In systemic lupus erythematosus anti-dsDNA antibodies can promote thrombosis through direct platelet activation

Highlights

• Anti-dsDNA Abs from the blood of SLE patients activate platelets trough FcγRIIA.

• Anti-dsDNA-Abs induce platelet degranulation and P-selectin expression.

• Immune platelet activation includes rearrangement of cytoskeletal proteins.

• Anti-dsDNA-Abs cause platelet microvesiculation and cytoplasmic disintegration.

• Immune platelet activation is a prothrombotic mechanism in SLE.

Abstract

Systemic lupus erythematosus (SLE) is associated with a high risk of venous and arterial thrombosis, not necessarily associated with prothrombotic antiphospholipid antibodies (Abs). Alternatively, thrombosis may be due to an increased titer of anti-dsDNA Abs that presumably promote thrombosis via direct platelet activation. Here, we investigated effects of purified anti-dsDNA Abs from the blood of SLE patients, alone or in a complex with dsDNA, on isolated normal human platelets. We showed that anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes induced strong platelet activation assessed by enhanced P-selectin expression and dramatic morphological and ultrastructural changes. Electron microscopy revealed a significantly higher percentage of platelets that lost their discoid shape, formed multiple filopodia and had a shrunken body when treated with anti-dsDNA Abs or anti-dsDNA Ab/dsDNA complexes compared with control samples. In addition, these platelets activated with anti-dsDNA Ab/dsDNA complexes typically contained a reduced number of secretory α-granules that grouped in the middle and often merged into a solid electron dense area. Many activated platelets released plasma membrane-derived microvesicles and/or fell apart into subcellular cytoplasmic fragments. Confocal microscopy revealed that platelets treated with anti-dsDNA Ab/dsDNA complex had a heterogeneous distribution of septin2 compared with the homogeneous distribution in control platelets. Structural perturbations were concomitant with mitochondrial depolarization and a decreased content of platelet ATP, indicating energetic exhaustion. Most of the biochemical and morphological changes in platelets induced by anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes were prevented by pre-treatment with a monoclonal mAb against FcγRIIA. The aggregate of data indicates that anti-dsDNA Abs alone or in a complex with dsDNA strongly affect platelets via the FcγRIIA receptor. The immune activation of platelets with antinuclear Abs may comprise a prothrombotic mechanism underlying a high risk of thrombotic complications in patients with SLE.

Introduction

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease associated with immune inflammation of various organs and tissues (joints, kidney, skin, central nervous system, etc.). In SLE the immune system works against various autologous cellular antigens, including cytoplasmic proteins, cell surface, and nuclear components such as DNA and histones [1,2].

Elevated levels of anti-dsDNA antibodies (Abs) in the blood is a characteristic sign of SLE, but the titer of anti-dsDNA Abs can vary during the disease depending on the disease activity [3]. Anti-dsDNA Abs form complexes with DNA that stimulate the peripheral blood mononuclear cells through toll-like receptors 9 followed by production of interferon-α, a strong pro-inflammatory factor [[4], [5], [6]]. Platelets also can be potentially activated by DNA-containing immune complexes through the FcγRIIA receptor [7] or by free anti-DNA Abs through the β3 subunit of the integrins αIIbβ3 and αvβ3 [8].

SLE patients have an increased risk of venous and arterial thrombosis [9], which is one of the major causes of mortality in SLE. Cerebrovascular accidents, coronary occlusions, and pulmonary embolisms are the most common thrombotic events during SLE [10]. It has been shown that thrombosis during SLE is strongly associated with the presence of antiphospholipid Abs in the blood [11]. However, about 20% of SLE patients with thrombosis are negative for antiphospholipid Abs [12], suggesting that antiphospholipid Abs are not the only pathogenic factor responsible for the thrombosis during SLE. It is likely that the predisposition to thrombosis in SLE can be due to chronic platelet activation in the blood stream [13] followed by expression of procoagulant phosphatidylserine [14] and secretion of prothrombotic compounds assessed by increased expression of P-selectin [13]. Platelet activation in SLE is associated with complement deposition on cells that can activate platelets in combination with inflammatory cytokines [7,14]. Activated platelets can be a source of the soluble CD40 ligand, which can secondarily promote activation of antigen-presenting cells, including monocytes and plasmacytoid dendritic cells. These cellular reactions are critically important for the pathogenesis of SLE because they lead to the production of autoantibodies, including anti-DNA Abs that can aggravate the disease [7]. Importantly, platelet-mediated activation of plasmacytoid dendritic cells promotes the secretion of interferon-α [7] that may influence megakaryocytes, so that platelets derived from these megakaryocytes are pre-activated [15]. In the blood of SLE patients, platelet-monocyte complexes are formed [16], suggesting that activated platelets bearing the CD40 ligand (CD154) can trigger monocytes through CD154-CD40 interaction. Moreover, activated platelets can cross-activate neutrophils that form prothrombotic neutrophil extracellular traps (NETs) [17,18]. Despite strong evidence for involvement of platelets and anti-DNA Abs in the pathogenesis of SLE, many aspects of platelet activation and its relation to DNA-containing immune complexes remain unknown. Very little is known about ultrastructural changes of platelets in SLE and whether anti-dsDNA Abs or their complexes with dsDNA can induce these changes.

Here we studied functional and structural alterations of normal isolated human platelets induced by purified SLE-related anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes to investigate mechanisms of immune platelet activation, including a role of the FcγRIIA receptor. The results show that anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes affect platelet morphology, ultrastructure, septin 2 intracellular distribution, and lead to the α-granule secretion, indicating strong platelet activation followed by their energetic exhaustion. These changes are lessened substantially in the presence of an anti-FcγRIIA Ab, implying that the FcγRIIA receptors mediate the pathogenically important pro-thrombotic interactions between platelets and anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes.