A single high-fat meal provokes pathological erythrocyte remodeling and increases myeloperoxidase levels: implications for acute coronary syndrome.

High-fat meal (HFM) consumption can produce acute lipemia and trigger myocardial infarction in patients with atherosclerosis, but the mechanisms are poorly understood. Erythrocytes (red blood cells, RBCs) intimately interact with inflammatory cells and blood vessels and play a complex role in regulating vascular function. Chronic high-fat feeding in mice induces pathological RBC remodeling, suggesting a novel link between HFM, RBCs, and vascular dysfunction. However, whether acute HFM can induce RBC remodeling in humans is unknown. Ten healthy individuals were subjected to biochemical testing and assessment of endothelial-dependent flow-mediated dilation (FMD) before and after a single HFM or iso-caloric meal (ICM). Following the HFM, triglyceride, cholesterol, and free fatty acid levels were all significantly increased, in conjunction with impaired post-prandial FMD. Additionally, peripheral blood smears demonstrated microcytes, remodeled RBCs, and fatty monocytes. Increased intracellular ROS and nitration of protein band 3 was detected in RBCs following the HFM. The HFM elevated plasma and RBC-bound myeloperoxidase (MPO), which was associated with impaired FMD and oxidation of HDL. Monocytic cells exposed to lipid in vitro released MPO, while porcine coronary arteries exposed to fatty acids ex vivo took up MPO. We demonstrate in humans that a single HFM induces pathological RBC remodeling and concurrently elevates MPO, which can potentially enter the blood vessel wall to trigger oxidative stress and destabilize vulnerable plaques. These novel findings may have implications for the short-term risk of HFM consumption and alimentary lipemia in patients with atherosclerosis.

Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related Atherosclerosis.

BACKGROUND: High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). METHODS AND RESULTS: A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC(-/-) mice. In RBCs from HFD-fed wild-type and DARC(-/-) mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. CONCLUSIONS: RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.

Pulmonary platelet thrombi and vascular pathology in acute chest syndrome in patients with sickle cell disease.

A growing body of evidence suggests a role for platelets in sickle cell disease (SCD). Despite the proinflammatory, occlusive nature of platelets, a role for platelets in acute chest syndrome (ACS), however, remains understudied. To provide evidence and potentially describe contributory factors for a putative link between ACS and platelets, we performed an autopsy study of 20 SCD cases-10 of whom died from ACS and 10 whose deaths were not ACS-related. Pulmonary histopathology and case history were collected. We discovered that disseminated pulmonary platelet thrombi were present in 3 out of 10 of cases with ACS, but none of the matched cases without ACS. Those cases with detected thrombi were associated with significant deposition of endothelial vWF and detection of large vWF aggregates adhered to endothelium. Potential clinical risk factors were younger age and higher platelet count at presentation. However, we also noted a sharp and significant decline in platelet count prior to death in each case with platelet thrombi in the lungs. In this study, neither hydroxyurea use nor perimortem transfusion was associated with platelet thrombi. Surprisingly, in all cases, there was profound pulmonary artery remodeling with both thrombotic and proliferative pulmonary plexiform lesions. The severity of remodeling was not associated with a severe history of ACS, or hydroxyurea use, but was inversely correlated with age. We thus provide evidence of undocumented presence of platelet thrombi in cases of fatal ACS and describe clinical correlates. We also provide novel correlates of pulmonary remodeling in SCD.

Coagulation activation in sickle cell trait: an exploratory study.

Recent epidemiologic data suggest that sickle cell trait (HbAS; AS) is a risk factor for venous thromboembolism. We conducted an exploratory study of healthy subjects with AS under baseline conditions to determine whether a chronic basal hyperactivation of coagulation exists, and if so, what mechanism(s) contribute to this state. Eighteen healthy AS individuals were compared to 22 African-American controls with a normal haemoglobin profile (HbAA; AA) and 17 patients with sickle cell disease (HbSS; SS). Plasma thrombin-antithrombin complexes and D-dimer levels were elevated in AS relative to AA patients (P = 0·0385 and P = 0·017, respectively), and as expected, were much higher in SSversusAA (P < 0·0001 for both). Thrombin generation in platelet poor plasma was indistinguishable between AA and AS subjects, whereas a paradoxical decrease in endogenous thrombin potential was observed in SS (P ≤ 0·0001). Whole blood tissue factor was elevated in SS compared to AA (P = 0·005), but did not differ between AA and AS. Plasma microparticle tissue factor activity was non-significantly elevated in AS (P = 0·051), but was clearly elevated in SS patients (P = 0·004) when compared to AA controls. Further studies in larger cohorts of subjects with sickle cell trait are needed to confirm the results of this preliminary investigation.

Prior Spontaneous or Induced Abortion Is a Risk Factor for Cervical Dysfunction in Pregnant Women: a Systematic Review and Meta-analysis.

A history of abortion is associated with cervical dysfunction during pregnancy, but there remains uncertainty about whether risk can be stratified by the abortion type, the abortion procedure, or number of previous abortions. The objective of this study was to verify the relationship between cervical dysfunction measures in pregnancies with and without a history of termination. Embase and Medline databases were searched from 01 January 1960 to 01 March 2022 resulting in a full-text review of 28 studies. The Newcastle-Ottawa Scale (NOS) was used to assess the quality and risk of bias for non-randomized studies. The meta-analysis consisted of 6 studies that met all inclusion and exclusion criteria and included a combined total of 2,513,044 pregnancies. Cervical dysfunction was defined as either cervical insufficiency/incompetence in 4 of the studies and as short cervix in the others. Results from a random-effects model using reported adjusted odds ratios (aOR) estimated an increase in the odds of 2.71 (95% CI 1.76, 4.16) for cervical dysfunction in the current pregnancy related to a history of induced or spontaneous abortion. Subgroup analyses with only induced abortions (surgical/medical) estimated an aOR of 2.54 (95% CI 1.41, 4.57), while studies limited to surgical abortions had an aOR of 4.08 (95% CI 2.84, 5.86). The risk of cervical dysfunction in the current pregnancy was also found to be dependent on the number of previous abortions. In this meta-analysis, a prior history of abortion, and specifically induced abortions, was associated with cervical dysfunction. The protocol was registered in PROSPERO (CRD42020209723).

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.

Association of soluble fms-like tyrosine kinase-1 with pulmonary hypertension and haemolysis in sickle cell disease.

The pathophysiology of pulmonary hypertension (PHT) in sickle cell disease (SCD) is probably multifactorial. Soluble fms-like tyrosine kinase-1 (sFLT-1) is a member of the vascular endothelial growth factor receptor (VEGFR) family. By adhering to and inhibiting VEGF and placenta growth factor, it induces endothelial dysfunction. We sought to evaluate the association of sFLT-1 with clinical complications of SCD. We confirmed that sFLT-1 was significantly elevated in SCD patients compared to healthy, race-matched control subjects. The level of sFLT-1 was significantly higher in patients with PHT, but no association was observed between sFLT-1 and the frequency of acute pain episodes or history of acute chest syndrome. sFLT-1 was correlated with various measures of haemolysis, erythropoietin and soluble vascular cell adhesion molecule-1. By inducing endothelial dysfunction, sFLT-1 may contribute to the pathogenesis of SCD-associated PHT, although this effect does not appear to be independent of haemolysis.

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. DESIGN AND METHODS: This cross-sectional study was performed using a cohort of adult patients with SCD and control subjects without SCD. Spot urine for microalbumin/creatinine ratio, measures of hemolysis, inflammation and other laboratory studies were obtained. Pulmonary artery systolic pressure was determined by Doppler echocardiography, and the diagnosis of PHT was defined using age-, sex- and body mass index-adjusted reference ranges. RESULTS: Seventy-three patients with SCD and 21 healthy, race-matched control subjects were evaluated. In patients with SCD, normoalbuminuria was observed in 34 patients (46.6%), microalbuminuria in 24 patients (32.9%) and macroalbuminuria in 15 patients (20.5%). There was a significant correlation between urine albumin excretion and age. In patients with HbSS and Sbeta(0) thalassemia, the levels of sFLT-1, soluble VCAM and NT pro-BNP were significantly higher in those with macroalbuminuria, compared to patients with microalbuminuria and normoalbuminura, but no significant differences were observed in the levels of laboratory measures of hemolysis. Urine albumin excretion was associated with PHT and a history of stroke. CONCLUSIONS: Our study confirms the high prevalence of albuminuria in SCD. The association of urine albumin excretion with sFLT-1 suggests that this vascular endothelial growth factor receptor family member may contribute to the development of albuminuria in SCD. By inducing endothelial activation and endothelial dysfunction, sFLT-1 appears to be a link between glomerulopathy and PHT in SCD.

Fibronectin bridges monocytes and reticulocytes via integrin alpha4beta1.

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 alpha4beta1 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 alpha4beta1, 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.

Deletion of the Duffy antigen receptor for chemokines (DARC) promotes insulin resistance and adipose tissue inflammation during high fat feeding.

OBJECTIVE: Inflammation in adipose tissues in obesity promotes insulin resistance and metabolic disease. The Duffy antigen receptor for chemokines (DARC) is a promiscuous non-signaling receptor expressed on erythrocytes and other cell types that modulates tissue inflammation by binding chemokines such as monocyte chemoattractant protein-1 (MCP-1) and by acting as a chemokine reservoir. DARC allelic variants are common in humans, but the role of DARC in modulating obesity-related metabolic disease is unknown. METHODS: We examined body weight gain, tissue adiposity, metabolic parameters and inflammatory marker expression in wild-type and DARC knockout mice fed a chow diet (CD) and high fat diet (HFD). RESULTS: Compared to wild-type mice, HFD-fed DARC knockout mice developed glucose intolerance and insulin resistance independent of increases in body weight or adiposity. Interestingly, insulin sensitivity was also diminished in lean male DARC knockout mice fed a chow diet. Insulin production was not reduced by DARC gene deletion, and plasma leptin levels were similar in HFD fed wild-type and DARC knockout mice. MCP-1 levels in plasma rose significantly in the HFD fed wild-type mice, but not in the DARC knockout mice. Conversely, adipose tissue MCP-1 levels were higher, and more macrophage crown-like structures were detected, in the HFD fed DARC knockout mice as compared with the wild-type mice, consistent with augmented adipose tissue inflammation that is not accurately reflected by plasma levels of DARC-bound MCP-1 in these mice. CONCLUSIONS: These findings suggest that DARC regulates metabolic function and adipose tissue inflammation, which may impact obesity-related disease in ethnic populations with high frequencies of DARC allelic variants.

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease.

Sickle cell disease (SCD) results from a point mutation in the ß-globin gene forming hemoglobin S (HbS), which polymerizes in deoxygenated erythrocytes, triggering recurrent painful vaso-occlusive crises and chronic hemolytic anemia. Reactivation of fetal Hb (HbF) expression ameliorates these symptoms of SCD. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a transcription factor that triggers cytoprotective and antioxidant pathways to limit oxidative damage and inflammation and increases HbF synthesis in CD34+ stem cell-derived erythroid progenitors. We investigated the ability of dimethyl fumarate (DMF), a small-molecule Nrf2 agonist, to activate γ-globin transcription and enhance HbF in tissue culture and in murine and primate models. DMF recruited Nrf2 to the γ-globin promoters and the locus control region of the ß-globin locus in erythroleukemia cells, elevated HbF in SCD donor-derived erythroid progenitors, and reduced hypoxia-induced sickling. Chronic DMF administration in SCD mice induced HbF and increased Nrf2-dependent genes to detoxify heme and limit inflammation. This improved hematological parameters, reduced plasma-free Hb, and attenuated inflammatory markers. Chronic DMF administration to nonanemic primates increased γ-globin mRNA in BM and HbF protein in rbc. DMF represents a potential therapy for SCD to induce HbF and augment vasoprotection and heme detoxification.

Association of pro-inflammatory high-density lipoprotein cholesterol with clinical and laboratory variables in sickle cell disease.

Background Although cholesterol levels are known to be decreased in sickle cell disease (SCD), the level of pro-inflammatory high-density lipoprotein cholesterol (proHDL) and its association with clinical complications and laboratory variables has not been evaluated. Design and methods Plasma levels of total cholesterol, high-density lipoprotein cholesterol (HDL), proHDL, and selected clinical and laboratory variables were ascertained in a cohort of SCD patients and healthy African American control subjects in this single-center, cross-sectional study. Results Although total cholesterol was significantly lower in SCD patients compared with control subjects, HDL and proHDL levels were similar in both the SCD and control groups. In univariate analyses, proHDL was correlated with echocardiography-derived tricuspid regurgitant jet velocity. ProHDL was higher in SCD patients with suspected pulmonary hypertension (PHT) compared to patients without suspected PHT. ProHDL was positively correlated with lactate dehydrogenase, total bilirubin, direct bilirubin, indirect bilirubin, prothrombin fragment 1+2, D-dimer, and thrombin-antithrombin complexes. In multivariable analyses, only higher lactate dehydrogenase and direct bilirubin levels were associated with higher levels of proHDL. Conclusions SCD is characterized by hypocholesterolemia. Although proHDL is not increased in SCD patients compared with healthy controls, it is significantly associated with markers of liver disease. In addition, proHDL is associated with tricuspid regurgitant jet velocity and markers of coagulation, although these associations are not significant in multivariable analyses.

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 αIIbß3 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, Sß(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.

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/Sß° thalassemia and SC/Sß+ 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/Sß° 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.

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.

Cytokines and plasma factors in sickle cell disease.

PURPOSE OF REVIEW: Plasma 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 FINDINGS: New 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-beta 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. SUMMARY: Increased understanding of the origin and pathology of cytokine levels in sickle cell disease may provide novel therapeutic approaches in the management of the disease.

Mechanism of CD47-induced alpha4beta1 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 alpha4beta1 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 alpha4beta1 on SS RBCs as indicated by an RGD-dependent interaction of this integrin with soluble, plasma fibronectin. However, CD47 engagement also induces an alpha4beta1-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 alpha4beta1-mediated adhesion via a protein kinase A-dependent, serine phosphorylation of the alpha4 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 alpha4beta1 from cytoskeletal restraints. In addition, CD47 co-immunoprecipitates with alpha4beta1 in a sickle reticulocyte-enriched fraction of SS RBCs. These studies therefore identify the alpha4beta1 integrin on SS RBCs as a CD47-activated receptor for TSP, VCAM-1, and plasma fibronectin, revealing novel binding characteristics of this integrin.