Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion. Blood 2003 Jul 15;102(2):718-24
Date
03/29/2003Pubmed ID
12663449DOI
10.1182/blood-2002-11-3497Scopus ID
2-s2.0-0038494616 (requires institutional sign-in at Scopus site) 16 CitationsAbstract
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 fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P <.001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P <.005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs 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 scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.
Author List
Watkins NA, Du LM, Scott JP, Ouwehand WH, Hillery CAMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnemia, Sickle Cell
Arterial Occlusive Diseases
Binding Sites
Biosensing Techniques
Calcium
Cell Adhesion
Cells, Cultured
Depression, Chemical
Endothelium, Vascular
Enzyme-Linked Immunosorbent Assay
Epitopes
Erythrocytes, Abnormal
Humans
Immunoglobulin Variable Region
Molecular Sequence Data
Peptide Library
Protein Structure, Tertiary
Recombinant Proteins
Sequence Alignment
Sequence Homology, Amino Acid
Thrombospondins
Tumor Necrosis Factor-alpha
