Julia E. Brittain

Activation of sickle red blood cell adhesion via integrin-associated protein/CD47–induced signal transduction

Peripheral human red blood cells (RBCs) are not generally known to become activated and adhesive in response to cell signaling. We show, however, that soluble thrombospondin via integrin-associated protein (IAP; CD47) increases the adhesiveness of sickle RBCs (SS RBCs) by activating signal transduction in the SS RBC. This stimulated adhesion requires occupancy of IAP and shear stress and is mediated by the activation of large G proteins and tyrosine kinases. Reticulocyte-enriched RBCs derived from sickle-cell disease (SCD) patients are most responsive to IAP-induced activation. These studies therefore establish peripheral SS RBCs as signaling cells that respond to a novel synergy between IAP-induced signal transduction and shear stress, suggesting new therapeutic targets in SCD.

Association of coagulation activation with clinical complications in sickle cell disease.

Background The contribution of hypercoagulability to the pathophysiology of sickle cell disease (SCD) remains poorly defined. We sought to evaluate the association of markers of coagulation and platelet activation with specific clinical complications and laboratory variables in patients with SCD. Design and Methods Plasma markers of coagulation activation (D-dimer and TAT), platelet activation (soluble CD40 ligand), microparticle-associated tissue factor (MPTF) procoagulant activity and other laboratory variables were obtained in a cohort of patients with SCD. Tricuspid regurgitant jet velocity was determined by Doppler echocardiography and the presence/history of clinical complications was ascertained at the time of evaluation, combined with a detailed review of the medical records. Results No significant differences in the levels of D-dimer, TAT, soluble CD40 ligand, and MPTF procoagulant activity were observed between patients in the SS/SD/Sβ0 thalassemia and SC/Sβ+ thalassemia groups. Both TAT and D-dimer were significantly correlated with measures of hemolysis (lactate dehydrogenase, indirect bilirubin and hemoglobin) and soluble vascular cell adhesion molecule-1. In patients in the SS/SD/Sβ0 thalassemia group, D-dimer was associated with a history of stroke (p = 0.049), TAT was associated with a history of retinopathy (p = 0.0176), and CD40 ligand was associated with the frequency of pain episodes (p = 0.039). In multivariate analyses, D-dimer was associated with reticulocyte count, lactate dehydrogenase, NT-proBNP and history of stroke; soluble CD40 ligand was associated with WBC count and platelet count; and MPTF procoagulant activity was associated with hemoglobin and history of acute chest syndrome. Conclusions This study supports the association of coagulation activation with hemolysis in SCD. The association of D-dimer with a history of stroke suggests that coagulation activation may contribute to the pathophysiology of stroke in clinically severe forms of SCD. More research is needed to evaluate the contribution of coagulation and platelet activation to clinical complications in SCD.

Calcium-dependent properties of CIB binding to the integrin αIIb cytoplasmic domain and translocation to the platelet cytoskeleton

The alphaIIbbeta3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation. Fibrinogen binding to alphaIIbbeta3 subsequently induces a cascade of intracellular signalling events. The molecular mechanisms of this bi-directional alphaIIbbeta3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. We reported previously the sequence of a novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein) that interacts specifically with the alphaIIb cytoplasmic domain in the yeast two-hybrid system. Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are widely expressed. In addition, isothermal titration calorimetry indicated that CIB binds to an alphaIIb cytoplasmic-domain peptide in a Ca(2+)-dependent manner, with moderate affinity (K(d), 700 nM) and 1:1 stoichiometry. In aggregated platelets, endogenous CIB and alphaIIbbeta3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel manner, demonstrating that the cellular localization of CIB is regulated, potentially by alphaIIbbeta3. Thus CIB may contribute to integrin-related functions by mechanisms involving Ca(2+)-modulated binding to the alphaIIb cytoplasmic domain and changes in intracellular distribution.

Mechanism of CD47-induced α4β1 Integrin Activation and Adhesion in Sickle Reticulocytes

We recently reported that CD47 (integrin-associated protein) on sickle red blood cells (SS RBCs) activates G-protein-dependent signaling, which promotes cell adhesion to immobilized thrombospondin (TSP) under relevant shear stress. These data suggested that signal transduction in SS RBCs may contribute to the vaso-occlusive pathology observed in sickle cell disease. However, the CD47-activated SS RBC adhesion receptor(s) that mediated adhesion to immobilized TSP remained unknown. Here we demonstrate that the α4β1 integrin (VLA-4) is the receptor that mediates CD47-stimulated SS RBC adhesion to immobilized TSP. This adhesion requires both the N-terminal heparin-binding domain and the RGD site of TSP. CD47 signaling induces an “inside-out” activation of α4β1 on SS RBCs as indicated by an RGD-dependent interaction of this integrin with soluble, plasma fibronectin. However, CD47 engagement also induces an α4β1-mediated, RGD-independent adhesion of SS RBCs to immobilized vascular cell adhesion molecule-1 (VCAM-1). CD47 signaling in SS RBCs appears to be independent of large scale changes in cAMP formation but nonetheless promotes α4β1-mediated adhesion via a protein kinase A-dependent, serine phosphorylation of the α4 cytoplasmic domain. CD47-activated SS RBC adhesion absolutely requires the Src family tyrosine kinases and is also enhanced by treatment of SS RBCs with low concentrations of cytochalasin D, which may release α4β1 from cytoskeletal restraints. In addition, CD47 co-immunoprecipitates with α4β1 in a sickle reticulocyte-enriched fraction of SS RBCs. These studies therefore identify the α4β1 integrin on SS RBCs as a CD47-activated receptor for TSP, VCAM-1, and plasma fibronectin, revealing novel binding characteristics of this integrin.

Cytokines and plasma factors in sickle cell disease

Purpose of reviewPlasma cytokines and related factors represent a burgeoning area of inquiry related to the pathogenesis in sickle cell disease. Cytokines derived from platelets, white blood cells and endothelial cells have all been implicated in the development of several sequelae of this disease. In this review, we seek to provide an overview of the noted and potentially novel roles for several key plasma factors in sickle cell disease. We also consider the putative role for those cytokines implicated by genetic analysis in sickle cell disease, but where the pathogenic, or ameliorative, role has yet to be determined. Recent findingsNew roles for the platelet as a key mediator in the release of cytokines in sickle cell disease have recently been demonstrated. Angiogenic and inflammatory factors are also being explored in this illness. Members of the vascular endothelial growth factor and transforming growth factor-β superfamilies have been suggested to contribute to several key events in pathogenesis of sickle cell disease, but with the promise of nitrous oxide therapy in this disorder, these cytokines merit a fresh perspective in the context of sickle cell disease. SummaryIncreased understanding of the origin and pathology of cytokine levels in sickle cell disease may provide novel therapeutic approaches in the management of the disease.

Urinary albumin excretion is associated with pulmonary hypertension in sickle cell disease: potential role of soluble fms-like tyrosine kinase-1.

Background:  Pulmonary hypertension (PHT) is reported to be associated with measures of renal function in patients with sickle cell disease (SCD). The purpose of this exploratory study was to determine the relationship between albuminuria and both clinical and laboratory variables in SCD.

Placenta growth factor in sickle cell disease: association with hemolysis and inflammation

Placenta growth factor (PlGF) is released by immature erythrocytes and is elevated in sickle cell disease (SCD). Previous data generated in vitro suggest that PlGF may play a role in the pathophysiology of SCD-associated pulmonary hypertension (PHT) by inducing the release of the vasoconstrictor, endothelin-1. In this cross-sectional study of 74 patients with SCD, we confirm that PlGF is significantly elevated in SCD compared with healthy control subjects. We found significantly higher levels of PlGF in SCD patients with PHT but observed no association of PlGF with the frequency of acute pain episodes or history of acute chest syndrome. The observed correlation between PlGF and various measures of red cell destruction suggests that hemolysis, and the resultant erythropoietic response, results in the up-regulation of PlGF. Although relatively specific, PlGF, as well as N-terminal pro-brain natriuretic peptide and soluble vascular cell adhesion molecule, has low predictive accuracy for the presence of PHT. Prospective studies are required to conclusively define the contribution of PlGF to the pathogenesis of PHT and other hemolytic complications in SCD.

Fibronectin bridges monocytes and reticulocytes via integrin α4β1

Leucocytes are emerging as critical determinants in the severity of the pathology associated with sickle cell disease (SCD) and recent studies have shown that they can bind to sickle red blood cells (SS RBCs). However, the mechanism of this interaction is unclear. The α4β1 integrin on monocytes and SS reticulocytes was found to mediate the interaction of these cells in in‐vitro adhesion assays and in the blood of SCD patients. Plasma fibronectin (Fn), a ligand for α4β1, could link SS RBCs to monocytes, as peptides derived from both the Arg‐Gly‐Asp‐Ser (RGDS) and CS‐1 site in Fn disrupted the reticulocyte/monocyte interaction. It was further shown in whole blood that 70% of the interacting monocytes were also bound to platelets, suggesting the existence of multi‐cellular aggregates in SCD. Platelet inclusion in these aggregates was mediated by a P‐selectin/P‐selectin glycoprotein ligand‐1 interaction, which has been demonstrated to activate the monocyte. These results suggest a new model for understanding the mechanism of attachment of SS RBCs to monocytes and implicate the platelet as a component and contributor to potentially occlusive aggregates that circulate in the blood of SCD patients.

A Pilot Study of Eptifibatide for Treatment of Acute Pain Episodes in Sickle Cell Disease

INTRODUCTION The contribution of platelet activation to the pathogenesis of sickle cell disease (SCD) remains uncertain. We evaluated the safety and efficacy of eptifibatide, a synthetic peptide inhibitor of the αIIbβ3 receptor, in SCD patients during acute painful episodes. MATERIALS AND METHODS In this single site, double-blind, placebo-controlled trial, eligible patients with SCD admitted for acute painful episodes were randomized to receive eptifibatide or placebo at a ratio of 2:1. RESULTS Thirteen patients (SS - 10, Sβ(0) - 2, SC - 1) were randomized to receive either eptifibatide (N=9; 6 females; median age - 25years) or placebo (N=4; 3 females; median age - 31years). In the intent-to-treat analysis, there were no major bleeding episodes in either the eptifibatide or placebo arms (point estimate of difference: 0.00, 95% CI; -0.604, 0.372). There was one minor bleeding episode in the eptifibatide arm (point estimate of difference for any bleeding: 0.11, 95% CI: -0.502, 0.494). There was no significant difference in the proportion of patients with thrombocytopenia between the treatment groups (point estimate of difference: 0.11, 95% CI: -0.587, 0.495). There were no differences in the median times to discharge, median times to crisis resolution or the median total opioid use. CONCLUSIONS In this small study, eptifibatide appeared to be safe, but did not improve the times to crisis resolution or hospital discharge. Adequately powered studies are required to evaluate the safety and efficacy of eptifibatide in SCD. Clinicaltrials.gov Identifier: NCT00834899.

Midgestation Maternal Serum 25-Hydroxyvitamin D Level and Soluble Fms-Like Tyrosine Kinase 1/Placental Growth Factor Ratio as Predictors of Severe Preeclampsia

Recent studies have shown that low serum 25-hydroxyvitamin D (25[OH]D) level is a risk factor for preeclampsia. The clinical significance of in vitro findings that vitamin D regulates vascular endothelial growth factor production is unclear. We sought to determine whether there is an association between midgestation serum 25(OH)D levels and angiogenic factor activity and to compare their predictive value for the development of severe preeclampsia. We conducted a nested case-control study of women with severe preeclampsia (n=41) versus women with uncomplicated term birth (n=123) who had second trimester genetic screening (15–20 weeks). Using banked frozen serum, we measured levels of 25(OH)D, vascular endothelial growth factor, soluble fms-like tyrosine kinase 1, and placental growth factor and compared their correlations and predictive values. We found no correlation between serum 25(OH)D and angiogenic factors levels. 25(OH)D alone was comparable to vascular endothelial growth factor and soluble fms-like tyrosine kinase 1/placental growth factor ratio as a predictive marker for severe preeclampsia. A composite of both 25(OH)D level and soluble fms-like tyrosine kinase 1/placental growth factor ratio was more predictive than either alone (area under curve: 0.83 versus 0.74 and 0.67, respectively). In conclusion, combining midpregnancy 25(OH)D level with soluble fms-like tyrosine kinase 1/placental growth factor ratio provides a better prediction for the development of severe preeclampsia.