Sickle Cell Adhesion Research Articles

Role of Rap1 in promoting sickle red blood cell adhesion to laminin via BCAM/LU

AbstractVaso-occlusion is a hallmark of sickle cell disease. Agonist-induced activation of sickle red blood cells (SS RBCs) promotes their adhesion to vascular proteins, potentially contributing to vasoocclusion. Previously, we described a cyclic adenosine monophosphate (cAMP)-dependent increase in SS RBC adhesion to laminin. Here, we investigated whether Rap1, a small guanosine triphosphatase (GTPase) known to promote integrin-mediated adhesion in other cells, was involved in this signaling pathway. We found that agonists known to induce cAMP signaling promoted the GTP-bound, active state of Rap1 in SS RBCs. The cAMP-dependent exchange factor Epac (exchange protein directly activated by cAMP) is a likely upstream activator of Rap1, since Epac is present in these cells and the Epac-specific cAMP analog 8CPT-2-Me (8-(4-cholorophenylthio)-2′-O-methyl-cAMP) activated Rap1 and promoted SS RBC adhesion to laminin. This 8CPT-2-Me-stimulated adhesion was integrin independent, since it was insensitive to RGD peptide or antibodies against the only known integrin on SS RBCs, α4β1. However, this adhesion was completely inhibited by either a soluble version of basal cell adhesion molecule/Lutheran (BCAM/LU) or a BCAM/LU adhesion-blocking anti-body. Surprisingly, 8CPT-2-Me-activated Rap1 did not promote SS RBC adhesion to a known α4β1 ligand, vascular cell adhesion molecule 1 (VCAM-1). These results demonstrate that Epac-induced Rap1 activation in SS RBCs promotes BCAM/LU-mediated adhesion to laminin. Thus, Epac-mediated Rap1 activation may represent an important signaling pathway for promoting SS RBC adhesion. (Blood. 2005;105:3322-3329)

Transgenic Sickle Mice Are Markedly Sensitive to Renal Ischemia-Reperfusion Injury

Ischemic injury is invoked as a mechanism contributing to end-organ damage and other complications of sickle cell disease (SCD). However, the intrinsic sensitivity of tissues in SCD to ischemic insults has never been addressed. We examined the effect of renal ischemia in a transgenic mouse expressing human sickle hemoglobin. Twenty-four hours after bilateral, total renal artery occlusion for 15 minutes, transgenic sickle mice exhibited worse renal function and more marked histological injury. With bilateral renal ischemia of greater duration (22.5 minutes), and after 6 hours, transgenic sickle mice exhibited massive vascular congestion, sickling of red blood cells, more marked histological injury in the kidney, and more prominent congestion in the capillary beds in the lungs and heart. Additionally, serum amyloid P-component, the murine homologue of C-reactive protein, was markedly increased in transgenic sickle mice as compared to wild-type mice. Twenty-four hours after bilateral renal ischemia for 22.5 minutes, transgenic sickle mice exhibited 28% mortality, with no mortality observed in any other group. With bilateral renal ischemia of short or long duration, renal expression of caspase-3 was most prominent in transgenic sickle mice subjected to ischemia. Thus, renal ischemia in this murine model induces more severe renal injury and extrarenal complications. We conclude that tissues in SCD exhibit heightened vascular congestion and sensitivity to ischemia and that clinically apparent or silent episodes of ischemia may contribute to the complications of SCD.

Epinephrine acts through erythroid signaling pathways to activate sickle cell adhesion to endothelium via LW-αvβ3 interactions

The possible role of physiologic stress hormones in enhancing adhesion of sickle erythrocytes (SS RBCs) to endothelial cells (ECs) in sickle cell disease (SCD) has not been previously explored. We have now found that up-regulation of intracellular cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) by epinephrine significantly increased sickle but not normal erythrocyte adhesion to both primary and immortalized ECs. Inhibition of serine/threonine phosphatases also enhanced sickle erythrocyte adhesion at least partially through a PKA-dependent mechanism. Adhesion was mediated through LW (intercellular adhesion molecule-4 [ICAM-4], CD242) blood group glycoprotein, and immunoprecipitation studies showed that LW on sickle but not on normal erythrocytes undergoes increased PKA-dependent serine phosphorylation as a result of activation. The major counter receptor for LW was identified as the alphavbeta3 integrin on ECs. These data suggest that adrenergic hormones such as epinephrine may initiate or exacerbate vaso-occlusion and thus contribute to the association of vaso-occlusive events with physiologic stress.

Antioxidants Inhibit Sickle Cell Adhesion to Activated Endothelium in an Ex Vivo Preparation.

In sickle cell anemia (SS), intravascular sickling and attendant transient occlusive events are implicated in the generation of reactive oxygen species (ROS) and vascular endothelial activation. Oxidants (as well as cytokines) result in up-regulation of endothelial adhesion molecules that may lead to increased adhesion of sickle red blood cells (SS RBC) and contribute to vaso-occlusion. We hypothesize that endothelial activation will augment the expression of adhesion molecules involved in SS RBC-endothelium interaction, and that antioxidants will have an inhibitory effect on this interaction. To this end, we have tested selected antioxidants for their efficacy to inhibit SS RBC adhesion in the ex vivo mesocecum vasculature of the rat stimulated by platelet-activating factor (PAF). PAF, a potent inflammatory agent, results in endothelial ROS generation (Suematsu et al. Am. J. Physiol. 1993), as well as in increased adhesion of SS RBCs (Kaul et al. Blood, 2000). Here, we have tested the efficacy of superoxide dismutase (SOD), catalase and polynitroxylated albumin (PNA). While SOD and catalase remove superoxide (O2 ·−) and H2O2, respectively, nitroxides covalently attached to albumin (i.e., PNA) act as intra-vascular SOD mimetic and also facilitate heme-mediated catalytic removal of H2O2. To directly demonstrate the involvement of ROS (O2 •- and H2O2), the ex vivo vasculature was pre-treated with PAF (200 pg/ml in Ringer-albumin solution) followed by infusion with SOD (1500 U in 5 ml Ringer-albumin) or catalase (10,000 U in 5 ml) (each 15-min incubation). The control preparations were incubated with PAF and Ringer-albumin. For experiments involving PNA, the vasculature was first treated with PAF and followed by infusion of PNA or control human serum albumin (HSA) (each 33 mg/ml) diluted in Ringer-albumin solution (incubation 30 min). In PAF or PAF/HSA-treated preparations, infusion of SS RBCs (Hct 30 in Ringer-albumin) caused widespread adhesion of these cells exclusively in postcapillary venules. The adhesion was inversely correlated with vessel diameter, resulting in frequent blockage of small-diameter venules. In contrast, preparations treated with PAF/SOD or PAF/catalase showed a marked decrease in adhesion in venules of all diameters resulting in a significantly lower Y-intercept of the regression line as compared with the control group (p<0.00001). On the other hand, PNA caused almost complete inhibition of SS RBC adhesion in PAF-treated preparation, demonstrating even a greater anti-adhesive efficacy as evidenced by lower Y-intercept (p<0.001–0.0001 vs. SOD and catalase). The decrease in adhesion was accompanied by a significantly lower peripheral resistance (p<0.03–0.018). These results show that antioxidants inhibit SS RBC adhesion to endothelium stimulated by PAF, probably by scavenging and/or blocking PAF-generated oxidants. Thus, blockade of oxidant generation may constitute an effective therapeutic strategy to prevent SS RBC adhesion.

Protein Kinases Associated with Activation of Sickle Red Blood Cell Adhesion.

We have previously found that activation of adenylate cyclase in sickle red cells (SS RBCs) results in increased RBC adhesion to cultured endothelial cells (EC) through activation of protein kinase A (PKA). Protein tyrosine phosphorylation by src family kinases, known to be present in normal RBCs, is also involved in enhancing SS RBC adhesion to EC, as inhibition of these kinases also inhibits activation of adhesion. However, in normal RBCs neither activation of adenylate cyclase nor cAMP analogs elicits a similar increase in cell adhesion. We therefore investigated whether normal and SS RBCs differed with respect to the protein kinases downstream of cAMP, such as PKA catalytic and regulatory subunits and protein tyrosine kinases p56lck, p56lyn and p72syk. We also investigated the extracellular signal regulated kinases p44 and p42 (ERK 1 and 2 respectively), which can be activated downstream of tyrosine kinases. PKA catalytic and regulatory subunits and ERK1/2 were studied after SS and normal RBCs were sham-treated or treated for 30 min with 80 μM forskolin, which activates adenylate cyclase. For protein tyrosine kinases, both SS and normal RBCs were sham-treated or treated with 1μg/ml Pertussis toxin, which suppresses Gαi-mediated inhibition of adenylate cyclase. Equal amounts of total membrane proteins were separated by polyacrylamide gel electrophoresis before transfer to nitrocellulose membrane. Protein kinases were detected in isolated membranes by immunoblotting using specific antibodies. Immunoblots showed that the levels of membrane-associated PKAcβ subunit were similar in both SS and normal RBCs and did not change after forskolin stimulation. However, treatment of SS RBCs with forskolin induced a significant reduction in membrane-bound PKARIIβ subunit. In general, dissociation of PKAR (regulatory) subunits is associated with activation of the catalytic (c) domain. In contrast, no change in the amount of membrane-bound PKARIIβ was observed when normal RBCs were treated with forskolin. These data demonstrate that PKARIIβ dissociated from PKAcβ after adenylate cyclase activation in SS RBCs, consistent with activation of PKAcβ. Our results thus suggest that activation of PKAcβ might be one of the mechanisms contributing to increased SS RBC adhesion. The amount of membrane-bound PKARIIα in SS RBCs did not change with stimulation, suggesting that PKAcα may not undergo activation as a result of forskolin stimulation. Immunoblots also showed no change in the levels of membrane-bound p56lck, p56lyn and p72syk detected before and after normal or SS RBCs were stimulated with Pertussis toxin. However, normal RBCs contained lower levels of protein tyrosine kinases, including p56lyn, p56lck and p76syk. We also found that ERK1/2 are bound to the membrane in normal and SS RBCs. The total amount of membrane-associated ERK1 (p44) is markedly higher in SS RBCs than in normal RBCs, and membrane ERK1 levels increased only in SS RBCs after stimulation with forskolin. Taken together, our results suggest that PKAcβ is activated in SS RBCs but not in normal RBCs in response to elevation of cAMP. The higher levels of src kinases in SS RBCs may also contribute to an increased adhesive response to adenylate cyclase activation compared to normal RBCs. In addition, we have obtained the first evidence that ERK1/2 is conserved in enucleated human erythrocytes and that ERK1 is recruited to bind to SS RBC membranes as a result of adenylate cyclase activation, suggesting that it might be involved in regulating cell adhesion.

Modulation of Erythrocyte Adhesion by Changes in Cellular Tonicity and Volume.

The sickle red blood cell (SS-RBC) membrane manifests many abnormal properties, including oxidative damage of membrane proteins and lipids, disruption of the lipid bilayer, abnormal clustering of surface proteins, dehydration, and increased adhesive properties. Conditions that alter the RBC membrane may modify or abnormally expose its native components and contribute to the adhesive properties of the RBC. We have previously reported that sickle RBCs have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. Since a significant proportion of sickle RBCs are inherently dehydrated, in part due to intracellular K+ loss via the Gardos and K-Cl co-transport channels, we test the hypothesis that dehydration-induced alterations in membrane organization or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilized TSP under flow conditions. Expanding on our previous preliminary results, we found that sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (p<0.005), suggesting that cellular dehydration may contribute to its adhesive phenotype. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (p<0.01). Furthermore, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides chondroitin sulfate A and high molecular weight dextran sulfate, but not by the anionic polysaccharide chondroitin sulfate C. TSP adhesion of sickle and dehydrated normal RBCs in our in vitro system was also not inhibited by competitors of CD47-, band 3-, or RBC phosphatidylserine-meditaed adhesion. Increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers; this adhesion was also inhibited by chondroitin sulfate A and high molecular weight dextran sulfate but not by chondroitin sulfate C. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs, as compared to untreated normal murine RBCs, to kidney capillaries following reinfusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, and suggest that changes in volume are more important than changes in intracellular tonicity in the adhesion of RBCs to TSP. There are striking similarities in the adhesive properties of sickle and dehydrated normal RBCs, supporting the theory that adhesion of sickle RBCs arises as the result of exposure or modification of normal erythroid membrane components. Characterization of the membrane epitope(s) responsible for adhesion of dehydrated normal erythrocytes to TSP may thus provide a parallel avenue to identify reagents that can inhibit RBC adhesion and resultant vasoocclusion in SCD.

The contribution of endothelial cell P-selectin to the microvascular flow of mouse sickle erythrocytes in vivo

AbstractMicrovascular occlusion in sickle cell disease can be initiated by adhesion of sickle red blood cells (RBCs) to the endothelium. Our objective in this study was to verify the relevance in vivo of our discovery that sickle RBCs adhere abnormally to endothelial P-selectin in vitro. We used computer-assisted intravital microscopy to characterize RBC flow velocity (VRBC) in mice. We found faster VRBC of sickle RBCs in P-selectin knock-out and control mice than in sickle cell mice, which have increased endothelial cell P-selectin expression. Agonist peptide for murine protease-activated receptor-1 (PAR-1), which selectively activates mouse endothelial cells but not platelets, was used to assess the effects of endothelial cell P-selectin on microvascular flow. Suffusion of venules with this agonist stopped flow promptly in normal and sickle mice but not in P-selectin knock-out mice or in control mice pretreated with anti-P-selectin monoclonal antibody or unfractionated heparin (UFH). Agonist-induced slowing of flow was reversed rapidly by suffusion with UFH, provided flow had not already stopped. We conclude that endothelial cell P-selectin contributes to the microcirculatory abnormalities in sickle cell disease and that blocking P-selectin may be useful for preventing painful vasoocclusion in sickle cell disease. (Blood. 2004;104:3378-3385)

Sickle cell adhesion depends on hemodynamics and endothelial activation

Venular microvascular circulation in patients with sickle cell anemia exhibits reduced and episodic blood flow. Sickle erythrocyte adhesion to postcapillary venule endothelium is postulated to initiate and propagate episodes of vasoocclusive pain. Hemodynamics likely mediate the adherence of sickle cells to endothelium, controlling delivery of potentially adherent erythrocytes and removal of loosely adherent erythrocytes on the endothelium. This study found a high dependence on shear stress of sickle erythrocyte adhesion to vascular cell adhesion molecule-1 (VCAM-1) on endothelium stimulated by tumor necrosis factor-alpha (TNF-alpha). Shear stress varied from 1.0 dyne/cm 2 (microvascular venular flow), in which VCAM-1 ligand interactions induced by TNF-alpha primarily controlled adherence, to 0.1 dyne/cm 2 (low flow), in which stimulation had little effect on adherence. At shear stresses analogous to in vivo velocities from laser Doppler ultrasound studies (0.8 and 0.6 dyne/cm 2 ), TNF-alpha promoted 1.9- and 2.7-fold increases in adhesion compared with unstimulated (baseline) adherence. These findings suggest a dynamic vasoocclusive process that depends on both receptor expression and shear stress. These results indicate that, in the microvasculature, slightly reduced inflow rate, increased receptor expression, or both may result in large increases in sickle erythrocyte adhesion.

In Vivo Studies of Sickle Red Blood Cells

The defining clinical feature of sickle cell anemia is periodic occurrence of painful vasoocclusive crisis. Factors that promote trapping and sickling of red cells in the microcirculation are likely to trigger vasoocclusion. The marked red cell heterogeneity in sickle blood and abnormal adhesion of sickle red cells to vascular endothelium would be major disruptive influences. Using ex vivo and in vivo models, the authors show how to dissect the relative contribution of heterogeneous sickle red cell classes to adhesive and obstructive events. These studies revealed that (1) both rheological abnormalities and adhesion of sickle red cells contribute to their abnormal hemodynamic behavior, (2) venules are the sites of sickle cell adhesion, and (3) sickle red cell deformability plays an important role in adhesive and obstructive events. Preferential adhesion of deformable sickle red cells in postcapillary venules followed by selective trapping of dense sickle red cells could result in vasoocclusion. An updated version of this 2-step model is presented. The multifactorial nature of sickle red cell adhesion needs to be considered in designing antiadhesive therapy in vivo.

Sickle red cell microrheology and sickle blood rheology.

The hallmark of the phenotypic expression of sickle cell disease is the remarkable degree of heterogeneity in the clinical manifestations. They vary latitudinally among patients and longitudinally in the same patient. The pathogenesis of sickle cell anemia centers on the sequence of events that occur between polymerization of deoxy hemoglobin S and increased red cell destruction, vasoocclusion, and end organ damage. Cellular dehydration, changes in sickle red blood cell rheology, adhesion of sickle red cells to vascular endothelium, inflammatory response, and tissue injury are some of the factors that contribute to hemolytic anemia, vasoocclusion, and eventual multiorgan damage. The focus of this review is on the rheology of sickle blood and microrheology of sickle RBC. Determinants of sickle RBC rheology and the factors that modulate its severity are discussed.

Diminished sickle cell adhesion molecules following bone marrow transplantation (BMT)

Diminished sickle cell adhesion molecules following bone marrow transplantation (BMT)

Vaso-occlusion in sickle cell anemia: role of interactions between blood cells and endothelium.

Sickle cell anemia (SCA) is one of the most common inherited blood disorder. It has been defined as a molecular disease more than 50 years ago and since then has given rise to an enormous amount of investigations. SCA is usually characterized by three major features, namely hemolytic anemia, painful vaso-occlusive crises (VOC) and susceptibility to infections. Other potentially life-threatening complications include splenic sequestration, acute chest syndrome, and stroke. Finally, evolution during adulthood, now commonly observed, is complicated by chronic organ damage. Despite a unique mutation in the beta-globin gene and a common basic mechanism based upon the polymerization of deoxyhemoglobin S (Hb-S) and vaso-occlusion by sickled red blood cells, its phenotypic expression is highly variable, and the occurrence of the major complications is unpredictable. Most particularly, events triggering vaso-occlusion still remain largely unknown. In the recent years, the discovery of an abnormal adhesion of sickle red blood cells to the vascular endothelium has provided important new insights. It is clear now that all the circulating cells as well as the endothelium are part of the phenomenon and that cell activation and inflammatory processes play a major role in the pathogenesis of the disease.

Inflammatory mediators promote strong sickle cell adherence to endothelium under venular flow conditions.

Adherence of sickle erythrocytes to endothelium in venules is thought to initiate or propagate vaso-occlusive episodes. Because of blood shear forces with normal microvascular flow, adherence in post-capillary venules requires binding via high-affinity receptor-mediated pathways. Microvascular flow in sickle patients is episodic, even in asymptomatic patients, so adherence may also occur at low shear not requiring high-affinity binding. Sickle cell binding to endothelium was quantified under flow or static incubation with unusually large vWF, thrombospondin, alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/fibronectin (FN). Adherence under flow at 0.5 dyne/cm(2) shear stress leads to the greatest number of adherent sickle cells. Adherence under flow at 1.0 dyne/cm(2) leads to the strongest adherence. Static incubation conditions promote weak adherence of low numbers of sickle cells to endothelium. Following attachment at 1.0 dyne/cm(2), adherence strength was 2.5 +/- 0.1 or 2.6 +/- 0.2 dynes/cm(2) for alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/FN pathways, a level 50% greater than adherence strength mediated by thrombospondin or ULvWF (1.7 +/- 0.08 or 1.6 +/- 0.07 dynes/cm(2), respectively). Sickle cell adhesion promoted by simultaneous activation of alpha(4)beta(1)/VCAM-1 and alpha(4)beta(1)/FN pathways is the strongest at 6.2 +/- 0.2 dynes/cm(2) and adherent red cells resist detachment shear stresses up to 10 dynes/cm(2). These data demonstrate that sickle cell adhesion to endothelium is regulated both by receptor/ligand affinity and flow conditions. Thus, both microvascular flow conditions and receptor-ligand interactions may regulate sickle cell adherence in vivo.

Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion

AbstractThe enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.

Hydroxyurea downregulates endothelin-1 gene expression and upregulates ICAM-1 gene expression in cultured human endothelial cells.

The clinical efficacy of oral hydroxyurea (HU) in adults and children with sickle cell anemia (SCA) cannot solely be explained by its ability to enhance fetal hemoglobin (HbF) expression. Since increased adherence of sickle red blood cells to vascular endothelium is a possible contributing factor to vaso-occlusive crisis (VOC), we explored the effect of HU on human endothelial cell (EC) lines (TrHBMEC and EA-hy 926). We demonstrated that HU, in a dose-dependent and reversible manner, significantly decreased (up to three-fold) the release of endothelin-1 (ET-1), a vasoconstrictor peptide through downregulation (up to three-fold) of ET-1 gene expression. This finding is of therapeutic relevance as SCA patients exhibit elevated serum levels of ET-1 during episodes of VOC and levels correlate with disease severity. Unexpectedly, HU upregulated (up to three-fold) the expression of membrane-bound intercellular cell adhesion molecule 1 (mbICAM-1) and its soluble form (sICAM-1) with a parallel increase in ICAM-1 mRNA expression. Although ICAM-1 does not appear to be involved in the sickle cell adhesion to vascular endothelium, it may exacerbate vaso-occlusion by promoting leukocyte adhesion. The HU-induced increase in mbICAM-1 may appear inconsistent with the clinical benefits confered by HU. However, both the increase in sICAM-1- and HU-induced leukocyte reduction in patients, may counteract the potentially detrimental effect of elevated mbICAM-1 expression. Also HU reduces the expression of vascular cell adhesion molecule (VCAM-1) on EC. Since HU reduces the very late antigen 4-positive reticulocytes in SCA patients, a ligand for VCAM-1, HU-induced downregulation of VCAM-1 on EC will very likely decrease the reticulocyte-endothelium adhesion. Thus, HU, apart from inducing HbF expression in the red cell, also affects the expression profile of EC compartment.

Heparin inhibits the flow adhesion of sickle red blood cells to P-selectin

The adhesion of sickle erythrocytes to vascular endothelium is important to the generation of vascular occlusion. Interactions between sickle cells and the endothelium use several cell adhesion molecules. We have reported that sickle cell adhesion to endothelial cells under static conditions involves P-selectin. Others have shown that sickle cell adhesion is decreased by unfractionated heparin, but the molecular target of this inhibition has not been defined. We postulated that the adhesion of sickle cells to P-selectin might be the pathway blocked by unfractionated heparin. In this report we demonstrate that the flow adherence of sickle cells to thrombin-treated human vascular endothelial cells also uses P-selectin and that this component of adhesion is inhibited by unfractionated heparin. We also demonstrate that sickle cells adhere to immobilized recombinant P-selectin under flow conditions. This adhesion too was inhibited by unfractionated heparin, in a concentration range that is clinically attainable. These findings and the general role of P-selectin in initiating adhesion of blood cells to the endothelium suggest that unfractionated heparin may be useful in preventing painful vascular occlusion. A clinical trial to test this hypothesis is indicated.

Sickle erythrocyte adherence to endothelium at low shear: role of shear stress in propagation of vaso-occlusion.

Under venular flow conditions, sickle cell adherence to endothelium is mediated by cell adhesion molecules and adhesive proteins associated with inflammation, coagulation, and endothelial perturbation. Periodic and reduced blood flow are observed in sickle microcirculation during hematologic steady state, suggesting that blood flow is compromised in sickle microcirculation. We tested the hypothesis that low blood flow enhances adherence by quantifying sickle cell adhesion to endothelium under venular flow (1.0 dyne/cm(2) shear stress) and low flow (0.1 dyne/cm(2) shear stress), with and without addition of adhesion promoting agonists. Under low flow, sickle cell adherence to endothelium increases with contact time in the absence of endothelial activation or adhesive protein addition. In contrast, at venular shear stress, sickle cell adherence only occurs following endothelial activation with TNF-alpha or addition of thrombospondin. Analysis of these data with a mathematical model reveals that at low flow adherence is "transport-controlled," meaning that contact time between sickle cells and endothelium is a more important determinant of adherence than high-affinity receptor-ligand interactions. Low-affinity interactions are sufficient for adhesion at low flow. In contrast, at venular flow (1 dyne/cm(2) shear stress) adherence is "affinity-controlled," meaning that adherence requires induction of specific high-affinity receptor-ligand interactions. These findings demonstrate that in addition to activating factors and adherence proteins, microvascular shear stress is an important determinant of sickle cell adhesion to endothelium. This suggests that in vivo, erythrostasis is an important determinant of adhesion that can act either independently or concurrently with ongoing acute events to induce adhesive interactions and vaso-occlusion.

P-selectin mediates the adhesion of sickle erythrocytes to the endothelium.

The adherence of sickle red blood cells (RBCs) to the vascular endothelium may contribute to painful vaso-occlusion in sickle cell disease. Sickle cell adherence involves several receptor-mediated processes and may be potentiated by the up-regulated expression of adhesion molecules on activated endothelial cells. Recent results showed that thrombin rapidly increases the adhesivity of endothelial cells for sickle erythrocytes. The current report presents the first evidence for the novel adhesion of normal and, to a greater extent, sickle RBCs to endothelial P-selectin. Studies of the possible interaction of erythrocytes with P-selectin revealed that either P-selectin blocking monoclonal antibodies or sialyl Lewis tetrasaccharide inhibits the enhanced adherence of normal and sickle cells to thrombin-treated endothelial cells. Both RBC types also adhere to immobilized recombinant P-selectin. Pretreating erythrocytes with sialidase reduces their adherence to activated endothelial cells and to immobilized recombinant P-selectin. Herein the first evidence is presented for the binding of normal or sickle erythrocytes to P-selectin. This novel finding suggests that P-selectin inhibition be considered as a potential approach to therapy for the treatment of painful vaso-occlusion in sickle cell disease.

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.

Integrin-associated protein is an adhesion receptor on sickle red blood cells for immobilized thrombospondin

AbstractThe adhesive protein thrombospondin (TSP) potentially mediates sickle (SS) red blood cell (RBC) adhesion to the blood vessel wall, thereby contributing to vaso-occlusive crises in sickle cell disease. We previously reported that SS RBCs bind to immobilized TSP under flow conditions, whereas normal (AA) red cells do not. However, the SS RBC receptors that mediate this interaction are largely unknown. Here it is reported that integrin-associated protein (IAP), or CD47, mediates the adhesion of these cells to immobilized TSP under both flow and static conditions. A peptide derived from the C-terminal IAP binding site of TSP also supports sickle cell adhesion; adhesion to this peptide or to TSP is inhibited specifically by the anti-IAP monoclonal antibody, 1F7. Furthermore, these data suggest that IAP on SS RBCs is structurally different from that expressed on AA RBCs but that IAP expression levels do not vary between AA and SS RBCs. This structural difference may contribute to the enhanced adhesion of SS RBCs to immobilized TSP. These results identify IAP as a TSP receptor on SS RBCs and suggest that this receptor and its binding site within TSP represent potential therapeutic targets to decrease vaso-occlusion.