Antigenic characterization of endothelial cell-derived microparticles and their detection ex vivo

Abstract number: P1734

Abid Hussein^* M., Meesters† E. W., Osmanovic* N., Romijn F. P. H., Th‡ M., Nieuwland^* R., Sturk^* A.

*Academic Medical Center, Netherlands ^*Academic Medical Center, Netherlands ‡Leiden University, Netherlands †Slotervaart Hospital, Netherlands

Introduction  

Microparticles (MP) from various blood cells occur in the circulation. Recently, MP from endothelial cells (EC; EMP) were reported in various patient groups and healthy individuals. However, the antibodies used for the EMP detection by flow cytometry were mainly directed against antigens without EC specificity.

Aim  

Characterize the antigens on EC and EMP to establish reliable markers for EMP detection.

Methods  

EMP were isolated from supernatants of unstimulated and interleukin-1a activated human umbilical vein EC (HUVEC; n = 3; 0–72 h), stained with annexin V plus antibodies against CD31 (PECAM-1), CD51 (a_V), CD61 (b₃), CD62E (E-selectin) or CD142 (Tissue Factor [TF]), and analyzed by flow cytometry. Expression of these antigens was also determined on unstimulated and activated HUVEC. Human platelet-MP (PMP), the main MP population in plasma, were prepared in vitro. EMP and PMP were studied in plasma from SLE patients (n = 11) and healthy individuals (n = 10).

Results  

HUVEC constitutively exposed PECAM-1, a_v and b₃ 0. These antigens were (almost) absent on EMP (< 15% positive for a_V and b₃), or exposed only on a subpopulation (PECAM-1; 30–60%). Activated HUVEC (> 80%) and (subpopulations of) EMP exposed E-selectin and TF. PMP strongly exposed PECAM-1, b₃, but not a_v or E-selectin. Plasma samples contained 0.5% MP staining for E-selectin and/or a_V 0. Plasma from one SLE-patient contained E-selectin exposing MP (21%), but little a_V – positive MP.

Conclusions  

EC release EMP in vitro. The antigenic phenotype of EMP differs from EC and is activation dependent, and E-selectin is a valid marker for EMP detection ex vivo to establish endothelial cell activation.