Binding and endocytosis of heparin by human endothelial cells in culture

Abstract

Binding of heparin and low molecular weight heparin fragments (CY 222, M r range 1500–8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [ 125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [ 3H]heparin could be distinguished: one with high affinity ( K d = 0.12 μM) and another with lower affinity ( K d = 1.37 μM) and a relative large capacity of binding (1.16·10 7 molecules/cell) was calculated. The K d for unlabeled heparin, as calculated from competition experiments, was 0.23 μM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 ( K d = 4.3 μM) from competition experiments with [ 125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25–30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4°C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37°C, but not at 4°C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.