Platelets secrete a low-molecular-weight protein, platelet factor four (PF-4), which binds to and neutralizes heparin and related sulfated glycosaminoglycans (GAGs). To examine the interactions of PF-4 with the GAGs present on endothelial cell surfaces, we incubated 125I-PF-4 with cell suspensions derived from confluent monolayers of cultured bovine aortic endothelium. Binding of 125I-PF-4 was inhibited by a 100-fold excess of nonradioactive PF-4 and varied with duration and temperature of incubation. At 4 degrees C, binding reached equilibrium at 20 minutes with kd = 2.87 mumol/L and Bmax of 63.83 pmol/10(5) cells. Binding capacity was reduced 83.4% by brief incubation of endothelial cells with trypsin and 46.67% by incubation with Flavobacterium heparinase, but was unchanged by chondroitin-ABCase treatment. At 37 degrees C, PF-4 was internalized by confluent monolayer of bovine aortic endothelial cells primarily through low-affinity adsorptive endocytosis. The internalized PF-4 was degraded to amino acids and small peptides with 50% conversion after 18-hour incubation. These studies demonstrate that a secreted platelet protein can bind to and enter endothelial cells. Binding may explain the rapid clearance of released PF-4 from plasma and could have important local effects on endothelial structure and function.