Characterization of a neutrophil cell surface glycosaminoglycan that mediates binding of platelet factor 4

Abstract

Platelet factor 4 (PF-4) is a platelet-derived α-chemokine that binds to and activates human neutrophils to undergo specific functions like exocytosis or adhesion. PF-4 binding has been shown to be independent of interleukin-8 receptors and could be inhibited by soluble chondroitin sulfate type glycosaminoglycans or by pretreatment of cells with chondroitinase ABC. Here we present evidence that surface-expressed neutrophil glycosaminoglycans are of chondroitin sulfate type and that this species binds to the tetrameric form of PF-4. The glycosaminoglycans consist of a single type of chain with an average molecular mass of ~23 kDa and are composed of ~85-90% chondroitin 4-sulfate disaccharide units type CSA (→GlcAβ1→3GalNAc(4-O- sulfate)-β1→) and of ~10-15% di-O-sulfated disaccharide units. A major part of these di-O-sulfated disaccharide units are CSE units (→4GlcAβ1→3GalNAc(4,6-O-sulfate)β1→). Binding studies revealed that the interaction of chondroitin sulfate with PF-4 required at least 20 monosaccharide units for significant binding. The di-O-sulfated disaccharide units in neutrophil glycosaminoglycans clearly promoted the affinity to PF- 4, which showed a K(d) ~ 0.8 μM, as the affinities of bovine cartilage chondroitin sulfate A, porcine skin dermatan sulfate, or bovine cartilage chondroitin sulfate C, all consisting exclusively of monosulfated disaccharide units, were found to be 3-5-fold lower. Taken together, our data indicate that chondroitin sulfate chains function as physiologically relevant binding sites for PF-4 on neutrophils and that the affinity of these chains for PF-4 is controlled by their degree of sulfation.