Attachment of cultured human endothelial cells is promoted by specific association with S protein (vitronectin) as well as with the ternary S protein-thrombin-antithrombin III complex.
The interaction of the multifunctional S protein (vitronectin) with cultured human endothelial cells of macrovascular and microvascular origin was investigated. Purified S protein, coated on polystyrene Petri dishes, induced dose-dependent and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVECs) as well as human omental tissue microvascular endothelial cells (HOTMECs) at 37 degrees C. Not only isolated S protein, but also the ternary S protein-thrombin-antithrombin III (STAT) complex promoted attachment of approximately 90% of the cells within 2 hours at an S protein concentration of 0.13 mumol/L. Inhibition of attachment in these experiments was achieved by the addition of the cell-attachment pentapeptide Gly-Arg-Gly-Asp-Ser and by monospecific antibodies against S protein, whereas nonrelated peptides or antibodies against fibronectin, fibrinogen, or von Willebrand factor (vWF) were ineffective. Direct binding of S protein to HUVECs and HOTMECs was studied with cells in suspension at a density of 1 x 10(6) cells/mL and was maximal after 120 minutes. S protein bound to both cell types in a dose-dependent fashion with an estimated dissociation constant Kd = 0.2 mumol/L. At a 200-fold to 500-fold molar excess of unlabeled S protein, greater than 80% of bound radiolabeled S protein was displaceable, whereas binding was reduced to 30% to 50% by addition of the pentapeptide, the STAT complex, or by physiologic concentrations of fibrinogen or vWF as well as Fab fragments of anti(human S protein)IgG, but not by Fab rabbit IgG. These findings present evidence for the specific association of S protein with endothelial cells ultimately leading to attachment and spreading of cells. Moreover, a novel function for the ternary STAT complex, which induced endothelial cell attachment and spreading virtually identical to free S protein, is described. These data further suggest a possible role for S protein during coagulation as major vessel wall-related adhesive protein at sites of vascular injury.
D000954:Antigens, Surface / Q000502:physiology*; D000990:Antithrombin III / Q000378:metabolism; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology*; D006023:Glycoproteins / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D011971:Receptors, Immunologic / Q000378:metabolism; D019030:Receptors, Vitronectin; D013917:Thrombin / Q000378:metabolism; D019096:Vitronectin
D000954:Antigens, Surface; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D046911:Macromolecular Substances; D011971:Receptors, Immunologic; D019030:Receptors, Vitronectin; D019096:Vitronectin; D000990:Antithrombin III; D013917:Thrombin