Circulating microparticles in patients with antiphospholipid antibodies: characterization and associations

Abstract

The antiphospholipid syndrome is characterized by venous or arterial thrombosis and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies. These antibodies cause activation of endothelial and other cell types leading to the release of microparticles with procoagulant and pro-inflammatory properties. The aims of this study were to characterize the levels of endothelial cell, monocyte or platelet derived, and tissue factor-bearing microparticles in patients with antiphospholipid antibodies, to determine the association of circulating microparticles with anticardiolipin and anti-β2-glycoprotein antibodies, and to define the cellular origin of microparticles that express tissue factor. Microparticle content within citrated blood from 47 patients with antiphospholipid antibodies and 144 healthy controls was analyzed within 2hours of venipuncture. Levels of Annexin-V, CD105 and CD144 (endothelial derived), CD41 (platelet derived) and tissue factor positive microparticles were significantly higher in patients than controls. Though levels of CD14 (monocyte-derived) microparticles in patient plasma were not significantly increased, increased levels of CD14 and tissue factor positive microparticles were observed in patients. Levels of microparticles that stained for CD105 and CD144 showed a positive correlation with IgG (R=0.60, p=0.006) and IgM anti-beta2-glycoprotein I antibodies (R=0.58, p=0.006). The elevation of endothelial and platelet derived microparticles in patients with antiphospholipid antibodies and their correlation with anti-β2-glycoprotein I antibodies suggests a chronic state of vascular cell activation in these individuals and an important role for β2-glycoprotein I in development of the pro-thrombotic state associated with antiphospholipid antibodies.

Keywords: Antiphospholipid; Endothelial cell; Microparticles; Platelet; Thrombosis.

Figure 1. Levels of circulating microparticles in patients and controls

Levels of microparticles derived from endothelial cells (CD105 and CD144), platelets (CD41), monocytes (CD14) or those staining with an anti-tissue factor antibody were measured in fresh platelet-free plasma by flow cytometry as described in Materials and Methods.

Figure 2. Correlations between endothelial cell-derived microparticles and anti-β2GPI and anti-cardiolipin antibody levels

The correlations between endothelial cell-derived microparticles and IgG anti-β2GPI or anticardiolipin antibody levels, or annexin V staining, were determined. Significant correlations were seen between microparticle levels and anti-β2GPI antibodies, and microparticle levels and annexin V staining. Similar correlations between microparticle levels and IgM anti-β2GPI antibodies were observed (see text).

Figure 3

Levels of tissue factor positive microparticles. (A) Microparticles from patients and controls were stained with an antibody to tissue factor and analyzed by flow cytometry. This figure depicts the tissue factor positivity of microparticles from individual patients and controls. (B) Levels of tissue factor microparticles by cell type. Microparticles were double stained with a cell-specific antibody and antibody against tissue factor, as described in Materials and Methods, and analyzed by flow cytometry. This figure depicts the total as well as the percentages of tissue factor positive microparticles derived from endothelial cells, platelets or monocytes. (C) Confocal micrograph (× 63) of a single microparticle in patient plasma staining positively for tissue factor (green, FITC), CD14 (red-PE) or overlay after double staining. For size comparison, two fluorescent 1 µM flow cytometry beads are depicted.