Platelet-Derived Thrombospondin-1 Is Necessary for the Vitamin D-Binding Protein (Gc-Globulin) to Function as a Chemotactic Cofactor for C5a

Abstract

The chemotactic activity of C5a and C5a des Arg can be enhanced significantly by the vitamin D-binding protein (DBP), also known as Gc-globulin. DBP is a multifunctional 56-kDa plasma protein that binds and transports several diverse ligands. The objective of this study was to investigate the mechanisms by which DBP functions as a chemotactic cofactor for C5a using neutrophils and U937 cells transfected with the C5aR (U937-C5aR cells). The results demonstrate that U937-C5aR cells show C5a chemotactic enhancement only to DBP in serum, but, unlike mature neutrophils, this cell line cannot respond to DBP in plasma or to purified DBP. Analysis by SDS-PAGE and isoelectric focusing revealed no structural difference between DBP in serum compared with DBP in plasma. However, plasma supplemented with either serum, DBP-depleted serum, or activated platelet releasate provides a required factor and permits DBP to function as a chemotactic cofactor for C5a. Fractionation of activated platelet releasate revealed that the additional factor possessed the properties of thrombospondin-1 (TSP-1). Finally, purified TSP-1 alone could reproduce the effect of serum or platelet releasate, whereas Abs to TSP-1 could block these effects. These results provide clear evidence that TSP-1 is needed for DBP to function as a chemotactic cofactor for C5a.

CD44 and Annexin A2 Mediate the C5a Chemotactic Cofactor Function of the Vitamin D Binding Protein

“…Recently, we have speculated that formation of a DBP binding site complex is a dynamic, multistep, and transient process involving perhaps several distinct cell surface macromolecules (15). However, the precise components that form the binding site complex as well as the mechanism(s) by which DBP enhances chemotaxis to the C5-derived peptides are not known.…”

“…The DBP binding site complex has been inferred by functional, structural, and kinetic cell binding studies (10,(13)(14)(15). Recently, we have speculated that formation of a DBP binding site complex is a dynamic, multistep, and transient process involving perhaps several distinct cell surface macromolecules (15).…”

Selective inhibition of the C5a chemotactic cofactor function of the Vitamin D binding protein by 1,25(OH)2 Vitamin D3

“…However, very little mechanistic information is available concerning how DBP enhances chemotaxis to the C5 derived peptides. Several studies from our laboratory have demonstrated that the binding of DBP to the neutrophil plasma membrane, and subsequent protease-mediated shedding of the binding site, are essential for the chemotaxis enhancement of C5a (DiMartino and Kew, 1999;DiMartino et al, 2001;Kew et al, 1995a;Kew et al, 1995b) More recently, we have demonstrated that DBP requires platelet-derived thrombospondin-1 for maximal cochemotactic activity (Trujillo and Kew, 2004). In addition, cell surface CD44 and annexin A2 are part of a DBP binding site complex and mediate the C5a chemotactic cofactor function of DBP (McVoy and Kew, 2005).…”

“…2) but obviously does not. Moreover, the chemotactic activity in complement-activated serum is DBP-dependent because immunodepletion of DBP from activated serum reduces the chemotactic activity by almost 75% without altering the levels of C5a (Kew and Webster, 1988;Trujillo and Kew, 2004). This apparent paradox may be explained by how and when 1,25(OH) 2 D 3 interacts with neutrophils.…”

“…Both plasma membrane binding and subsequent shedding of DBP are essential for the protein to function as a chemotactic cofactor for C5a. Recent studies from our lab also have shown that DBP requires platelet-derived thrombospondin-1 for maximal co-chemotactic activity (Trujillo and Kew, 2004), and that cell surface CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP (McVoy and Kew, 2005 …”