Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C.
An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of protein S toward factor Va inactivation by activated protein C (APC). The initial rate of endothelial cell-mediated factor Va inactivation was 21.7 pM factor Va/50 pM APC min-1, which could be enhanced twice at a protein S concentration of 5 nM. This increase appeared to be specific for protein S because it could be inhibited by C4b-binding protein and polyclonal antibodies against protein S. Furthermore, thrombin-cleaved protein S did not accelerate factor Va inactivation by APC on endothelial cells. The binding of 125I-protein S to endothelial cells was time-dependent, specific, saturable, and required the presence of calcium ions. Scatchard analysis revealed (8.0 +/- 0.3) x 10(5) binding sites per cell with an apparent Kd of 24.4 +/- 2.2 nM. To study the physiological importance of the binding of protein S to human endothelial cells, seven monoclonal antibodies were examined for their ability to influence the protein S cofactor activity and binding capacity. Monoclonal antibodies directed against the gamma-carboxyglutamic acid domain and the thrombin-sensitive region of protein S completely inhibited the protein S cofactor function in factor Va inactivation by APC on HUVECs. These monoclonal antibodies also inhibited 125I-protein S binding to HUVECs. Another monoclonal antibody, directed against an epitope on the third and/or fourth epidermal growth factor-like region, did not influence either protein S cofactor activity or binding of protein S to HUVECs. We conclude that binding of protein S to HUVECs is essential for the expression of its cofactor activity for APC. At least two regions in protein S, the gamma-carboxyglutamic acid domain and the thrombin-sensitive region, are involved in the expression of cofactor activity.
Hackeng
T M
TM
Department of Haematology, University Hospital Utrecht, The Netherlands.
Hessing
M
M
van 't Veer
C
C
Meijer-Huizinga
F
F
Meijers
J C
JC
de Groot
P G
PG
van Mourik
J A
JA
Bouma
B N
BN
D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015943:Factor Va / Q000037:antagonists & inhibitors; D006801:Humans; D007457:Iodine Radioisotopes; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D017293:Protein S / Q000378:metabolism*; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D011486:Protein C; D017293:Protein S; D015943:Factor Va