Submitted November 19, 2002
Accepted March 20, 2003
Single-chain antibody fragments derived from a human synthetic phage display library bind thrombospondin and inhibit sickle cell adhesion
Nicholas A Watkins, Lily M Du, J Paul Scott, Willem H Ouwehand, and Cheryl A Hillery*
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI, USA
Department of Haematology, University of Cambridge and National Blood Service, Cambridge, United Kingdom
* Corresponding author; email: chillery{at}bcsew.edu.
The enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single chain Fv (scFv) fragments displayed on filamentous phage. Six unique scFv that bound purified TSP by ELISA were isolated following 3 rounds of phage selection of increasing stringency. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFv inhibited the adhesion of sickle RBCs to immobilized TSP by greater than 40% compared to control scFv (p<0.001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (p<0.005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFv bound an epitope in the calcium binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFv that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface bound TSP under flow conditions. These scFv will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.
