HIGH-AFFINITY BINDING-SITES FOR ACTIVATED PROTEIN-C AND PROTEIN-C ON CULTURED HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS - INDEPENDENT OF PROTEIN-S AND DISTINCT FROM KNOWN LIGANDS

Activated protein C (APC) is an antithrombotic serine proteinase havin g anticoagulant, profibrinolytic and anti-inflammatory activities. Des pite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized t he interaction of APC and its active site blocked forms with human umb ilical vein endothelial eels (HUVEC). ht 4 degrees C I-125-APC bound t o HUVEC in a specific, time dependent, saturable and reversible manner . Scatchard analysis of the binding, isotherm demonstrated a K-d value of 6.8 nM and total number of binding sites per cell of 359,000. Simi lar binding isotherms were obtained using radioiabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competi tion studies showed that the binding of APC, PPACK-APC and PC were mut ually exclusive suggesting that they bound to the same site(s). Proteo lytic removal of the N-terminal gamma-carboxyglutamic acid (gla) domai n of PC abolished its ability to compete indicating that the gla-domai n was essential for cell binding. Surprisingly, APC binding to these c ells appeared to be independent of protein S, a cofactor of APC genera lly thought to be required for its high affinity binding to cell surfa ces. The identity of the cell binding site(s), for the most part, appe ared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, th rombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caus ed partial inhibition of I-125-APC binding indicating that factor VIII or a homolog accounted for similar to 30% of APC binding. Studies of the properties of surface bound I-125-APC or I-125-PC and their fate a t 4 degrees C compared to 37 degrees C were consistent with associatio n of similar to 25% of the initially bound radioligand with an endocyt ic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37 degrees C in a n intact and functional state. These data indicate the presence of spe cific, high affinity binding sites for APC and PC on the surface of HU VEC. While a minor proportion of binding sites may be involved in endo cytosis, the identity and function of the major proportion is presentl y unknown. It is speculated that this putative receptor may be a furth er mechanisms of localizing the PC antithrombotic system to the vascul ar endothelium.