We have previously reported that 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 several binding domains including: 1) the amino-terminal heparin-binding domain, 2) the CSVTCG sites within the type 1 repeats that may interact with CD36, 3) the RGD integrin-binding site within the last type 3 repeat and 4) the carboxy-terminal cell-binding domain. The goal of this study was to map the SS-RBC binding site(s) on TSP. In order to evaluate the heparin-binding domain of TSP, the 25 kDa N-terminal fragment of TSP was produced by thermolysin digestion and purified by heparin affinity chromatography. When the purified 25 kDa N-terminal TSP fragment was immobilized on the surface of a flow adhesion assay, it supported only 9.5±1.8% (mean±SE, N=4) of the binding activity of SS-RBCs to intact TSP at 1 dyne/cm2 flow. The 70 kDa chymotrypsin digest TSP fragment that contains the type I repeats did not support SS-RBC adhesion (<1% adhesion, N=4). In addition, both OKM-5, a murine anti-CD36 mAb that blocks TSP binding to CD36, and the TSP peptide CSVTCG, which blocks the interaction of TSP with CD36, failed to inhibit the binding of SS-RBCs to intact TSP (95±13% and 121±37%, respectively). However, the 140 kDa TSP thermolysin fragment containing the C-terminal cell binding domain and Type I, II, and III repeats of TSP, fully supported the adhesion of SS-RBCs under flow conditions(126±25%, N=4). These data provide evidence for the role of the carboxy-terminal domain(s) of TSP in the adhesion of SS-RBCs to the subendothelial matrix protein TSP under conditions of flow.
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Hillery, C., Du, M., French, J. et al. SICKLE RED BLOOD CELLS BIND TO THE 140 kDa C-TERMINAL DOMAIN OF THROMBOSPONDIN. ▴ 921. Pediatr Res 39 (Suppl 4), 156 (1996). https://doi.org/10.1203/00006450-199604001-00943
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DOI: https://doi.org/10.1203/00006450-199604001-00943