Microvascular Endothelial Cells Express Phosphatidylserine Receptor: A Functionally Active Receptor for Phosphatidylserine-Positive Erythrocytes.

Abstract

Phosphatidylserine (PS)-positive erythrocytes adhere to both endothelial cells and the sub-endothelial matrix components. While thrombospondin mediates these interactions, it is not known whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control erythrocytes, we demonstrate that PS-positive erythrocytes adhered directly to human lung micro-endothelial cells in the absence of plasma ligands, that this adhesion was further enhanced following endothelial activation with IL-1α, TNF-α, LPS, and hypoxia (2.5- to 8-fold increase), and that this adhesive interaction was selective to erythrocyte-PS. We next explored whether micro-endothelial cells express an adhesion receptor that recognizes cell surface expressed PS (PSR) similar to that expressed on activated macrophages. Using RT-PCR and Western blotting, we demonstrate constitutive expression of both PSR mRNA and protein which were up-regulated in a time-dependent manner following endothelial activation (with maximal increases of 3-fold in mRNA and 2-fold in protein noted at 4 and 6 hour, respectively). While minimal PSR expression was noted on un-stimulated cell surface (8% positivity), endothelial activation up-regulated surface expression of this receptor (35–40% positivity in IL-1α and TNF-α activated cultures). In antibody blocking studies, using both artificially generated PS-positive erythrocytes and also using PS-positive erythrocytes from patient with sickle cell disease, we demonstrate that PSR was functionally active supporting PS-mediated erythrocyte adhesion to activated endothelial cells. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the micro-endothelium which is up-regulated by such pathologically relevant agonists as hypoxia and cytokines.

Heme induces endothelial tissue factor expression: potential role in hemostatic activation in patients with hemolytic anemia

“…RT‐PCR products of TF mRNA from THP‐1 cells (a positive control for TF induction) incubated in the absence (lane 5) or presence (lane 6) of 100 ng mL −1 lipopolysaccharide (LPS) are shown for comparison. β‐Actin mRNA, a constitutively expressing transcript, was coamplified with TF mRNA as an endogenous control for TF mRNA quantitation, using previously described primers [14]. The RT‐PCR products of TF mRNA (380 bp) and β‐actin mRNA (687 bp) from a representative gel are shown at the top.…”

“…Cell surface TF expression was assessed using two complementary methods, including an enzyme‐linked immunosorbent assay (ELISA)‐based assay (intact cell monolayer) and flow cytometry (cell suspension), as previously described for endothelial adhesion receptors [14]. Total TF protein level was assessed by using western blotting of cellular proteins.…”

Insight into the complex pathophysiology of sickle cell anaemia and possible treatment

“…The exposure of PS is also the basis of MP generation . Setty et al demonstrated the existence of a specific phosphatidylserine receptor (PSR) being present on endothelial cells, thereby facilitating sickle red cell adhesion to the endothelium. Ongoing inflammatory processes are enhanced by sickling and may further contribute to expression of adhesion molecules; for example, tumour necrosis factor alpha (TNF‐α) and interleukin‐18 (IL‐18) can increase vascular cell adhesion molecule‐1 (VCAM‐1) on endothelial cells .…”

Pleiotropic effects of intravascular haemolysis on vascular homeostasis

“…In part, this phosphatidylserine exposure mediates binding and phagocytosis by macrophages. However, it also seems capable of inducing binding to endothelial cells with unknown consequences (Haynes et al, 2006;Setty & Betal, 2008). Microparticles displaying phosphatidylserine derived from erythrocytes (Allan et al, 1982), platelets (Wun et al, 1998) and endothelial cells (Shet et al, 2003) have been observed in sickle cell disease and thalassaemia (Westerman et al, 2008).…”