Visualization of macro-immune complexes in the antiphospholipid syndrome by multi-modal microscopy imaging

The antiphospholipid syndrome (APS) is an autoimmune thrombotic condition that is marked by autoantibodies against phospholipid-binding proteins. The mechanism(s) of thrombogenesis has (have) resisted elucidation since its description over thirty years ago. Nevertheless, a defining aspect of the disorder is positivity for clinical laboratory tests that confirm antibody binding to anionic phospholipids. It is remarkable that, to our knowledge, the binding of proteins from plasmas of APS patients to phospholipid has not been previously imaged. We therefore investigated this with high resolution microscopy-based imaging techniques that have not been previously used to address this question, namely atomic force microscopy and scanning electron microscopy. Atomic force microscopy imaging of APS plasmas incubated on an anionic planar phospholipid layer revealed the formation of distinct complex three-dimensional structures, which were morphologically dissimilar to structures formed from control plasmas from healthy patients. Likewise, scanning electron microscopy analysis of phospholipid vesicles incubated with APS plasmas in suspension showed formation of layered macro-immune complexes demonstrated by the significant agglomeration of a complex proteinaceous matrix from soluble plasma and aggregation of particles. In contrast, plasmas from healthy control samples bound to phospholipid vesicles in suspension generally displayed a more flattened, mat-like appearance by scanning electron microscopy. Scanning electron microscopy of plasma samples incubated on planar phospholipid layers and previously imaged by atomic force microscopy, corroborated the results obtained by mixing the plasmas with phospholipids in solution. Analysis of the incorporated proteins by silver stained SDS-polyacrylamide gel electrophoresis indicated considerable heterogeneity in the composition of the phospholipid vesicle-adsorbed proteins among APS patients. To our knowledge, these results provide the first images of plasma-derived APS immune complexes at high resolution, and show their consistent presence and heterogeneous compositions in APS patients. These findings demonstrate how high resolution microscopic techniques can contribute to advancing the understanding of an enigmatic disorder and may lay additional groundwork for furthering mechanistic understanding of APS.