Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain

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 heparinbinding 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. (Translational Research 2008;152:165-177)