Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of blood coagulation factor Xa (fXa) and factor VIIa. We have recently shown that fXa binding stimulates the uptake and degradation of cell surface-bound 125I-TFPI (Ho, G., Toomey, J. R., Broze, G. J., Jr., and Schwartz, A. L. (1996) J. Biol. Chem. 271, 9497-9502). In the present study we examined the role of cell surface glycosaminoglycans (GAGs) in this process. Removal of cell surface GAG chains by treatment of cells with heparinase or heparitinase but not chondroitinase markedly reduced fXa-stimulated 125I-TFPI uptake and degradation. Inhibition of GAG sulfation by growth of cells in chlorate-containing medium similarly decreased fXa-stimulated 125I-TFPI degradation. These results suggest that heparan sulfate proteoglycans (HSPGs) are required for the uptake and degradation of 125I-TFPI.fXa complexes. Chemical cross-linking/immunoprecipitation analyses revealed that 125I-TFPI was directly associated with HSPGs on the cell surface and that fXa binding increased the amount of 125I-TFPI bound. Of the several cell lines evaluated, bend endothelial cells demonstrated the greatest fXa stimulation of 125I-TFPI uptake and degradation. Cross-linking/immunoprecipitation analyses on bend cells also revealed that HSPGs were specifically associated with TFPI and fXa. These data suggest that HSPGs may directly act as the uptake and degradation receptor for TFPI.fXa complexes.