PO-47 - Microparticles derived from ovarian cancer cell line contained genomic and biologically active proteins, including tissue factor involved in coagulation.

Introduction: The microparticles (MPs) are sized vesicles of less than 1 μm, from different cell types upon activation or subsequent to apoptosis. They are involved in the thrombotic process, particularly in cancer. The role of MPs in ovarian cancer and their involvement in thrombosis being poorly understood.

Aim: The aim of this study was to identify in vitro the generation of MPs by an human ovarian adenocarcinoma cell line (OVCAR-3).

Materials And Methods: OVCAR-3 cells were cultured in three conditions [without stimulation, with protein C (PC), and with activated protein C (APC)]. Then, the MPs present in the supernatant, were isolated by ultracentrifugation and were analyzed for their shape and properties by flow cytometry, electron microscopy, cryofracture analysis, DNA and RNA, and proteomic analysis. The level of tissue factor (TF) on MP was evaluated by TF-induced shortening of Ca(2+) plasma coagulation time.

Results: Our results demonstrated that 1) 92% of MPs derived from OVCAR-3 were less than 100 nm. 2) As tested by flow cytometry, the MPs contained b2 microglobulin, annexin, DNA fragments and TF that induces a shortening of Ca(2+) -induced plasma coagulation time. When OVCAR-3 were cultured for 18H with PCA, MPs were generated in greater amount than those generated by OVCAR-3 in its absence and their level of TF was increased of 20%. Curiously, in contrast with intact OVCAR-3 cells, the endothelial protein C receptor (EPCR) was not detected in MPs 3) Proteomic analysis show that the MPs contain proteins involved in cancer progression such as mucins (5A and 5B), keratin type-1, actin, annexin (A1, A2, A4), CD44, glypican, heat shock (70kDa and HS90a) proteins, Agrin associated with heparan sulfate proteoglycan abundant in the tumor-specific basement membrane, Ephrin type A receptor, coronin-1C, catenin α, integrin β-1 and also p-selectin responsible of platelet activation. They also express several DNA associated proteins includingtranscription factors, various polymerases, nucleases, and histones involved in chromosome packaging and transcription in the cell nucleus.

Conclusions: MPs derived from OVCAR-3 have an apoptotic character. They expressed several biologically active proteins, DNA and their associated proteins. Despite the absence of EPCR expression on MPs that was expressed on intact OVCAR-3 cells, they expressed procoagulant TF activity already found on intact ovarian cancer cells. This activity is greater extent in the presence of APC.