title: Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion.
abstract: 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.
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, Milwaukee, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Watkins
authors.forename: Lily M
authors.initials: LM
authors.lastname: Du
authors.forename: J Paul
authors.initials: JP
authors.lastname: Scott
authors.forename: Willem H
authors.initials: WH
authors.lastname: Ouwehand
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Hillery
mesh_terms: D000595:Amino Acid Sequence; D000755:Anemia, Sickle Cell / Q000097:blood* / Q000150:complications; D001157:Arterial Occlusive Diseases / Q000097:blood / Q000209:etiology; D001665:Binding Sites; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D003864:Depression, Chemical; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004913:Erythrocytes, Abnormal / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019699:Thrombospondins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000939:Epitopes; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D019699:Thrombospondins; D014409:Tumor Necrosis Factor-alpha; D002118:Calcium