Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain

Abstract

Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and sub-endothelial matrix components is mediated in part via thrombospondin (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Since a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red cells (PS +ve -RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50u/ml) inhibited PS +ve -RBC adhesion to immobilized TSP in a concentration-dependent manner (21-77% inhibition, P<0.05). Pre-incubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS +ve -RBC adhesion to TSP. Antibodies that recognize the collagen-and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. While pre-incubation of PS +ve -RBCs with TSP-peptides from the heparin-binding domain containing the specific heparin-binding motif KKTRG inhibited PS +ve -erythrocyte adhesion to matrix TSP (P<0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide lacking the binding-motif neither inhibited nor supported PS +ve -RBC adhesion. Additional experiments show that soluble-TSP also interacted with PS +ve -RBCs via its heparinbinding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited Pselectin-and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results taken together with the previously documented findings provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease. Brief Statement of Key Findings: Besides serving as a signal for phagocytic recognition and removal of apoptotic cells, cell surface PS can also function as an adhesion ligand. PS-dependent erythrocyte adhesion to endothelium and sub-endothelial matrix components is mediated in part via TSP. We demonstrate that PS-positive erythrocytes bind to TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Our results taken together with previously documented heparin inhibitory effects on P-selectin-and soluble-TSP-mediated sickle erythrocyte-endothelial adhesion suggest that heparin and its low-molecular-weight derivatives may prove beneficial as anti-adhesive therapeutics targeting various pathways in the erythrocyte adhesion-cascade.

Sevuparin binds to multiple adhesive ligands and reduces sickle red blood cell‐induced vaso‐occlusion

“…Thrombospondin A had the lowest dissociation constant. Similar Kd values for heparinoids have been documented elsewhere (Koenig et al , ; Barabino et al , ; Wang et al , ; Wang & Geng, ; Brittain et al , ; Simonis et al , ; Gayen Betal & Setty, ).…”

“…Several non‐membrane bound proteins, such as von Willebrand factor (VWF) (Krishnan et al , ; Zhou et al , ; Chen et al , ) and thrombospondin (TSP) (Brittain et al , , ,b; Novelli et al , ), may contribute by serving as anchoring or bridging proteins, and several of these proteins have heparin‐binding sites. Furthermore, both heparin and low molecular weight heparins (LMWHs) can bind to several adhesion proteins (Koenig et al , ; Gupta et al , ; Polanowska‐Grabowska et al , ; Burnette et al , ) to inhibit SS‐RBC adhesion (Barabino et al , ; Matsui et al , , ; Gayen Betal & Setty, ) and to restore blood flow in animal models of SCD (Embury et al , ).…”

Pathophisiology of Sickle Cell Disease and New Drugs for the Treatment

“…A double blind randomized trial with tinzaparin in SCD patients during acute VOCs has documented a reduction of severity and duration of the VOCs51,60.…”

Prothrombotic mechanisms and anticoagulant therapy in dogs with immune‐mediated hemolytic anemia

“…Endothelial cell PS receptor expression is upregulated in response to inflammatory cytokines, hypoxia, and free hemoglobin . Erythrocyte PS also binds to thrombospondin, a protein that is present in subendothelial matrix . The importance of PS in normal hemostasis is illustrated by Scott syndrome.…”

“…In addition, in vitro studies using RBCs taken from people with sickle cell disease have shown that PS binds to the heparin‐binding domain of the endothelial cell matrix protein, thrombospondin. Clinically relevant doses of heparin inhibit this binding in a concentration‐dependent manner . Heparin also inhibits complement.…”