Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926.
Protein C activation by alpha-thrombin on the surface of endothelial cells depends on an essential membrane-glycoprotein cofactor, thrombomodulin. In the present study we have monitored the activity of thrombin-thrombomodulin complexes on human saphenous-vein endothelial cells (HSVEC) or on the endothelial cell line EA.hy 926. Cell monolayers were exposed for 5 min to 8.5 nM human alpha-thrombin and then washed to remove unbound thrombin. The cells were then incubated at 37 degrees C for 5-180 min. At the end of the respective incubation periods, purified human protein C (120 nM) was added in order to assay the activity of the thrombin-thrombomodulin complexes present on the cell surface. HSVEC pre-exposed to thrombin retained their full capacity to promote protein C activation up to 90 min after free thrombin was removed. This capacity then decreased slowly to reach 56% of control value after 180 min of incubation. Original activity was 3.8 +/- 0.9 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 5). The capacity of protein C activation of EA.hy 926 cells remained constant for 120 min after free thrombin was removed, then decreased to 76% of control after 180 min. Original activity was 2.0 +/- 0.4 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 3). Similar results were obtained with cells fixed with 3% paraformaldehyde. However, during the 5-180 min incubation period, non-fixed cells of both types were capable of significantly internalizing fluorescent acetylated low-density lipoprotein. In the experimental protocol used here, an eventual inhibition of thrombin internalization by protein C can be excluded, as protein C is only added at the end of the incubation period. We conclude that there is no evidence of rapid internalization of thrombin-thrombomodulin complexes on HSVEC or the EA.hy 926 cell line, as assessed by the ability of membrane-bound thrombin to activate protein C.
Beretz
A
A
INSERM U.311, Service d'Hémostase et de Thrombose, Centre Régional de Transfusion Sanguine, Strasbourg, France.
Freyssinet
J M
JM
Gauchy
J
J
Schmitt
D A
DA
Klein-Soyer
C
C
Edgell
C J
CJ
Cazenave
J P
JP
D002460:Cell Line; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D011486:Protein C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D012501:Saphenous Vein / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013917:Thrombin