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 CAAuthor
John Paul Scott MD Emeritus Professor in the Pediatrics department at Medical College of WisconsinMESH 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
