Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and subendothelial matrix components is mediated in part via thrombospondin (TSP). Although TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Because a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red blood cells (PS(+ve)-RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50 u/mL) inhibited PS(+ve)-RBC adhesion to immobilized TSP in a concentration-dependent manner (21% to 77% inhibition, P < 0.05). Preincubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS(+ve)-RBC adhesion to TSP. Antibodies that recognize the collagen- and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. Although preincubation of PS(+ve)-RBCs with TSP peptides from the heparin-binding domain that contained the specific heparin-binding motif KKTRG inhibited PS(+ve)-erythrocyte adhesion to matrix TSP (P < 0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide that lacks the binding motif neither inhibited nor supported PS(+ve)-RBC adhesion. Additional experiments show that soluble TSP also interacted with PS(+ve)-RBCs via its heparin-binding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited P-selectin- and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results, taken together with the previously documented findings, provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease.