The most likely explanation for the enhanced binding of CCL5 after exposure to low concentrations of CSA or serum is the occurrence of CCL5-CSA complexes and their subsequent binding to surface glycosaminoglycans via their CCL5 part or to surface receptors for CSA via their CSA part. when lower concentrations of serum were used, CCL5-presentation on endothelial cells was markedly enhanced. This enhancement was neutralized if serum was digested with chondroinitase ABC. Using different chondroitinsulfate-subtypes we demonstrate that chondroitinsulfate A mediates the enhanced presentation of CCL5 on endothelial cells, whereas chondroitinsulfate B/C even at low concentrations block CCL5 binding. CCR5 downregulation on CCR5-transfected CHO cells or human monocytes is increased by preincubation of CCL5 with serum or chondroitinsulfate A. == Conclusion == We show that chondroitinsulfate A released from platelets increases the binding of chemokines to endothelial cells and supports receptor internalization in a dose dependent manner. These data help to understand the proinflammatory effects of activated platelets. == Background == The adhesion and transendothelial migration of leukocytes is largely dependent on chemokines and adhesion molecules. In order to support leukocyte recruitment chemokines need to be immobilized on the luminal surface of the endothelial cell wall. Within tissues leukocytes are also directed by gradients of chemokines [1]. By interacting with different chemokine receptors (CCR1 and CCR5) the chemokine CCL5 (RANTES) has been shown to be involved in several steps of leukocyte recruitment [2]. Chemokines can gain access to the luminal site of the endothelium by transcytosis through endothelial cells [3], after release from circulating leukocytes or after secretion from activated endothelial cells [4]. Platelets have been identified as important source of chemoattractant factors such as CCL5 [5], but also release substantial amounts of chondroitinsulfate A [6]. In vivo, platelet activation and adhesion occurs at sites of vascular injury and facilitates leukocyte recruitment [7-10]. It has been shown that membrane bound glycosaminoglycans are critically involved in immobilization and presentation of chemokines [11-14]. Different patterns of glycosaminoglycan expression on cells may favor the binding of certain chemokines and thereby influence the cellular composition of the inflammatory response. However, chemokines also interact with soluble glycosaminoglycans that compete with the binding Protosappanin A of chemokines to cell surfaces. Heparin has the highest affinity to CCL5, followed by heparansulfate, chondroitinsulfate C, dermatansulfate (chondroitinsulfate B) and chondroitinsulfate A [15]. We could demonstrate that human Protosappanin A serum inhibits CCL5 binding on CHO cells and cultured human endothelial cells and could identify the responsible serum factor as chondroitinsulfate A (CSA) released from platelets after activation [16]. Glycosaminoglycans also alter the ability of chemokines to interact with chemokine receptors. Soluble Glycosaminoglycans have been shown to inhibit binding of IL-8 to CXCR1 and CXCR2 Mouse monoclonal to CD152 and CCL3 to CCR1 [15]. It was also shown that CCL5/glycosaminoglycan complexes are able to bind to deglycated PBMC and thereby block HIV-1 infection more effectively than CCL5 alone [17]. Activated platelets have been identified as a major source of CSA in human serum [6]. In addition, release of chondroitinsulfate A was shown in activated T cells [18,19]. It is commonly thought that interaction of chemokines with soluble glycosaminoglycans reduces their ability to bind to cell surfaces and interferes with leukocyte recruitment. However, these results do not fit to the proinflammatory effects caused by intravascular activation of platelets. Therefore we analyzed in more detail the influence of serum and various glycosaminoglycans on the binding of CCL5 to endothelial cells and on the ability of CCL5 to activate CCR5. == Results and discussion == == Influence of serum and glycosaminoglycans on CCL5 binding to endothelial cells == In previous experiments we have shown a reduced binding of CCL5 to cell surfaces after preincubation of CCL5 with human serum and have identified the responsible serum factor as Protosappanin A chondroitinsulfate A (CSA) released from activated platelets. In these experiments we used undiluted or moderately diluted serum and high concentrations of CSA [16]. The use of undiluted serum or high concentrations of glycosaminoglycans may not reflect in vivo settings of local inflammation where only a minor proportion of total platelets are activated and release CSA. We therefore analyzed the effects of serum or chondroitinsulfate A over a wide range of concentrations. CCL5 was preincubated with decreasing concentrations of human serum and then Protosappanin A incubated with microvascular human endothelial cells (MVEC) (Fig.1). High concentrations of serum block CCL5 binding to the surface of microvascular endothelial cells confirming our previous results [16]. However, Protosappanin A preincubation of CCL5 with lower concentrations of serum resulted in a threefold higher.