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

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\u3c0.05). 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\u3c0.001), 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

Microvascular endothelial cells express a phosphatidylserine receptor: a functionally active receptor for phosphatidylserine-positive erythrocytes

Phosphatidylserine (PS)-positive erythrocytes adhere to endothelium and subendothelial matrix components. While thrombospondin mediates these inter-actions, it is unknown whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing 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-1alpha, TNF-alpha, 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 PS-positive erythrocytes generated either artificially via ionophore treatment of control erythrocytes or from patients with sickle cell disease, we demonstrate that PSR was functional, supporting PS-mediated 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

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

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 \u3c 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

Quantitative sensory testing in children with sickle cell disease: additional insights and future possibilities.

Quantitative sensory testing (QST) is used in a variety of pain disorders to characterize pain and predict prognosis and response to specific therapies. In this study, we aimed to confirm results in the literature documenting altered QST thresholds in sickle cell disease (SCD) and assess the test-retest reliability of results over time. Fifty-seven SCD and 60 control subjects aged 8-20 years underwent heat and cold detection and pain threshold testing using a Medoc TSAII. Participants were tested at baseline and 3 months; SCD subjects were additionally tested at 6 months. An important facet of our study was the development and use of a novel QST modelling approach, allowing us to model all data together across modalities. We have not demonstrated significant differences in thermal thresholds between subjects with SCD and controls. Thermal thresholds were consistent over a 3- to 6-month period. Subjects on whom hydroxycarbamide (HC) was initiated shortly before or after baseline testing (new HC users) exhibited progressive decreases in thermal sensitivity from baseline to 6 months, suggesting that thermal testing may be sensitive to effective therapy to prevent vasoocclusive pain. These findings inform the use of QST as an endpoint in the evaluation of preventative pain therapies

Isolation and identification of prostaglandins from the reproductive organs of male silkmoth, Bombyx mori L.

The presence of a fat soluble material in the reproductive organs of male silkmoth, Bombyx mori L., which stimulates the contraction of the smooth muscle has been demonstrated to be due to prostaglandins. The active material isolated and separated by thin layer chromatography has been shown to be a mixture of two types of prostaglandins, PGE and PGFα. PGE could be resolved into PGE1 and PGE2 and PGFα into PGF1α and PGF2α. The identifications have been made by comparing the Rf values with standard compounds on TLC, by u.v. absorption and fluorescence emission spectroscopic analyses and by enzymic assay procedures