title: Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C.
abstract: 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.
authors.affiliation: Department of Haematology, University Hospital Utrecht, The Netherlands.
authors.forename: T M
authors.initials: TM
authors.lastname: Hackeng
authors.forename: M
authors.initials: M
authors.lastname: Hessing
authors.forename: C
authors.initials: C
authors.lastname: van 't Veer
authors.forename: F
authors.initials: F
authors.lastname: Meijer-Huizinga
authors.forename: J C
authors.initials: JC
authors.lastname: Meijers
authors.forename: P G
authors.initials: PG
authors.lastname: de Groot
authors.forename: J A
authors.initials: JA
authors.lastname: van Mourik
authors.forename: B N
authors.initials: BN
authors.lastname: Bouma
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D011486:Protein C; D017293:Protein S; D015943:Factor Va