Insights into the Procoagulant Profile of Patients with Systemic Lupus Erythematosus without Antiphospholipid Antibodies

Abstract

We aimed to identify the key players in the prothrombotic profile of patients with systemic lupus erythematosus (SLE) not mediated by antiphospholipid antibodies, as well as the potential utility of global coagulation tests to characterize hemostasis in these patients. Patients with SLE without antiphospholipid antibodies and without signs of thrombosis were included. The kinetics of clot formation were determined by ROTEM^®. Platelet activation markers were determined by flow cytometry. Thrombin generation associated with Neutrophil Extracellular Traps (NETs) and microparticles (MPs) was measured by calibrated automated thrombogram (CAT). The plasma levels of PAI-1 were also determined. ROTEM^® showed a procoagulant profile in SLE patients. SLE patients had activated platelets and more leukocyte/platelet aggregates at basal conditions. The plasma PAI-1 and platelet aggregates correlated with several ROTEM^® parameters. The thrombin generation associated withthe tissue factor (TF) content of MPs and with NETs was increased. Our results suggest the utility of global tests for studying hemostasis in SLE patients because they detect their procoagulant profile, despite having had neither antiphospholipid antibodies nor any previous thrombotic event. A global appraisal of hemostasis should, if possible, be incorporated into clinical practice to detect the risk of a thrombotic event in patients with SLE and to consequently act to prevent its occurrence.

Keywords: neutrophil extracellular traps; systemic lupus erythematosus; thrombin generation; thromboelastometry.

Figure 1

Procoagulant profile in patients with SLE. ROTEM^® thromboelastography was performed in whole blood. Detailed procedures and measured parameters are shown in “Materials and Methods”. Student’s t-test or Mann–Whitney test was performed, and p ≤ 0.05 was set as significant. SLE, systemic lupus erythematosus; CT, clotting time; A15, amplitude at 15 min; MCF, maximum clot firmness.

Figure 2

Basal activity of fibrinogen receptor, surface exposition of P-selectin, platelet/leukocyte aggregate formation and PS exposure. (A) PRP from healthy controls and patients with SLE was incubated with either FITC-PAC1, FITC anti-P-selectin mAb, or FITC anti-CD63 mAb. (B) To test the platelet/leukocyte aggregates, whole blood was incubated with PE anti-CD41 mAb and FITC-anti-CD45 mAb. (C) Annexin V binding was tested in washed platelets resuspended in the adequate buffer (see Methods). Samples were analyzed by flow cytometry. The Mann–Whitney test was performed, and p ≤ 0.05 was considered significant. Data are expressed as arbitrary units (mean fluorescence ×% of positive cells (A), mean fluorescence of leukocytes positive for CD41 (B), or percentage of positive cells (C)).

Figure 3

Plasminogen activator inhibitor-1 (PAI-1) levels in plasma and its correlation with ROTEM^® parameters. Plasma levels of PAI-1 (A) measured with enzyme-linked immunosorbent assay correlated with A15 and MCF parameters (B). A Mann–Whitney test and Spearman’s correlation were performed, and p < 0.05 was considered significant.

Figure 4

Formation of NETs.NETs were evaluated in basal conditions and after stimulation with 100 nMof PMA. DNA (DAPI, blue), neutrophil myeloperoxidase (MPO, FITC, green), and DAPI/FITC merge images areshown. Original magnification ×10.

Figure 5

Thrombin generation associated with NETs. The effect of NETs on the thrombin generation was tested in either non-stimulated (A) or 100 nM of PMA-stimulated neutrophils (B) in the presence of PRP from healthy controls adjusted to 1 × 10⁵ platelets/µL, with (+) or without (−) CTI. Detailed procedures are explained in “Materials and Methods”. A Mann–Whitney test was performed, and p < 0.05 was considered significant.