Erythrocyte adhesion is modified by alterations in cellular tonicity and volume

We tested the hypothesis that dehydration-induced alterations in red blood cell (RBC) membrane organisation 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 immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P<0.005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P<0.01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47-, band 3-, or RBC phosphatidylserine-mediated adhesion. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs to kidney capillaries following re-infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.     

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

The 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.     

Glycoprotein IV-independent adhesion of sickle red blood cells to immobilized thrombospondin under flow conditions

The abnormal adherence of red blood cells (RBC to the blood vessel wall is believed to contribute to the vascular occlusion observed in patients with sickle call anemia. The cell adhesion receptors GPIV (CD36) and integrin alpha 4 beta 1 (CD49d/CD29) were previously identified on circulating sickle reticulocytes, and shown to mediate sickle RBC adhesion to the endothelium. The presence of damaged endothelium in these patients suggests that exposed extracellular matrix proteins could provide a potential substrate for sickle RBC adhesion. To determine whether RBC adhesion receptors could mediate adhesion to extracellular matrix proteins, we tested their ability to adhere to a variety of immobilized, purified proteins under flow conditions. Neither sickle nor normal RBC adhered to fibronectin, vitronectin, fibrinogen, or collagen. In contrast, we observed substantial adhesion of sickle but not normal RBC to thrombospondin (TSP). The adhesion was not inhibited with known antagonists of the GPIV-TSP interaction, nor by inhibitors of several other known binding domains in TSP. Moreover, the adhesion was resistant to inhibition by soluble TSP, suggesting that immobilization of TSP exposes an adhesive site that is cryptic on TSP in solution. However, the glycosaminoglycans, chondroitin sulfate A, and dextran sulfate were potent inhibitors of this adhesion. These results suggest that a mechanism distinct from GPIV is responsible for sickle RBC adhesion to immobilized TSP under flow conditions.     

Increased erythrocyte adhesion in mice and humans with hereditary spherocytosis and hereditary elliptocytosis

Mice with disruptions of the red blood cell (RBC) cytoskeleton provide severe hemolytic anemia models in which to study multiorgan thrombosis and infarction. The incidence of cerebral infarction ranges from 70% to 100% in mice with alpha-spectrin deficiency. To determine whether mutant RBCs abnormally bind adhesive vascular components, we measured adhesion of mouse and human RBCs to immobilized human thrombospondin (TSP) and laminin (LM) under controlled flow conditions. Mutant RBCs had at least 10-fold higher adhesion to TSP compared with normal RBCs (P <.006). Mutant relative to unaffected RBC adhesion to LM was significantly (P <.01) increased as well. Treatment of RBCs with the anionic polysaccharide dextran sulfate inhibited mutant RBC adhesion to TSP (P <.001). Treatment of RBCs with antibodies to CD47 or the CD47-binding TSP peptide 4N1K did not inhibit TSP adhesion of RBCs. Previously, we have shown that infarcts in alpha-spectrin-deficient sph/sph mice become histologically evident beginning at 6 weeks of age. TSP adhesion of RBCs from 3- to 4- and 6- to 8-week-old sph/sph mice was significantly higher than RBCs from adult mice (> 12 weeks old; P <.005). While the mechanism of infarction in these mice is unknown, we speculate that changes in RBC adhesive characteristics contribute to this pathology.     

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

The 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. (Blood. 2001;97:2159-2164)     

Hydroxyurea therapy decreases the in vitro adhesion of sickle erythrocytes to thrombospondin and laminin

The adhesion of sickle erythrocytes to the vascular endothelium and subendothelial matrix probably contributes to the pathogenesis of vaso-occlusive disease. The chemotherapeutic agent hydroxyurea (HU) decreases the frequency of vaso-occlusive crises in patients with sickle cell disease. However, the exact mechanism(s) of HU's effect on vaso-occlusive crises is not fully understood. The goal of this study was to determine the effect of HU therapy on the adhesion of sickle erythrocytes to the subendothelial matrix proteins thrombospondin (TSP) and laminin under conditions of flow in vitro. Erythrocytes from patients with severe sickle cell disease on HU therapy (n = 14) had significantly less adhesion to TSP (687 +/- 92 erythrocytes/mm2, mean +/- SE) than untreated patients with severe disease (n = 18, 1176 +/- 117 erythrocytes/mm2, P = 0.003). In addition, there was significantly less adhesion of erythrocytes to immobilized laminin in patients treated with HU (1695 +/- 293 erythrocytes/mm2) than in the untreated patients (2590 +/- 296 erythrocytes/mm2, P = 0.02). Erythrocytes from an additional nine patients with severe sickle cell disease were studied both before and after initiation of HU therapy. Erythrocytes from these patients became less adhesive to both TSP (P = 0.001) and laminin (P = 0.01), a change that was sustained in most patients throughout the duration of the study (2 months to > 12 months). This study suggests that HU modulates the adhesive phenotype of sickle erythrocytes, an effect that may be in addition to, or independent of, other known effects of HU, such as an increase in fetal haemoglobin level.     

The carboxy-terminal cell-binding domain of thrombospondin is essential for sickle red blood cell adhesion.

Sickle red blood cells (SS-RBCs) have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. TSP has at least four domains that mediate cell adhesion. The goal of this study was to map the site(s) on TSP that binds SS-RBCs. Purified TSP proteolytic fragments containing either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (<10% adhesion). However, a 140-kD thermolysin TSP fragment, containing the carboxy-terminal cell-binding domain in addition to the type 1, 2, and 3 repeats fully supported the adhesion of SS-RBCs (126% +/- 25% adhesion). Two cell-binding domain adhesive peptides, 4N1K (KRFYVVMWKK) and 7N3 (FIRVVMYEGKK), failed to either inhibit or support SS-RBC adhesion to TSP. In addition, monoclonal antibody C6. 7, which blocks platelet and melanoma cell adhesion to the cell-binding domain, did not inhibit SS-RBC adhesion to TSP. These data suggest that a novel adhesive site within the cell binding domain of TSP promotes the adhesion of sickle RBCs to TSP. Furthermore, soluble TSP did not bind SS-RBCs as detected by flow cytometry, nor inhibit SS-RBC adhesion to immobilized TSP under conditions of flow, indicating that the adhesive site on TSP that recognizes SS-RBCs is exposed only after TSP binds to a matrix. We conclude that the intact carboxy-terminal cell-binding domain of TSP is essential for the adhesion of sickle RBCs under flow conditions. This study also provides evidence for a unique adhesive site within the cell-binding domain that is exposed after TSP binds to a matrix.     

Thrombospondin mediates adherence of CD36+ sickle reticulocytes to endothelial cells.

Initiation of vasocclusion in sickle disease pathophysiology may involve abnormal red blood cell (RBC) adhesivity to endothelium, a phenomenon influenced by both RBC and plasma factors. Using human umbilical vein endothelial cells and a gravity sedimentation adherence assay, we have examined thrombospondin (TSP) as a plasma factor in this adhesive event. The already-abnormal adherence of sickle RBCs in buffer/albumin is significantly augmented (P < .001) by the addition of TSP, with half-maximal effect at about 0.3 microgram/mL. This effect is abolished by antibodies to either TSP or glycoprotein (GP) IV (CD36), as well as peptides RGDS and CSVTCG. The even greater adherence (P < .005) of sickle RBCs in autologous platelet-rich plasma (without added TSP) is dramatically inhibited by alpha CD36 antibodies (OKM5 and alpha GPIV) and significantly diminished by alpha TSP, by peptides RGDS and CSVTCG, and by two antibodies to the vitronectin receptor (7E3 and LM609). Studies of density-separated subpopulations and of RBC adhesion to immobilized proteins, as well as analysis of sickle RBCs using fluorescence-activated cell sorting and single cell microfluorometry, show that TSP responsiveness is a feature of the immature sickle "stress" reticulocytes, which carry CD36 (and not GPIIbIIIa-like receptors) as the TSP-receptive moiety. The endothelial cell's participation in this phenomenon appears to be more complex, and the data are consistent with the notion that it involves TSP interaction with other plasma proteins and/or multiple receptor structures. Other potential adhesogenic proteins (plasma von Willebrand factor, vitronectin, fibrinogen, and fibronectin) neither exhibited an affinity for reticulocytes nor supported increased sickle RBC adherence when added to buffer/albumin in these assay systems. In aggregate, our results indicate that TSP may be the major promoter of RBC adhesivity in plasma, and they suggest that therapeutic benefit might derive from interference with sickle reticulocyte CD36, as achieved by antibodies and CSVTCG in these studies.     

Novel epinephrine and cyclic AMP-mediated activation of BCAM/Lu-dependent sickle (SS) RBC adhesion

The vasoocclusive crisis is the major clinical feature of sickle cell anemia, which is believed to be initiated or sustained by sickle (SS) red blood cell (RBC) adhesion to the vascular wall. SS RBCs, but not unaffected (AA) RBCs, adhere avidly to multiple components of the vascular wall, including laminin. Here we report a novel role for epinephrine and cyclic adenosine monophosphate (cAMP) in the regulation of human SS RBC adhesiveness via the laminin receptor, basal cell adhesion molecule/Lutheran (BCAM/Lu). Our data demonstrate that peripheral SS RBCs contain greater than 4-fold more cAMP than AA RBCs under basal conditions. Forskolin or the stress mediator epinephrine further elevates cAMP in SS RBCs and increases adhesion of SS RBCs to laminin in a protein kinase A (PKA)-dependent manner, with the low-density population being the most responsive. Epinephrine-stimulated adhesion to laminin, mediated primarily via the beta 2-adrenergic receptor, occurred in SS RBC samples from 46% of patients and was blocked by recombinant, soluble BCAM/Lu, implicating this receptor as a target of cAMP signaling. Thus, these studies demonstrate a novel, rapid regulation of SS RBC adhesion by a cAMP-dependent pathway and suggest that components of this pathway, particularly PKA, the beta 2-adrenergic receptor, and BCAM/Lu, should be further explored as potential therapeutic targets to inhibit SS RBC adhesion.     

Adherence of phosphatidylserine-exposing erythrocytes to endothelial matrix thrombospondin

Phospholipid asymmetry is well maintained in erythrocyte (RBC) membranes with phosphatidylserine (PS) exclusively present in the inner leaflet. The appearance of PS on the surface of the cell can have major physiologic consequences, including increased cell-cell interactions. Because increased adherence of PS-exposing RBCs to endothelial cells (ECs) may be pathologically important in hemoglobinopathies such as sickle cell disease and thalassemia, we studied the role of PS exposure in calcium ionophore-treated normal RBC adherence to human umbilical vein endothelial cell (HUVEC) monolayers. When HUVEC monolayers were incubated with these PS-exposing RBCs, the ECs retracted and the RBCs adhered primarily in the gaps opened between the ECs. A linear correlation was found between the number of PS-exposing RBCs in the population and the number of adhering RBCs to the monolayer. Pretreatment of RBCs with annexin V significantly decreased adherence by shielding PS on the RBCs. Similarly, PS-containing lipid vesicles decreased RBC binding by competing for the PS binding sites in the monolayer. PS-exposing RBCs and PS-containing lipid vesicles adhered to immobilized thrombospondin (TSP) and matrix TSP, respectively, and adherence of PS-exposing RBCs to EC monolayers was reduced by antibodies to TSP and to its EC receptor, alpha(v)beta(3). Together, these results indicate a role for PS and matrix TSP in the adherence of PS-exposing RBCs to EC monolayers, and suggest an important contribution of PS-exposing RBCs in pathologies with reported vascular damage, such as sickle cell anemia. (Blood. 2000;95:1293-1300)     

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

Vaso-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, alpha4beta1. 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 alpha4beta1 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.     

Adhesion of sickle neutrophils and erythrocytes to fibronectin

The pathophysiology of vaso-occlusive crisis in sickle cell disease involves interactions among blood cells, plasma proteins, and vessel wall components. The initial goal of this work was to quantify the adhesion of sickle red blood cells (RBCs) to fibronectin immobilized on glass under both static and dynamic shear stress conditions. High-power microscopic inspection of static assay plates showed striking numbers of adherent neutrophils as well as RBCs. Sickle neutrophils and RBCs were significantly more adherent to fibronectin than the corresponding normal cells in static adhesion assays. Adhesion of both sickle neutrophils and sickle RBCs in dynamic adhesion assays was promoted by a period of static incubation preceding initiation of shear stress conditions. Adherent neutrophils remained attached at shear stresses up to 51 dyne/cm2; most adherent RBCs were attached at shear stresses up to 13 dyne/cm2, but detached at a shear stress of 20 dyne/cm2. Sickle neutrophil adhesion was enhanced significantly by autologous plasma. Elevated levels of plasma interleukin-6 (IL-6; but not IL-1 or IL-8) were found in 6 of 9 sickle cell disease samples examined, and elevated levels of tumor necrosis factor were found in 2 of 9 samples. Plasma IL- 6 levels correlated positively with both the number of sickle neutrophils adherent to fibronectin and the ability of sickle plasma to enhance adhesion of normal neutrophils to fibronectin. These data suggest possible roles for neutrophil activation and for fibronectin in mediating sickle neutrophil and RBC adhesion.     

Oxidative stress activates red cell adhesion to laminin in sickle cell disease

Vaso-occlusive crises are the hallmark of sickle cell disease (SCD). They are believed to occur in two steps, starting with adhesion of deformable low-dense red blood cells (RBC), or other blood cells such as neutrophils, to the wall of post-capillary venules, followed by trapping of denser RBC or leukocytes in the areas of adhesion because of reduced effective lumen-diameter. In SCD, RBC are heterogeneous in terms of density, shape, deformability and surface proteins, which accounts for the differences observed in their adhesion and resistance to shear stress. Sickle RBC exhibit abnormal adhesion to laminin mediated by Lu/BCAM protein at their surface. This adhesion is triggered by Lu/BCAM phosphorylation in reticulocytes but such phosphorylation does not occur in mature dense RBC despite firm adhesion to laminin. In this study, we investigated the adhesive properties of sickle RBC subpopulations and addressed the molecular mechanism responsible for the increased adhesion of dense RBC to laminin in the absence of Lu/BCAM phosphorylation. We provide evidence for the implication of oxidative stress in post-translational modifications of Lu/BCAM that impact its distribution and cis-interaction with glycophorin C at the cell surface activating its adhesive function in sickle dense RBC.     

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca²⁺) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1–10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca²⁺ influx. Yoda1 induced Ca²⁺-dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo−/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.     

Erythrocyte adhesion in sickle cell disease

The vaso-occlusive process in patients with sickle cell disease is complex and is likely to involve interactions between hemoglobin S red blood cells (SS RBCs) and vascular endothelium, as well as between SS RBCs and leukocytes. Vaso-occlusive events lead to recurrent pain and a wide spectrum of end-organ damage, including pulmonary hypertension and renal failure. However, the triggers inducing adhesion and vaso-occlusion are only now being elucidated. Investigators have characterized the ability of a number of RBC surface structures to adhere to both endothelial cells and components of the subendothelial extracellular matrix. In addition, evidence is accumulating to suggest that SS RBC adhesion receptors undergo activation under physiologic conditions. An understanding of these mechanisms at the molecular level should ultimately allow development of new preventive and treatment strategies to abrogate vaso-occlusive events.     

Adhesion of normal erythrocytes at depressed venous shear rates to activated neutrophils,activated platelets,and fibrin polymerized from plasma

Deep vein thrombosis (DVT) is a low flow pathology often prevented by vascular compression to increase blood movement. We report new heterotypic adhesive interactions of normal erythrocytes operative at low wall shear rates (gamma(w)) below 100 s(-1). Adhesion at gamma(w) = 50 s(-1) of washed red blood cells (RBCs) to fibrinogen-adherent platelets was 4-fold less (P <.005) than to collagen-adherent platelets (279 +/- 105 RBC/mm(2)). This glycoprotein VI (GPVI)-triggered adhesion was antagonized (> 80% reduction) by soluble fibrinogen (3 mg/mL) and ethylenediaminetetraacetic acid (EDTA). RBC-platelet adhesion was reduced in half by antibodies against CD36 or GPIb, but not by antibodies against GPIIb/IIIa, von Willebrand factor (VWF), thrombospondin (TSP), P-selectin, beta(1), alpha(v), or CD47. Adhesion of washed RBCs to fibrinogen-adherent neutrophils was increased 6-fold in the presence of 20 microM N-formyl-Met-Leu-Phe to a level of 67 RBCs per 100 neutrophils after 5 minutes at 50 s(-1). RBC-neutrophil adhesion was diminished by anti-CD11b (76%), anti-RBC Landsteiner-Wiener (LW) (ICAM4; 40%), or by EDTA (> 80%), but not by soluble fibrinogen or antibodies against CD11a, CD11c, CD36, TSP, beta(1), alpha(v), or CD47. RBC adhesion to activated platelets and activated neutrophils was prevented by wall shear stress above 1 dyne/cm(2) (at 100 s(-1)). Whereas washed RBCs did not adhere to fibrin formed from purified fibrinogen, adhesion was marked when pure fibrin was precoated with TSP or when RBCs were perfused over fibrin formed from recalcified plasma. Endothelial activation and unusually low flow may be a setting prone to receptor-mediated RBC adhesion to adherent neutrophils (or platelets/fibrin), all of which may contribute to DVT.     

Adherent leukocytes capture sickle erythrocytes in an in vitro flow model of vaso-occlusion

Recent in vivo studies suggest that adherent leukocytes bind RBCs and contribute to the microvascular pathology that characterizes sickle cell disease (SCD). A parallel-plate flow assay was used: to investigate the capture of RBCs by adherent neutrophils, monocytes, and T-lymphocytes; to examine whether RBC capture is elevated in patients with SCD; and to determine whether hydroxyurea (HU) therapy affects these interactions. Four measures of cell-cell adhesion were used: adhesion of leukocytes to TNF-alpha-treated human umbilical vein endothelial cells (HUVECs), percent of adherent leukocytes that captured RBCs, number of RBCs captured per interacting leukocyte, and duration of RBC capture. Leukocyte subpopulations from sickle patients were more adherent to activated ECs and captured more RBCs per interacting leukocyte than the corresponding subpopulations from healthy controls. While HU did not affect leukocyte adhesion to activated ECs, it reduced the proportion of adherent leukocytes that captured RBCs, as well as the number of RBCs captured per neutrophil. T-lymphocytes demonstrated elevated adhesion in all measures, and may be the leukocyte subpopulation whose behavior is most altered in SCD. Our findings suggest that neutrophils, monocytes, and T-lymphocytes could all be involved in adhesive interactions with autologous RBCs in patients with SCD.     

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.     

B-CAM/LU expression and the role of B-CAM/LU activation in binding of low- and high-density red cells to laminin in sickle cell disease

Red blood cells from patients with sickle cell disease (SS RBC) adhere to laminin and over-express the high-affinity laminin receptor basal cell adhesion molecule/Lutheran protein (B-CAM/LU). This receptor has recently been shown to undergo activation in vitro through a protein kinase A-dependent mechanism. Low-density SS RBC express two-thirds more B-CAM/LU than high-density SS RBC. However, high-density SS RBC have been identified as most adherent to laminin under flow conditions. We investigated the ability of low- and high-density SS RBC to interact with laminin under various conditions and explored factors that might be responsible for the differences in B-CAM/LU-laminin interaction between high- and low-density SS RBC. We confirmed that high-density SS RBC adhere to laminin more strongly than low-density SS RBC under flow conditions. However, low-density SS RBC bind soluble laminin most strongly and are the most adherent to laminin under static conditions. Soluble recombinant Lutheran extracellular domain protein completely blocked SS RBC adhesion to laminin under both static and flow conditions. The protein kinase A inhibitor 14-22 amide inhibited adhesion to laminin during flow by high-density SS RBC from patients with strongly adherent cells but had no effect on adhesion observed after a static phase. Deletion of the cytoplasmic domain of B-CAM as well as mutation of the juxtamembranous tyrosine residue failed to reduce B-CAM-mediated adhesion to laminin by transfected MEL cells. These studies confirm that B-CAM/LU is the most critical receptor mediating adhesion to laminin under both static and flow conditions. Dense SS RBC are most adherent to laminin despite bearing fewer laminin receptors, apparently due to a reversible protein kinase A-dependent process that is unlikely to involve direct phosphorylation of B-CAM/LU. Our results also suggest that the nature of the interaction of B-CAM/LU with laminin may be different under static and flow conditions.     

Whole blood viscosity and red blood cell adhesion: Potential biomarkers for targeted and curative therapies in sickle cell disease

Sickle cell disease (SCD) is a recessive genetic blood disorder exhibiting abnormal blood rheology. Polymerization of sickle hemoglobin, due to a point mutation in the β-globin gene of hemoglobin, results in aberrantly adhesive and stiff red blood cells (RBCs). Hemolysis, abnormal RBC adhesion, and abnormal blood rheology together impair endothelial health in people with SCD, which leads to cumulative systemic complications. Here, we describe a microfluidic assay combined with a micro particle image velocimetry technique for the integrated in vitro assessment of whole blood viscosity (WBV) and RBC adhesion. We examined WBV and RBC adhesion to laminin (LN) in microscale flow in whole blood samples from 53 individuals with no hemoglobinopathies (HbAA, N = 10), hemoglobin SC disease (HbSC, N = 14), or homozygous SCD (HbSS, N = 29) with mean WBV of 4.50 cP, 4.08 cP, and 3.73 cP, respectively. We found that WBV correlated with RBC count and hematocrit in subjects with HbSC or HbSS. There was a significant inverse association between WBV and RBC adhesion under both normoxic and physiologically hypoxic (SpO₂ of 83%) tests, in which lower WBV associated with higher RBC adhesion to LN in subjects with HbSS. Low WBV has been found by others to associate with endothelial activation. Altered WBV and abnormal RBC adhesion may synergistically contribute to the endothelial damage and cumulative pathophysiology of SCD. These findings suggest that WBV and RBC adhesion may serve as clinically relevant biomarkers and endpoints in assessing emerging targeted and curative therapies in SCD.