Watkins Nicholas A, Du Lily M, Scott J Paul, Ouwehand Willem H, Hillery Cheryl A
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, USA.
Blood. 2003 Jul 15;102(2):718-24. doi: 10.1182/blood-2002-11-3497. Epub 2003 Mar 27.
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.
镰状红细胞（RBCs）与血管内皮和内皮下基质的粘附增强，可能在镰状细胞病血管阻塞的发病机制中起重要作用。在体外流动条件下，镰状红细胞与血浆和细胞外基质蛋白血小板反应蛋白-1（TSP）的粘附增强。在本研究中，我们试图从展示在丝状噬菌体上的高度多样化的人单链Fv片段（scFvs）文库中开发与TSP结合的抗体。经过三轮严格度递增的噬菌体筛选，通过酶联免疫吸附测定法分离出6种与纯化的TSP结合的独特scFvs。使用体外流动粘附试验，与对照scFvs相比，6种分离出的scFvs中有3种抑制镰状红细胞与固定化TSP的粘附超过40%（P<.001）。此外，scFv TSP-A10部分抑制镰状红细胞与活化内皮细胞的粘附（P<.005）。使用TSP蛋白水解片段来定位结合位点，我们发现2种抑制性scFvs结合TSP钙结合域或近端细胞结合域中的一个表位，为这些域在镰状红细胞与TSP粘附中的作用提供了证据。总之，我们分离出一组scFvs，它们在流动条件下特异性结合TSP并差异抑制镰状红细胞与表面结合的TSP的粘附。这些scFvs将是研究TSP钙结合域和细胞结合域在镰状红细胞粘附中作用的有用试剂。
