Previous studies from our laboratories and others have documented a role for PS-positive red cells in erythrocyte-endothelial adhesion with data suggesting that adhesion was in part due to the PS-positive erythrocyte-TSP interaction. The PS-binding site on TSP has not been characterized to date. In this study using a dynamic flow adhesion assay and artificially generated PS-positive erythrocytes, we demonstrate that PS-positive erythrocytes bind to both immobilized and soluble thrombospondin via its heparin-binding domain located at the amino-terminus of the polypeptide. We show that the binding is PS specific and requires the presence of both divalent cations Ca2+ and Mg2+. Significant binding to TSP of erythrocytes from patients with SCD, who demonstrated high levels of percent PS-positive red cells, also occurred at the heparin-binding domain. In addition, both heparin and its low-molecular weight derivative enoxaparin inhibit this interaction.
TSP is a homotrimeric 450-kDa protein, and as shown in , each TSP subunit contains five distinct cell-binding domains, which interact with select protein(s), adhesion marker(s) or receptor(s).9-13
The heparin-binding domain which is located at the amino-terminus of the polypeptide interacts with heparin, heparansulfate proteoglycans, sulfatides, and β1-integrins.10-12
The WSPWS sequence, present within the type-I repeats, also interacts with heparansulfate proteoglycans.10,11
The CSVTCG motif that occurs twice within the type-I repeats binds to sulfatides, and CD36.10,11
The integrin-binding motif, RGDS, found in the last type-III repeat interacts with the fibrinogen receptor αIIbβ3, and the vitronectin receptor αVβ3.10,11,13
The RFYVVM sequence located at the carboxy-terminal of the polypeptide binds to CD47.10,11
While TSP can interact with a variety of cells via multiple cell-binding domains, the PS binding site has not been identified to date. PS-mediated cell binding may have potential implications not only in tethering of PS expressing apoptotic cells to phagocytes, but also in red cell-endothelial adhesion. Our interest in characterization of PS-binding site on TSP is due to previously documented work including the findings from this laboratory demonstrating that the PS is involved in erythrocyte-endothelial adhesion4
. Since heparin inhibits adhesion of sickle red cells to TSP,18,30,31
we hypothesized that PS-positive erythrocytes may bind to the heparin-binding domain on TSP. Using artificially generated PS-positive control erythrocytes, we demonstrate that PS-positive red cells bind to immobilized TSP via its heparin-binding domain. Our conclusion is based on the findings that heparin, the anti-TSP antibody recognizing the heparin-binding domain, and the specific TSP peptides containing the heparin-binding motif KKTRG inhibited PS-mediated erythrocyte adhesion to TSP (, , and ). Anti-TSP antibodies that recognize the collagen- or the CD47-binding domain, or the TSP peptide lacking the heparin-binding motif did not affect the binding process (, and ). Additional findings, demonstrating that immobilized TSP peptides containing the KKTRG motif supported PS-mediated erythrocyte adhesion, confirmed that PS interacted with TSP’s heparin-binding domain (). Pathophysiologic relevance of this work was documented in SCD patients, since HbSS erythrocyte binding to immobilized TSP matrix was also significantly inhibited by the anti-TSP antibody recognizing the heparin-binding domain (). Magnitude of the inhibitory effects produced by the latter anti-TSP antibody with sickle erythrocytes (a mean inhibition of 38%) was, however, smaller compared to that noted with artificially generated PS-positive erythrocytes (a mean inhibition of ~80%), which could be due to the differences in adhesion markers expressed on these cells as shown in . In contrast to sickle erythrocytes which express multiple cell adhesion markers28,32-38
with capabilities of interacting with multiple cell-binding domains on TSP,9-13
PS is the sole functional adhesion marker present on the ionophore-activated red cell and its interaction with TSP may, therefore, be limited to TSP’s heparin-binding domain. Pre-treatment of immobilized TSP with an antibody against the heparin-binding domain may, therefore, produce greater inhibition with the ionophore-activated control erythrocytes.
Besides PS, other relevant adhesion markers expressed on pathologic erythrocytes that are involved in red cell adhesion to endothelial cells and the sub-endothelial matrix components include CD36,32,33
To exclude an ancillary role for these latter adhesion molecules in PS-TSP adhesion, we first elected to use un-activated and ionophore-activated control HbAA erythrocytes where, as shown in , ionophore treatment did not alter the expression of other adhesion molecules except causing PS-positivity i.e. the ideal condition for our proposed experiments related to PS-TSP adhesion. In addition, pretreatment of TSP with an antibody that recognizes the CD47-binding domain on TSP had no effect on ionophore-activated erythrocyte adhesion (). Further, while it is not known whether erythrocyte activation with A23187 perturbs membrane sulfatide composition, pretreatment of immobilized TSP with bovine brain sulfatide had no effect on A23187-treated red cell binding (). These results demonstrate that the erythrocyte adhesion molecules including CD47, BCAM/LU and sulfatides do not appear to be involved in mediating ionophore-activated erythrocyte binding to the heparin-binding domain on TSP.
Protein conformational changes may affect the cell-binding characteristics of TSP.39
For example, studies have demonstrated that chelation of Ca2+
unfolds the globular cell-binding domain of TSP, destabilizes disulfide bonds and results in extensive thiol-disulfide exchange leading to altered cell-binding characteristics.40-42
We therefore evaluated the effects of divalent cations including Ca2+
, and found a 12-fold increase in PS-mediated erythrocyte binding to TSP in the presence of Ca2+
, although this effect also required the presence of Mg2+
. Additionally, both Ca2+
, by decreasing molecular motion of the PS head groups and narrowing the distance between the neighboring PS head groups,43
may maximize binding of PS-positive erythrocytes to TSP. Protein conformation of immobilized matrix TSP also appears to be different from that of soluble TSP as previously documented by differential adhesion of sickle erythrocytes to soluble verses immobilized matrix TSP.28,32,44
While soluble TSP-mediated sickle red cell adhesion to cultured endothelial cells was blocked by both OKM-5 (a murine monoclonal antibody against CD36) and by a TSP peptide containing the CD36-binding motif CSVTCG,32
these blocking agents had no effect on sickle erythrocyte adhesion to immobilized matrix TSP.28,44
In light of these documented differential effects of blocking agents on sickle erythrocyte adhesion to TSP, we elected to also evaluate soluble TSP interactions with PS-positive cells. We found that soluble TSP, like immobilized TSP, appears to interact with PS-positive erythrocytes via its heparin-binding domain. We demonstrate that erythrocytes acquire TSP-positivity following incubation of PS-positive red cells with soluble TSP. No TSP positivity was found on the surface of erythrocytes that were incubated in the absence of TSP, or PS-negative red cells incubated with soluble TSP (). In addition, TSP positivity was noted when an antibody recognizing the collagen-binding domain, but not an antibody recognizing the heparin-binding domain, was used as the detecting agent, likely due to preoccupancy of the heparin-binding domain, the binding site for anti-TSP-Ab9, on the TSP molecule by PS-positive erythrocytes. Since TSP is one of the components of the sub-endothelial matrix, in its immobilized form it facilitates adhesion of PS-positive red cells to sub-endothelium. In its soluble form TSP transforms a PS-positive red cell into a TSP-positive red cell which can interact not only with multiple adhesion receptors including CD36, CD47, α4β1, αvβ3 and αIIbβ3, but may also, potentially, play a role in the formation of hetero-cellular aggregates by facilitating adhesive interactions with other circulating cellular elements of blood including platelets and monocytes, as well as vascular endothelial cells. In this context it is interesting to note that previous studies in patients with SCD documented the presence of platelet-erythrocyte and neutrophil-erythrocyte aggreagates.45-47
Previous studies have evaluated the effects of several anionic polysaccharides including heparin, CSA and HDS on sickle erythrocyte adhesion to both immobilized matrix and soluble TSP and found differential inhibitory effects,18,28,31,44
in contrast to inhibition of a similar magnitude noted in this study with artificially generated PS-positive erythrocytes. These differences could be due to polysaccharides’ ability to recognize multiple GAG binding domains on TSP, as previously documented with CSA, which in addition to its interaction with the heparin binding domain, can also bind to regions within the type-I repeats and also to regions in the carboxy-terminal cell binding domain.48
Heparin has been used clinically as an anti-coagulant and anti-thrombotic agent.49
Major clinical concerns of prolonged heparin therapy, however, include heparin-induced thrombocytopenia, and abnormal bleeding. To overcome these adverse clinical effects of unfractionated heparin, several low molecular weight heparin derivatives have been developed, which have numerous advantages over unfractionated preparations including clinical safety and efficacy.50
In the present study we tested the effects on adhesion of heparin and enoxaparin, a low molecular weight heparin derivative, and demonstrate that both these polysaccharides inhibited PS-mediated erythrocyte adhesion to immobilized TSP not only at pharmacologic concentrations (5 to 50 U/ml) but also at levels (0.5 U/ml) achievable clinically in patients.49,51
Other investigators have demonstrated that heparin can inhibit soluble TSP-,18,31
mediated sickle erythrocyte adhesion to cultured human endothelial cells. In addition, a previous study has suggested that prophylactic heparin therapy reduced the frequency of vaso-occlusive pain in patients with SCD.52
Our results taken together with those previously published demonstrate that heparin can modulate multiple red cell adhesion pathways in patients with SCD and suggests that heparin and its low molecular weight derivatives may be useful as potential therapeutic agents targeting the enhanced pro-adhesive state in this patient group.
In summary, we have demonstrated that PS-positive erythrocytes bind to both immobilized and soluble thrombospondin via its heparin-binding domain located at the amino-terminus of the polypeptide. We have shown that soluble TSP by binding to PS-positive red cells can potentially generate a sub-population of red cells which are TSP-positive with potentially enhanced pro-adhesive capabilities. In addition, we have demonstrated that heparin and its low molecular weight derivative enoxaparin inhibit PS-mediated erythrocyte-TSP interaction. The therapeutic potential of these observations are germane not only to sickle cell disease pathogenesis, but to other disorders in which a proadhesive propensity due to cellular PS-thrombospondin interactions exacerbates disease pathophysiology.