THROMBOGENICITY OF MICROPARTICLES DERIVED FROM VASCULAR CELLS

Abstract Microparticles (MPs) are released from cells during processes such as apoptosis or during cell activation. These MPs contain phospholipids, proteins and even nucleic acids derived from their parent cells. They are found circulating in plasma but also in tissues such as atherosclerotic plaques. It is thought that MPs contain and transfer tissue factor and can thereby induce blood clotting. In this study we analyzed clot promoting properties of MPs generated from vascular cells in vitro. MPs were generated from endothelial cells (EC), smooth muscles cells (SMC), monocytes (U937), erythrocytes (RBC) or platelets (Pl) by inducing apoptosis or by calcium ionophore activation; they were subsequently isolated by differential centrifugation. Thrombogenicity of the MPs was evaluated using a thrombin generation assay (Technothrombin® TGA) and MP free plasma as substrate. MPs displayed a different thrombin generating potential depending on the parent cells. MPs derived from RBCs (~400nM peak thrombin/105 MPs/ml plasma), ECs (~300nM), SMCs (~300nM) and Pls (~300nM) were more thrombogenic than MPs derived from U937 (~200 nM). In addition EC, SMC and U937 MPs all expressed tissue factor but EC MPs induced thrombin generation in a tissue factor and FVII independent manner. EC MPs even expressed active tissue factor pathway inhibitor and functionally inhibited tissue factor dependent thrombin generation. Since the higher thrombin generation induced by MPs derived from EC as compared U937 derived MPs could not be explained by a different activity of tissue factor, we were interested whether lipids contained in the microparticles could account for the differences in thrombin generation. We therefore analyzed thrombin generation induced by lipids isolated from MPs and parent cells and could show that lipids from EC MPs and SMC MPs exhibited higher thrombin generation than those from U937 MPs. Upon analysis of lipids by thin layer chromatography and mass spectrometry we found that in general microparticles are enriched in cholesterol, sphingomyeline and phosphatidylserine over the parent cells and that EC and SMC MPs were enriched in negatively charged phospholipids (different species of phosphatidylserine and phosphatiylglycerol) as compared to MPs derived from U937 cells. When thrombogenicity was, however, evaluated in vivo by injecting MPs into mice it was found that the highest capability to induce thrombin-antithrombin (TAT) complexes had MPs derived from SMCs; also U937 MPs induced an increase in TAT levels, while EC MPs – although more thrombogenic than U937 MPs in vitro – did not induce TAT complex formation by themselves but were only synergistic in vivo. From these data we conclude that thrombin formation in vivo depends on the initiation of the tissue factor FVII pathway, while the extent of thrombin formation is dependent on negatively charged phospholipids contained to a higher extent in MPs derived e.g. from ECs.