APLS is an immune-inflammatory-thrombotic dysregulation distinguished by repeated thrombosis and provoked obstetric complications mostly rooted in the constant positivity of aPL. Neutrophil activation is considered another important player in the pathophysiology of both SLE and APLS through the formation of net-like structures in a process referred to as NETs formation. Assessment of NETosis-specific markers such as serum levels of MPO-DNA, citrullinated histones, and the neutrophil elastase-DNA complex is becoming essential for initiating NETs inhibitors. Histones proteins are a tectonic portion of chromatin built by DNA which, on the one hand, represent an autoantigen in SLE and APLS and, on the other hand, serve as an indicator for NETosis activation. Insufficient data is available on the clinical role for assessing the MPO and Histones level in APLS, especially the thrombotic type.

Given these data, we designed our study to explore the potential role of MPO and Histones in APLS Egyptian patients. We believe that this study is the first trial to analyze the prognostic effect of this combination in APLS patients.

In our study, we analyzed a group of demographic and laboratory parameters in APLS patients in comparison to normal volunteer subjects. Also, we involved a reactive inflammatory group with a high WBC count (>20x103μL with a high N/L ratio) to monitor the changes that are specific for NETosis activation in APLS since NETs formation can also occur in several other conditions such as malignancy, inflammation, and infection and plays a significant role in the body defense and innate immunity [13].

As expected, the APLS cases showed anemia and thrombocytopenia when compared to healthy control however, they had higher N/L ratio, INR, PTT, ESR, and CRP which is mostly related to the ongoing chronic inflammatory-thrombotic pathology in APLS. In comparing APLS patients with the reactive group, APLS patients showed also lower PLT count and higher ESR levels with no significant difference regarding Hb. However, the reactive group showed higher WBC count, neutrophils count, N/L ratio, and CRP when compared to APLS patients. These findings could be explained by the underlying pathology in each group and the chosen criteria for the reactive subjects. Also, According to Kourilovitch and Galarza–Maldonado, 2023 [14], neutrophil activation and NETs liberation lead to enhanced release of reactive oxygen species (ROS) and MPO that leads to cellular injury followed by chronic inflammation suppressing T- and B-cell immune functions, which may explain the higher N/L ratio in APLS and the reactive group. Furthermore, many studies stated the occurrence of thrombocytopenia and hemolytic anemia in APLS which are now included in the “Non-criteria features” of APLS [15]. Klack et al. 2013 found that APLS patients have a higher incidence of iron deficiency anemia when compared to healthy controls [16].

To demonstrate NETs formation in our studied groups, we explored the levels of MPO and Histones in all studied subject groups. MPO and Histones levels in APLS patients and reactive subjects were significantly higher than in the normal control group. In addition, the MPO and Histones levels were comparable with no significant difference between APLS patients and the reactive group. All these former data provided evidence for NETs formation. This was in agreement with de Moraes Mazetto et al. 2022 who stated that APLS patients especially thrombotic type showed higher serum MPO-DNA complex levels than in healthy controls and that its level correlated with the disease severity and poor outcome [11].

Threshold values were determined in our research for the MPO and Histones levels to predict activation of NETosis process. According to our results, MPO at a cutoff > 2.09 ng/ml was acknowledged as the best predictor for active NETosis in APLS patients which was slightly lower than that in the reactive group, (MPO at a cutoff > 3.62 ng/ml). In both APLS patients and the reactive group, Histones at a cutoff > 1.45 ng/ml were the best predictor for active NETosis. In recent published research, there was no determined cutoff for any of the NETs markers. We believe that determining a specific cutoff will help timely initiation of anti-NETosis therapy with subsequent prevention of unwanted sequelae of NETs formation.

In our work, we also studied the correlation of MPO and Histones levels with the other studied parameters in all studied subjects, normal controls group, reactive group, and APLS patients’ group to determine their prognostic role. In all studied groups, there was a significant positive correlation found between MPO levels, WBC count, and neutrophil count. This correlation was not found with Histones, which could be explained as neutrophils being very rich in MPO forming around 5% of its actual weight and stored in the azurophilic granules which are released upon neutrophils activation with subsequent NETs formation in response to ongoing inflammatory/immune dysregulation [17].

Additionally, in our study, there was a significant negative correlation found between MPO and Hb levels in all studied groups. The presence of anemia during NETs formation in a previous study on canines was found to be mostly attributed to the pathological inflammatory condition, organ damage, and autoimmune hemolytic process present as a part of the pathogenesis of APLS or from bleeding resulting from anticoagulant therapy used in APLS patient to prevent thrombosis [18].

MPO and Histones levels showed a significant negative correlation with CRP which indicated that NETs formation in APLS patients did not occur as an acute phase activation but rather due to another aetiology. In studies on NETs by Bravo-Barrera et al., 2017 [19], they have proved that autoantibodies against B2GP1 are the main culprit in NETs formation and its release is significantly augmented by ACL, in addition, they found a positive correlation between anti-B2GP1 IgG, LAC, ACL IgG and circulating MPO-DNA complexes. The same data was supported recently by Reshetnyak et al., 2023 [20].

In all our studied subjects, and in the APLS patients’ group alone, both MPO and Histones showed significant positive correlations with PT and INR which indicates an underlying coagulopathy found due to NETs formation hand in hand with immune-dysregulation specific in APLS since these findings were not observed in the reactive or normal control groups. The same findings were supported by Bravo-Barrera et al., 2017 [19], who highlighted the role of neutrophils in connecting the inflammation and coagulopathy induced by NETs formation which contributes to thrombosis (both venous and arterial) through various operations for instance binding and activating platelets, tissue factor (TF) and coagulation factor VII, which augment or initiate thrombosis. Besides, neutrophils release TF-bearing NETs which act as an activator for thrombus formation through thrombin generation. Also, Reshetnyak and Nurbaeva., 2023 [21], have also mentioned that NETs inside-constituent can suppress the fibrinolytic system and natural anticoagulants activity in the blood circulation. They also commented on the role of histones in interfering with the thrombomodulin-dependent activity of protein C. The use of anti-thrombotic treatment such as warfarin which inhibits coagulation factors synthesis especially thrombin plays an augmenting role.

There was a significant positive correlation found in all studied subjects between both MPO and Histones levels with ESR which may highlight increased NETs formation during APLS and SLE activity. Some Authors of previous research suggested that during flaring episodes of SLE activity, NETs levels are increased along with thrombotic complications furthermore, the MPO-DNA complex levels were elevated in patients with high anti-dsDNA, hypocomplementemia and glomerulonephritis indicating SLE activity [20].

Finally, upon studying the relation between NETs formation and the clinical characteristics of APLS patients, both MPO and Histones could not differentiate between 1ry and 2ry APLS which could be explained by common links in SLE and APLS pathophysiology. However, MPO and Histones were significantly higher in thrombotic APLS patients when compared with non-thrombotic ones. Besides, Histones levels were significantly increased in patients with aPL antibodies with double positivity (LAC, B2GB1or ACL) and triple positivity (LAC, B2GB1, ACL) when compared to those with single positivity (LAC only). Meanwhile, MPO level showed no significant difference among APLS patients based on their positivity for aPL antibodies. Our later findings suggest that NETs formation especially with a higher Histones level had a bad prognostic indication being associated with a more severe disease course. In concordance with our findings, Reshetnyak et al., 2023 [20] found that increased NETs formation namely MPO-DNA complex was more pronounced among thrombotic APLS patients with a poorer prognosis, such as those with multiple aPL antibodies activity and recurrent thrombosis. In addition, a large number of published researches highlighted the presence of elevated NETs in venous and arterial thrombi, lupus nephritis, immune-inflammatory mediated organ dysfunction, and pregnancy-related morbidity [19, 22].