Summary
Activated protein C (APC) is an antithrombotic serine proteinase having anticoagulant,
profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility,
relatively little is known about its clearance mechanisms. In the present study we
have characterized the interaction of APC and its active site blocked forms with human
umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner.
Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total
number of binding sites per cell of 359,000. Similar binding isotherms were obtained
using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone
(PPACK) inhibited APC indicating that a functional active site was not required. Competition
studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting
that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic
acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain
was essential for cell binding. Surprisingly, APC binding to these cells appeared
to be independent of protein S, a cofactor of APC generally thought to be required
for its high affinity binding to cell surfaces. The identity of the cell binding site(s),
for the most part, appeared to be distinct from other known APC ligands which are
associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin,
factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment
of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding.
Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25%
of the initially bound radioligand with an endocytic receptor. However, most of the
radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly
at 37° C in an intact and functional state. These data indicate the presence of specific,
high affinity binding sites for APC and PC on the surface of HUVEC. While a minor
proportion of binding sites may be involved in endocytosis, the identity and function
of the major proportion is presently unknown. It is speculated that this putative
receptor may be a further mechanisms of localizing the PC antithrombotic system to
the vascular endothelium.
