectin might lead to thrombocytopenia by binding GPIbα and activating platelets.

Except for serum calprotectin, age at the first occurrence of aPLs positivity, SLE, hypertension, hemolytic anemia, and anti-β2GPI antibody were also found to be independent risk factors for microvascular manifestations, which was similar to the results in other studies. Anti-β2GPI antibody, double aPL positivity, cerebrovascular events history, and hypertension were more prevalent in patients with PAPS and extra-criteria manifestations [32]. Huang et al. [33] found that SLE, anti-β2GPI antibody, LA, and triple aPL positivity were associated with extra-criteria manifestations in aPL-positive patients. Thrombocytopenia, elevated CRP, and anti-β2GPI antibody were found to be independent risk factors for microvascular manifestations in patients with PAPS [34]. The reason for anti-β2GPI antibody contributing to microvascular manifestations remains unclear. By binding to β2GPI, the antibody activates endothelial cells, which leads to inflammation, endothelial cell proliferation, and intimal hyperplasia [35]. This process might result in microvascular manifestations.

This study has several limitations. Firstly, the results should be validated in another APS cohort to confirm the relation between calprotectin and microvascular manifestations. Secondly, a single test of calprotectin could not reflect the whole picture of the disease for patients. Studies monitoring serum calprotectin levels continuously are needed to find out a more specific relation between calprotectin and disease progression. Thirdly, this study did not elucidate the mechanism that enables calprotectin to cause microvascular manifestations and thrombocytopenia. Cell and mouse experiments are needed to explore the pathogenesis of calprotectin. Fourthly, the treatment regimen had an impact on the occurrence of microvascular manifestations, but the influence could not be eliminated when we predict microvascular manifestations.