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Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C. 1993

T M Hackeng, and M Hessing, and C van 't Veer, and F Meijer-Huizinga, and J C Meijers, and P G de Groot, and J A van Mourik, and B N Bouma
Department of Haematology, University Hospital Utrecht, The Netherlands.

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.

UI MeSH Term Description Entries
D007457 Iodine Radioisotopes Unstable isotopes of iodine that decay or disintegrate emitting radiation. I atoms with atomic weights 117-139, except I 127, are radioactive iodine isotopes. Radioisotopes, Iodine
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D011486 Protein C A vitamin-K dependent zymogen present in the blood, which, upon activation by thrombin and thrombomodulin exerts anticoagulant properties by inactivating factors Va and VIIIa at the rate-limiting steps of thrombin formation.
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D004730 Endothelium, Vascular Single pavement layer of cells which line the luminal surface of the entire vascular system and regulate the transport of macromolecules and blood components. Capillary Endothelium,Vascular Endothelium,Capillary Endotheliums,Endothelium, Capillary,Endotheliums, Capillary,Endotheliums, Vascular,Vascular Endotheliums
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000911 Antibodies, Monoclonal Antibodies produced by a single clone of cells. Monoclonal Antibodies,Monoclonal Antibody,Antibody, Monoclonal
D013379 Substrate Specificity A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts. Specificities, Substrate,Specificity, Substrate,Substrate Specificities
D015943 Factor Va Activated form of factor V. It is an essential cofactor for the activation of prothrombin catalyzed by factor Xa. Coagulation Factor Va,Factor V, Activated,Blood Coagulation Factor V, Activated,Factor 5A,Factor Five A,Activated Factor V,Factor Va, Coagulation
D017293 Protein S The vitamin K-dependent cofactor of activated PROTEIN C. Together with protein C, it inhibits the action of factors VIIIa and Va. A deficiency in protein S; (PROTEIN S DEFICIENCY); can lead to recurrent venous and arterial thrombosis. Vitamin K-Dependent Protein S,Cofactor Protein S,Protein S, Vitamin K-Dependent,Protein S, Cofactor,Protein S, Vitamin K Dependent,Vitamin K Dependent Protein S

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