Soluble tissue factor released by cultured human umbilical vein endothelial cells is microparticle-associated and triggers thrombus formation in vivo

Abstract number: OC008

Abid Hussein^* M., Biró^* É., Meesters† E. W., Osmanovic^* N., Romijn‡ F. P. H. TH. M., Vogel§ G. M. T., Meuleman§ D. G., Sturk^* A., Nieuwland^* R.

^*Academic Medical Center, Netherlands; ‡Department of Clinical Chemistry, Leiden Universit, Nepal; §Section General Pharmacology, Organon, Netherlands †Slotervaart Hospital, Netherlands;

Introduction  

Human plasma contains noncell-bound soluble TF (sTF). In vitro, endothelial cells release microparticles (MP) exposing functional TF.

Aim  

In this study we investigated whether sTF is associated with MP of endothelial origin (EMP) and whether such EMP-associated TF triggers thrombus formation and occurs in vivo.

Methods  

Human umbilical vein endothelial cells (HUVEC; n= 3) were incubated with or without interleukin (IL)-1(a) (5 ng mL^-1) for 0–72 h and analyzed by flow cytometry for TF exposure. sTF in the culture supernatants was assessed by ELISA. Association between sTF and EMP in culture supernatants was determined using flow cytometry and Western blotting. The coagulant activity of sTF was studied in a thrombin generation assay in vitro and in a rat venous thrombosis model in vivo. The occurrence of EMP-associated TF was also studied in plasma samples from SLE patients (n = 11) and healthy individuals (n = 10).

Results  

Activated HUVEC transiently exposed TF. sTF appeared in the culture supernatants, which was entirely EMP associated. On Western blot, TF from both HUVEC and EMP showed as a single 47 kDa band. EMP-associated TF induced factor VII-dependent thrombin generation in vitro and thrombus formation in a rat thrombosis model (thrombus weight 35.1 ± 12.9 mg, n = 6; control 0.5 ± 0.7 mg, n = 6), which was inhibited by antihuman TF (5.6 ± 9.3 mg, P = 0.013; n = 6) but not by antihuman factor XII (26.9 ± 9.2 mg, P > 0.05; n = 6). Plasma from one SLE patient clearly contained EMP exposing TF.

Conclusions  

Endothelial cells release full-length TF which is EMP-associated, initiates thrombus formation and occurs in vivo.