Document Type

Article

Publication Date

Fall 10-1-2008

Comments

This article has been peer reviewed. It is the authors' final version prior to publication in Translational Research Volume 152, Issue 4, October 2008, Pages 165-177. The published version is available at . DOI: 10.1016/j.trsl.2008.07.007. Copyright © Elsevier Inc..

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 heparin-binding 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 P-selectin- 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.

TSP-Translational Res Submision Revised Version - Figures July 21 2008.doc (10142 kB)
6 Figures for "Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain"

TSP-Translational Res Submision Version - Table March 19 2008.doc (42 kB)
1 Table "Expression of adhesion molecules on HbAA control erythrocytes and HbSS red cells from patients with sickle cell disease"