Inverted Erythrocyte Membranes Are a Novel Platform for Expressing Phosphatidylserine and Investigating the Antiphospholipid Syndrome
Authors: Bloemen Saartje , Wu Xiao Xuan , De Laat Bas , Rand Jacob H. , Vasovic Ljiljana V. 
Published: October 2019

Abstract
Background: The antiphospholipid (aPL) syndrome (APS) is an acquired thrombophilic disorder in which autoantibodies target proteins bound to anionic phospholipids; the mechanisms involved have not yet been clearly established. Anionic phospholipids are normally present in the internal leaflets of cytoplasmic membranes and become expressed after cell injury or death. Current diagnosis of APS is based on a panel of non-mechanistic "criteria" tests, which consist of the paradoxical lupus anticoagulant (LA) and immunoassays for autoantibodies binding to anionic phospholipids (cardiolipin) or β2glycoprotein I. Also, there has been difficulty in identifying biologically relevant and robust phospholipid platforms for analyzing aPL-membrane interactions. We therefore decided upon a novel path by: 1) developing a cell-based model consisting of inverted erythrocyte membranes (iEMs) and 2) developing novel ways of detecting aPL-iEM interactions.
Materials & Methods: iEMs were prepared from erythrocytes using an established protocol, suspended in 20 mOsm Tris buffer and used fresh or stored at -80°C. iEMs were incubated with normal or patient plasmas for a minimum of 30 minutes and washed by centrifugation at 15,000g. Binding of IgG to iEMs was investigated by goat anti-human IgG-Alexa 633 (GαH IgG-Alexa 633) in flow cytometry. PS exposure on membranes was confirmed by microscopy with annexin A5 (A5)-Oregon green and A5-Alexa 647 binding in flow cytometry. Bright field images of aggregated membranes were obtained to visually assess aggregation. The potential of iEMs to serve as phospholipids necessary for coagulation was assessed through calibrated automated thrombinography.
Results: Incubation of iEMs with normal and aPL plasmas resulted in increased IgG binding to the iEMs, as determined by flow cytometry (figure 1A). The IgG staining was more pronounced with aPL compared to normal plasmas (mean fluorescence ± standard deviation; aPL (n = 3): 10004 ± 5416 versus healthy (n = 3): 4055 ± 1680). When studying these samples with bright field microscopy aggregate formation of iEMs was observed. The aggregates induced by aPL plasma were more numerous and larger than those forming in plasma of healthy donors (figure 2A-B). Amplified agglutination was observed in the presence of broad spectrum anti-hIgG/anti-hC3d reagent (figure 2C-D).
Fluorescence microscopy of iEMs incubated with A5-OG confirmed that a population of iEMs was exposing PS. Thrombin generation (TG) studies established that iEMs contribute to coagulation reactions by providing a source of anionic phospholipids. TG increased dose-dependently with higher levels of iEMs until a plateau concentration was reached. Additionally, A5 resistance was investigated by means of flow cytometry. The aPL plasmas induced a decrease in A5 binding to iEMs compared to normal plasma and the control conditions (figure 1B), consistent with the functional A5 resistance assay.
Discussion: We have developed a novel model for a more physiological approach to phospholipid based assays in APS using iEMs as a source of anionic phospholipids. They are easily prepared by hypotonic lysis of erythrocytes and can be frozen for storage until use.
We have thus far demonstrated that a higher level of iEM aggregation is induced by aPL samples compared to healthy plasmas. Moreover, iEMs expose PS and dose-dependently accelerate TG by functioning as a platform for coagulation. Previous findings on A5 resistance induced by aPL plasma are confirmed with this iEM model.
Conclusion: IEMs offer a novel and robust biologically-derived platform; they express PS, promote coagulation, bind aPL antibodies and open different approaches to investigate APS.
Figure 1. Flow cytometry analysis of iEMs incubated with patient (red) and plasmas from healthy donors (blue). (A) GαH IgG-Alexa 633 binding for iEMs incubated with APL versus healthy plasma; (B) A5 resistance with less A5-Alexa 647 binding iEMs incubated with APS plasma compared to healthy plasma. Figure 1. Flow cytometry analysis of iEMs incubated with patient (red) and plasmas from healthy donors (blue). (A) GαH IgG-Alexa 633 binding for iEMs incubated with APL versus healthy plasma; (B) A5 resistance with less A5-Alexa 647 binding iEMs incubated with APS plasma compared to healthy plasma. Figure 2. iEMs incubated with aPL plasma (B, D) and plasma from a healthy donor (A, C) visualized with bright field microscopy. Aggregate formation seen after incubation with aPL plasma (B) is hardly seen with normal plasma (A); Agglutinate formation after addition of anti-hIgG/anti-hC3d (C, D) is amplified in aPL plasma (D) compared to normal plasma (C). Examples of aggregates are indicated with white arrows. Figure 2. iEMs incubated with aPL plasma (B, D) and plasma from a healthy donor (A, C) visualized with bright field microscopy. Aggregate formation seen after incubation with aPL plasma (B) is hardly seen with normal plasma (A); Agglutinate formation after addition of anti-hIgG/anti-hC3d (C, D) is amplified in aPL plasma (D) compared to normal plasma (C). Examples of aggregates are indicated with white arrows.

Disclosures
De Laat: Synapse bv: Employment.