Thrombin Generation Related to Netosis in Patients with Systemic Lupus Erythematosus
Authors: Monzón Manzano Elena , Fernandez-Bello Ihosvany , Justo Sanz Raul , Robles Marhuenda Ángel , Acuña Paula , Alvarez Román María Teresa , G Arias-Salgado Elena , García Barcenilla Sara , Canales Miguel A , Jimenez-Yuste Victor , Butta Nora V. 
Published: November 2020

NETosis is a process suffered by neutrophils that consists in the loss of their function and the release of their nuclear material as large web-like structure called neutrophil extracelular traps (NETs). Many authors demonstrated that NETs participate in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), because the release of autoantigens amplifies inflammatory responses, perpetuating the exacerbation of autoimmunity. On the other hand, NETs may play a prominent role in thrombosis because they serve as a negative charge scaffold to trap platelets and coagulation factors, promoting blood clot formation.
Objetive: to determine participation of NETs in the hypercoagulable state of patients with SLE.
Methods: 32 patients with SLE without antiphospholipid antibodies and without history of thrombotic events were included after signing informed consent; 88 sex- and age-matched healthy controls were also recruited. Blood samples were drawn in citrate tubes (3.2%). Neutrophils were isolated by centrifugation of whole blood with a Percoll gradient at 500 g, 25 min, 5ºC. To induce NETs formation, 2.5x105 isolated neutrophils were incubated in RPMI-1640 medium with or without 100 nM phorbol 12-myristate 13-acetate (PMA) for 45 min, 37ºC.
To verify NETs formation, neutrophils were seeded on cover glasses pretreated with poly-L-lysine in RPMI-1640 medium with or without 100 nM PMA for 45 min, 37ºC. Samples were fixed and later incubated first, with an anti-human myeloperoxidase and then, with Alexa Fluor 488 goat anti-rabbit IgG. Finally, samples were embedded in mounting medium with DAPI and were observed by fluorescence microscopy with a Nikon Eclipse 90i microscope.
Cell free DNA (cfDNA) was determined in poor platelet plasma obtained by centrifugation of whole blood (2500 g for 15 min), using the Quant-iT™ Pico Green dsDNA assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.
To assess thrombin generation associated to NETs, 2.5x105 neutrophils from patients with SLE or from controls were incubated with either buffer or 100 nM PMA during 45 min. Then they were centrifuged at 5000g, 3 min and resuspended in 40-μL of rich platelet rich plasma (PRP) from healthy controls adjusted to 106 platelets/µL obtained from blood samples drawn either in citrate or citrate plus corn trypsin inhibitor (CTI) tubes. CTI is an inhibitor of FXIIa. Calibrated automated thrombogram (CAT) was performed without addition of any trigger.
Results: We observed that plasma from patients with SLE had increased free nucleic acids (cfDNA in fluorescence units, controls: 94.90±21.29, SLE patients: 112.4±26.59; P=0.0211). In accordance with this observation, analyses by fluorescence microscopy showed that neutrophils from SLE patients, but not from controls, had NETs even in basal conditions. Moreover, neutrophils from these patients generated more NETs in presence of 100 nM PMA (Figure 1).
To evaluate whether the increment of NETs observed in patients with SLE had consequences on the hemostasis of these patients, we tested thrombin generation of neutrophils from either patients with SLE or controls in the presence of platelets from healthy controls. Neutrophils from patients with SLE produced more thrombin than those from healthy controls under basal conditions and after stimulation with 100 nM PMA. These increments were avoided when PRP was collected from blood samples drawn with CTI (Figure 2).
Conclusions: Neutrophils from SLE patients without antiphospholipid antibodies and with no history of thrombotic seemed more prone to form NETs than those from healthy controls. NETs might be considered as a key element in the prothrombotic profile of patients with SLE and their analyses by thrombin generation test might be useful to detect risk of occurrence of thrombotic events in these patients and to prevent its occurrence by therapeutic management.
This work was supported by grants from FIS-FONDOS FEDER (PI19/00772). EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH).

Disclosures
Fernandez-Bello: Stago: Speakers Bureau; Pfizer: Speakers Bureau; SOBI,: Research Funding; Roche: Speakers Bureau; Novartis: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Current Employment, Research Funding, Speakers Bureau. Justo Sanz:Takeda: Current Employment. Alvarez Román:Bayer: Consultancy; Grifols: Research Funding; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau. García Barcenilla:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Speakers Bureau; NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Bayer: Speakers Bureau. Canales:Sandoz: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Karyopharm: Honoraria; Roche: Speakers Bureau; Takeda: Speakers Bureau; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Janssen: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Gilead: Honoraria; Janssen: Speakers Bureau; Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding. Butta:Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Grifols: Research Funding; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau.