Several lines of evidence suggest that sickle cell disease (SCD) is associated with a chronic inflammatory state. In this study of 70 children with SCD at steady state evaluated by a broad panel of biomarkers representing previously examined mechanisms of pathogenicity in SCD, high sensitivity C-reactive protein (hs-CRP), a marker of low-grade, systemic inflammation, emerged as the most significant laboratory correlate of hospitalizations for pain or vaso-occlusive (VOC) events. While markers of increased haemolytic status, endothelial activation and coagulation activation all correlated positively with VOC events by univariate analysis, baseline hs-CRP levels provided the most significant contribution to the association in multiple regression models (22%), and, hs-CRP, along with age, provided the best fit in negative binomial models. These data highlight the clinical relevance of the role of inflammation in paediatric VOC, providing both a rationale for future therapeutic strategies targeting inflammation in microvessel occlusive complications of SCD, and the potential clinical use of hs-CRP as a biomarker in childhood SCD.

Summary.  Objectives: We explored the possibility that heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease, could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Methods: Levels of TF mRNA, protein and procoagulant activity were measured in heme‐treated endothelial cells. Results: Heme induces TF expression on the surface of both macrovascular and microvascular endothelial cells in a concentration‐dependent manner, with 12‐fold to 50‐fold induction being noted (enzyme‐linked immunosorbent assay) between 1 and 100 μm heme (P < 0.05). Complementary flow cytometry studies showed that the heme‐mediated endothelial TF expression was quantitatively similar to that of tumor necrosis factor‐alpha (TNF‐α). Heme also upregulated the expression of TF mRNA (8‐fold to 26‐fold), protein (20‐fold to 39‐fold) and procoagulant activity (5‐fold to 13‐fold) in endothelial cells in a time‐dependent manner. The time‐course of heme‐mediated TF antigen expression paralleled the induction of procoagulant activity, with antibody blocking studies demonstrating specificity for TF protein. Interleukin (IL)‐1α, and TNF‐α are not involved in mediating the heme effect, as antibodies against these cytokines and IL‐1‐receptor antagonist failed to block heme‐induced TF expression. Inhibition of heme‐induced TF mRNA expression by sulfasalazine and curcumin suggested that the transcription factor nuclear factor kappaB is involved in mediating heme‐induced TF expression in endothelial cells. Conclusions: Our results demonstrate that heme induces TF expression by directly activating endothelial cells, and that heme‐induced endothelial TF expression may provide a pathophysiologic link between the intravascular hemolytic milieu and the hemostatic perturbations previously noted in patients with hemolytic anemia including sickle cell disease.

Phosphatidylserine (PS)-positive erythrocytes adhere to endothelium and subendothelial matrix components. While thrombospondin mediates these interactions, it is unknown whether PSassociated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PSexpressing control HbAA erythrocytes, we demonstrate that PS-positive erythrocytes adhered to human lung microendothelial cells in the absence of plasma ligands, that this adhesion was enhanced following endothelial activation with IL-1␣, TNF-␣, LPS, hypoxia, and heme, and that this adhesive interaction was selective to erythrocyte PS. We next explored whether microendothelial cells express an adhesion receptor that recognizes cell surface-expressed PS (PSR) similar to that expressed on activated macrophages. We demonstrate constitutive expression of both PSR mRNA and protein that were up-regulated in a time-dependent manner following endothelial activation. While minimal PSR expression was noted on unstimulated cells, endothelial activation up-regulated PSR surface expression. In antibody-blocking studies, using PSpositive erythrocytes generated either artificially via ionophore treatment of control erythrocytes or from patients with sickle cell disease, we demonstrate that PSR was functional, supporting PSmediated erythrocyte adhesion to activated endothelium. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the microendothelium that is up-regulated by such pathologically relevant agonists as hypoxia, cytokines, and heme. IntroductionThe anionic phospholipid phosphatidylserine (PS), present exclusively in the inner leaflet of the plasma membrane of a normal cell, is externalized following cell activation with both physiologic and pathologic stimuli such as activation of blood platelets resulting from vessel wall injury and cells that are undergoing apoptosis. 1,2 PS exposure on the red cell surface occurs in patients with various hemolytic anemias 3-9 activating numerous pathobiologic processes, of which one is red cell-endothelial cell adhesion. 10,11 The mechanism(s) underlying this PS-mediated adhesion process is not well defined. Interaction of PS with immobilized subendothelial matrix thrombospondin has been documented. 10 In addition, soluble thrombospondin-mediated adhesion of PS-positive erythrocytes to endothelium can occur via the constitutively expressed endothelial vitronectin receptor (VnR). 10 Whether PS-related erythrocyte adhesion to endothelium can occur in the absence of plasma ligands is not known. In the present study, we explore this possibility, and hypothesize that endothelial cells express novel adhesion receptor(s) that can interact directly with PS-positive erythrocytes in the absence of plasma ligands. One such candidate is a receptor that recognizes cell surface-expressed membrane-associated phosphatidylserine, referred hitherto as the phosphatidylserine receptor or PSR, originally described by Fadok et al on the surface of activated macrophages that recogni...

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.

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.