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)

Anionic polysaccharides inhibit adhesion of sickle erythrocytes to the vascular endothelium and result in improved hemodynamic behavior.

The abnormal adherence of sickle red blood cells (SS RBC) to vascular endothelium may play an important role in vasoocclusion in sickle cell anemia. Thrombospondin (TSP), unusually large molecular weight forms of von Willebrand factor, and laminin are known to enhance adhesion of SS RBC. Also, these endothelial proteins bind to sulfated glycolipids and this binding is inhibited by anionic polysaccharides. Reversible sickling may expose normally cryptic membrane sulfatides that could mediate this adhesive interaction. In this study, we have investigated the effect of anionic polysaccharides, in the presence or absence of TSP, on SS RBC adhesion to the endothelium, using cultured human umbilical vein endothelial cells (HUVEC) (for the adhesion assay) and the ex vivo mesocecum of the rat (for hemodynamic evaluation). The baseline adhesion (ie, without added TSP) of SS RBC to HUVEC was most effectively inhibited by high molecular weight dextran sulfate (HDS), whereas low molecular weight dextran sulfate (LDS) and the glycosaminoglycan chondroitin sulfate A (CSA) also had significant inhibitory effects. Heparin was mildly effective whereas other glycosaminoglycans (chondroitin sulfates B and C, heparan sulfate, and fucoidan) were ineffective. Similarly, HDS and CSA resulted in an improved hemodynamic behavior of SS RBC. Soluble TSP caused significant increases in SS RBC adhesion and in the peripheral resistance. Both HDS and CSA prevented TSP-enhanced adhesion and hemodynamic abnormalities. Thus, anionic polysaccharides can inhibit SS RBC-endothelium interaction in the presence or absence of soluble TSP. These agents may interact with RBC membrane component(s) and prevent TSP-mediated adhesion of SS RBC to the endothelium.

Inhibition of sickle erythrocyte adhesion to immobilized thrombospondin by von Willebrand factor under dynamic flow conditions.

Sickle red blood cell (RBC) adhesion to the blood vessel wall is hypothesized to be the initiating event in the periodic vaso-occlusive episodes that characterize sickle cell disease (SCD). Thrombospondin-1 (TSP) and von Willebrand factor (vWF) have each been implicated in the adhesion of sickle RBC to vascular endothelial cells (EC) and subendothelial matrices. To better understand the contributions of each of these adhesive glycoproteins, we examined the adhesion of sickle RBC to immobilized TSP and vWF using a parallel plate flow chamber. Under postcapillary venular shear stress (1 dyne/cm2), sickle RBC adhered preferentially to TSP. To explore potential interactive effects of vWF and TSP, we examined sickle RBC adhesion to mixtures of these proteins. Whether the proteins were first combined in solution or sequentially applied to the slide, the presence of vWF inhibited the binding of sickle RBC to TSP. The inhibition of adhesion by vWF was shown to be the result of specific and saturable binding of vWF to TSP. Furthermore, vWF in solution at normal plasma levels also inhibited RBC adhesion to immobilized TSP. These data indicate that sickle RBC adhesion in vivo may be significantly influenced by the relative concentrations of TSP and vWF in the vascular wall.

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.

Analysis of a vinyl pyrrolidone/poly(propylene fumarate) resorbable bone cement.

A resorbable bone cement was formulated from N-vinyl-2-pyrrolidinone (VP), the unsaturated polyester poly(propylene fumarate) (PPF), and the inorganic filler tribasic calcium phosphate (hydroxy apatite). Cure, initiated by benzoyl peroxide and accelerated by N,N-dimethyl-p-toluidine, resulted in the formation of VP crosslinks between polyester chains. During cure the cement hardened from a viscous moldable putty to a rigid structure with a shore D hardness of 50-60. The purpose of this study was to determine the fractions of PPF and VP incorporated into the crosslinked structure. Dissolution of the cured cement in water followed by extraction of the residue in tetrahydrofuran indicated that over 90% of the PPF was crosslinked over the range of PPF/VP ratios explored, but that the fraction of VP used in formation of crosslinks depended linearly on the PPF/VP ratio. Kinetic analysis of these data suggests that k'pp/kpf (the reactivity ratio) was approximately 2.0 where k'pp is the rate constant for the addition of VP radical to VP monomer leading to formation of poly(vinyl pyrrolidone), and kpf is for the addition of VP radical to PPF unsaturation.

Endothelial cell interactions with sickle cell, sickle trait, mechanically injured, and normal erythrocytes under controlled flow.

Increased adhesive forces between sickle erythrocytes and endothelial cells (EC) have been hypothesized to play a role in the initiation of vasoocclusion in sickle cell anemia. Erythrocyte/human umbilical vein EC interactions were studied under controlled flow conditions for normal (AA), homozygous sickle cell (SS), sickle cell trait (AS), mechanically injured normal, and "high-reticulocyte control" RBC by using video microscopy and digital image processing. The number of adherent RBC was determined at ten-minute intervals during a washout period. Results indicate that SS RBC were more adherent than AA RBC. Mechanically injured (sheared) AA RBC were also more adherent than control normal cells but less adherent than SS RBC. AS RBC did not differ significantly in their adhesive properties from normal RBC. Less-dense RBC were more adherent to EC than dense cells for normal, SS, and high-reticulocyte control RBC. The number of cells adherent at a given time during washout was a very strong function of wall shear rate. In addition, at all shear rates studied, the average velocity of individual SS RBC in the region near the EC surface was approximately half that of AA RBC at the same bulk volumetric flow rate through the flow chamber. These findings suggest that the increased adhesion of sickle RBC is at least partially related to the increased numbers of less-dense RBC present. Increased adherence of the less-dense cells to the EC lining vessel walls could contribute to microvascular occlusion by lengthening vascular transit times of other sickle cells.

Rheological studies of erythrocyte-endothelial cell interactions in sickle cell disease.

The abnormal adherence of sickle erythrocytes to endothelial cells (EC) has been hypothesized to play a role in the initiation of vaso-occlusion in sickle cell anemia. We studied erythrocyte/endothelial cell interactions under controlled flow conditions for normal (AA), homozygous sickle cell (SS), sickle cell trait (AS), mechanically injured normal, and "high reticulocyte control" red blood cells (RBC). Human umbilical vein endothelial cells grown to confluence on glass slides formed the base of a parallel plate flow chamber into which RBC suspensions were perfused at a constant flow rate, producing a wall shear stress of 1 dyne/cm2. Adhesion was monitored using video microscopy, and the number of adherent RBC was determined at ten-minute intervals during a wash out period. Results indicate that SS RBC were more adherent than AA RBC. Mechanically injured (sheared) RBC were also more adherent than control normal cells, but less adherent than SS RBC. AS RBC did not differ significantly in their adhesive properties from normal RBC. Less dense (younger) RBC were more adherent to EC than dense (older) cells for normal, SS and "high reticulocyte control" RBC. These findings suggest that the increased adhesion of sickle RBC is at least partially related to the increased numbers of young RBC present. Increased adherence of young cells to the EC lining vessel walls could contribute to microvascular occlusion by lengthening vascular transit times of other sickle cells. Microvascular occlusion is a major clinical problem in sickle cell anemia. This obstruction to blood flow could be due to decreased deformability of the cell and its inability to pass through small vessels. If this were the case it would be reasonable to expect that the most severely deformed sickle cells, the irreversibly sickled RBC (ISC), would play an important role in the initiation of vaso-occlusion. However, the number of circulating ISC is not well correlated with the frequency of painful crises and other microvascular occlusive phenomena. Recent evidence suggests that microvascular occlusion may be associated with increased adhesion of sickle cells to vascular endothelial cells. A strong correlation between erythrocyte adherence and clinical vaso-occlusive severity has been reported by Hebbel et al. Hoover et al. and Hebbel et al. demonstrated increased adhesion in static tests.(ABSTRACT TRUNCATED AT 400 WORDS)