The carboxy-terminal cell-binding domain of thrombospondin is essential for sickle red blood cell adhesion.
Sickle red blood cells (SS-RBCs) have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. TSP has at least four domains that mediate cell adhesion. The goal of this study was to map the site(s) on TSP that binds SS-RBCs. Purified TSP proteolytic fragments containing either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (<10% adhesion). However, a 140-kD thermolysin TSP fragment, containing the carboxy-terminal cell-binding domain in addition to the type 1, 2, and 3 repeats fully supported the adhesion of SS-RBCs (126% +/- 25% adhesion). Two cell-binding domain adhesive peptides, 4N1K (KRFYVVMWKK) and 7N3 (FIRVVMYEGKK), failed to either inhibit or support SS-RBC adhesion to TSP. In addition, monoclonal antibody C6. 7, which blocks platelet and melanoma cell adhesion to the cell-binding domain, did not inhibit SS-RBC adhesion to TSP. These data suggest that a novel adhesive site within the cell binding domain of TSP promotes the adhesion of sickle RBCs to TSP. Furthermore, soluble TSP did not bind SS-RBCs as detected by flow cytometry, nor inhibit SS-RBC adhesion to immobilized TSP under conditions of flow, indicating that the adhesive site on TSP that recognizes SS-RBCs is exposed only after TSP binds to a matrix. We conclude that the intact carboxy-terminal cell-binding domain of TSP is essential for the adhesion of sickle RBCs under flow conditions. This study also provides evidence for a unique adhesive site within the cell-binding domain that is exposed after TSP binds to a matrix.
Hillery
C A
CA
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, USA. chillery@bcsew.edu
Scott
J P
JP
Du
M C
MC
D000596:Amino Acids; D000755:Anemia, Sickle Cell / Q000097:blood / Q000473:pathology*; D001665:Binding Sites / Q000235:genetics; D002448:Cell Adhesion; D004912:Erythrocytes / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D017421:Sequence Analysis; D019699:Thrombospondins / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D019699:Thrombospondins