ALBERT

Description: Abstract 2587 Poster Board II-563 Background: SCD is considered to be a hypercoagulable state. Because of the great complexity of the hemostatic system it has not been easy to establish an etiological link between hypercoagulability and SCD vascular pathology. In this study we evaluate hemostatic perturbations in adult SCD patients by employing the computerized automated thrombogram (CAT), a novel thrombin generation assay that provides a global measure of coagulation potential and a direct assessment of the coagulation phenotype (Hemker HC et al.,Thromb Haemost. 2006; 96:553–61). We aim to characterize the coagulation phenotype of the SCD patient by a panel of CAT assay parameters. Methods: A total of 23 SCD patients (HbSS and HbSbeta0Thalassemia; 18 –58 years) were evaluated. Control group consisted of 6 age-matched controls (males=3, females=3). SCD plasma samples were obtained at baseline during routine clinic visits. Platelet poor plasma (PPP) ± corn trypsin inhibitor (CTI) - to minimize variability from activation of contact pathway during sample collection- was analyzed by CAT using 2 different triggers for initiation of thrombin generation: i) High trigger –5pM Tissue factor (TF) with 4uM phospholipid (PL) and ii) Low trigger- 1pM TF with 4uM PL. Five CAT assay parameters were studied – Lag time, Endogenous thrombin potential (ETP), Peak thrombin (Peak), Time to peak (ttPeak) and Start Tail. Students t-test was used to compare means of CAT assay parameters between SCD patients and controls. Lactate dehydrogenase (a biomarker of hemolysis-associated nitric oxide resistance and endothelial dysfunction) correlated with CAT assay parameters including lagtime (r= −0.35, p=0.05); ttPeak (r=−0.45, p

Description: Plasma levels of heme in the 20 to 600 μM range are found in clinical conditions associated with intravascular hemolysis including paroxysmal nocturnal hemoglobinuria and sickle cell disease, conditions also associated with a thrombotic tendency. Objectives: To investigate whether heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease (SCD), could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Additionally, in SCD patient-related studies, we assessed whether any association existed between whole blood TF activity (WBTF) and levels of surrogate markers of intra-vascular hemolysis including lactate dehydrogenase (LDH) and reticulocyte counts. Methods: Following incubation of human endothelial cells (from umbilical vein and/or lung microvasculature) with heme (1 to 100 μM) for various times (30 minutes to 8 hours), levels of TF protein were assessed using ELISA, flow cytometry and/or Western blotting; and TF mRNA by a semi-quantitative RT-PCR. An assay for TF functional activity was performed using a chromogenic tenase activity kit where specificity of TF activity was tested in antibody-blocking experiments. Three TF-specific antibodies including a rabbit polyclonal and two mouse monoclonal (clones hTF-1 and TF9-10H10) antibodies were used in assays involving TF protein analysis. All experiments were performed in media containing polymyxin B to neutralize any potential endotoxin contamination. In patient-related studies, 81 subjects with SCD (1 to 21 years) were evaluated for levels of WBTF, LDH, and reticulocyte counts and data analyzed for potential relationships. Results: Heme induced TF protein expression on the surface of both macro- and micro-vascular endothelial cells in a concentration-dependent manner with 12- to 50-fold induction noted (ELISA assays) between 1 and 100 μM heme (P

Description: Phosphatidylserine (PS)-dependent erythrocyte adhesion to both cultured endothelial cells and the components of sub-endothelial matrix appears to be mediated in part via thrombospondin-1 (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP has not been identified. Since a cell-binding domain for anionic heparin is located at the amino-terminal domain of TSP, we hypothesized that anionic PS-positive (PS+ve) red cells bind to this domain. In a recent preliminary study, using a flow adhesion system and PS+ve erythrocytes (prepared by treating control AA red cells with A23187), we have demonstrated that heparin inhibited PS+ve erythrocyte adhesion to immobilized TSP in a concentration-dependent manner with 58 to 77% inhibition noted at concentrations between 1 and 50 U/ml (n=9, P

Description: 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.

Description: Pulmonary function testing (PFT) is an important component for evaluating the outcome of experimental rodent models of respiratory diseases. Respiratory inductance plethysmography (RIP) provides a noninvasive method of PFT requiring minimal cooperation. RIP measures work of breathing (WOB) indices including phase angle (Ф), percent rib cage (RC %), breaths per minute (BPM), and labored breathing index (LBI) on an iPad. The aim of this study was to evaluate the utility of a recently developed research instrument, pneuRIP, for evaluation of WOB indices in a developmental rat model. Sprague Dawley rats (2 months old) were commercially acquired and anaesthetised with isoflurane. The pneuRIP system uses two elastic bands: one band (RC) placed around the rib cage under the upper armpit and another band (AB) around the abdomen. The typical thoracoabdominal motion (TAM) plot showed the abdomen and rib cage motion in synchrony. The plots of phase angle and LBI as a function of data point number showed that values were within the range. The distribution for phase angle and LBI was within a narrow range. pneuRIP testing provided instantaneous PFT results. This study demonstrated the utility of RIP as a rapid, noninvasive approach for evaluating treatment interventions in the rodent model.

Description: 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-1α, TNF-α, 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.