Increased adhesion of erythrocytes to components of the extracellular matrix: isolation and characterization of a red blood cell lipid that binds thrombospondin and laminin

Abstract

Red blood cell (RBC) adhesion to the vascular endothelium is increased in several pathologic conditions, including sickle cell disease and malaria. However, RBC interactions with components of the subendothelial matrix are not well-characterized. Under in vitro flow conditions of 1 dyne/cm2, washed RBCs bound to the purified adhesive molecules thrombospondin (TSP) and laminin. Sickle RBCs had the greatest adhesion of all tested RBCs. The adhesion of sickle RBCs to immobilized TSP was inhibited by the anionic polysaccharides high molecular weight (MW) dextran sulfate and chondroitin sulfate A, but not other anionic polysaccharides of similar structure and/or charge density. These data were consistent with the RBC adhesive molecule being a sulfated glycolipid. Therefore, TSP-binding lipids from normal and sickle RBCs were isolated and characterized. The TSP-binding lipid was purified by alkaline methanolysis, anion exchange chromatography and preparative thin layer chromatography (TLC). A homogeneous band on TLC was identified using a specific overlay TSP-binding assay. TSP binding to the purified lipid was stable to bass and neuraminidase treatment, labile to acid treatment, and was inhibited by high MW dextran sulfate, similar to that seen with intact RBCs binding to immobilized TSP under conditions of flow. In addition, soluble laminin bound to the purified RBC lipid. This acidic TSP- and laminin-binding lipid(s) isolated from both sickle and normal RBC membranes may contribute to erythrocyte interactions with the subendothelial matrix, hereby participating in the pathogenesis of vaso-occlusive diseases.

Erythrocyte adhesion is modified by alterations in cellular tonicity and volume

“…This hypothesis is supported by the increased adhesion of both NaCldehydrated and nystatin-dehydrated normal RBCs to TSP. In addition, inhibition of TSP adhesion to dehydrated normal RBCs with chondroitin sulphate A and high molecular weight dextran sulphate, but not chondroitin sulphate C, is similar to the inhibition of sickle RBC adhesion to TSP with the same treatments (Hillery et al, 1996). The mild augmentation of RBC adhesion upon pretreatment of dehydrated normal RBCs with the CD47 agonist peptide 4N1K parallels results with pretreatment of sickle RBCs with this peptide obtained by both our laboratory and others (Hillery et al, 1999;Brittain et al, 2001b).…”

“…Consistent with this behaviour, adhesion of NaCl-dehydrated (500 mOsM) or nystatin-dehydrated normal RBCs was also significantly (P < 0AE05, t-test) inhibited by pre-incubation of RBCs with HMWDS or CSA (Fig 4). In contrast, pre-incubation of RBCs with the related extracellular matrix polysaccharide CSC reduced, but did not significantly inhibit, dehydrated normal RBC adhesion to TSP (Fig 4), in agreement with similar findings with sickle RBCs (Hillery et al, 1996). When we further examined these data comparing the sickle, NaCltreated and nystatin-treated RBC groups using the Tukey method for multiple comparisons, we found that the sickle and nystatin-treated groups were significantly more inhibited by HMWDS compared with the NaCl-treated group.…”

“…We did not examine the adhesive pathways for CD36 or integrin a 4 b 1 as these receptors are primarily restricted to the reticulocyte subpopulation (Joneckis et al, 1993), normal RBCs are reticulocyte poor, and the adhesion of sickle RBCs to immobilised TSP under low shear flow conditions is not enriched for reticulocytes (Hillery et al, 1999). In addition, it has previously been shown that sickle RBC adhesion to immobilised TSP is probably not mediated by CD36 (Hillery et al, 1996;Joneckis et al, 1996). Our findings, that the adhesion of sickle RBCs to immobilised TSP was inhibited by the anionic polysaccharides HMWDS and CSA, but not with the anionic polysaccharide CSC that has a similar structure and charge density, is consistent with the RBC adhesive molecule being a sulphated glycolipid (Hillery et al, 1996;Joneckis et al, 1996).…”

“…Unlabelled annexin V was from BD Biosciences (San Jose, CA, USA) or Celsus (Cincinnati, OH, USA). TSP was purified from human platelets as previously described (Slayter, 1989;Hillery et al, 1996). Murine monoclonal anti-human CD47, clone B6H12, was purchased from Neomarkers (Freemont, CA, USA).…”

“…Donor and recipient mice were from the C57BL/6 J strain. RBCs were separated from platelet-rich plasma by centrifugation and washed three times with a citrate/glucose/saline buffer as previously described (Hillery et al, 1996).…”