Sickle erythrocytes adhere to fibronectin-thrombospondin-integrin complexes exposed by thrombin-induced endothelial cell contraction.

Vascular damage appears to be associated with sickle erythrocyte (SS RBC) adherence to the endothelium. Thrombin, which has been found in abnormal levels in many sickle patients, causes endothelial cell (EC) retraction and increased SS RBC adherence, and SS RBC adhere in the gaps opened between the EC. Our objective was to elucidate the mechanism of adherence to activated EC monolayers and to determine whether the matrix proteins thrombospondin (TSP) and fibronectin (FN) are mediators of this adherence. Thrombin activation elicited the same 2.5-fold increase in adherence whether 10 or 35% of the matrix was exposed, and the majority of the RBC adhered at the edges of the EC regardless of the extent of matrix exposed. Using static adherence assays we investigated whether TSP, FN, or the integrins alpha(v)beta(3) and alpha(5)beta(1) mediated adherence. Blocking antibodies to any of these four had no effect on adherence to untreated monolayers. However, all the increased adherence elicited by thrombin was abrogated by each one, whereas control antibodies had no effect. Immunofluorescent microscopy demonstrated that both integrins were present on the luminal surface of confluent EC. Neither TSP nor FN was exposed in confluent cultures but they both became available as receptors after EC retraction. These data suggest that SS RBC adhere to a complex of matrix TSP and FN maintained in an adhesive conformation by interactions with both integrins.

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)

Enhanced adherence of sickle erythrocytes to thrombin-treated endothelial cells involves interendothelial cell gap formation.

The adherence of sickle erythrocytes to vascular endothelium has the capacity to initiate vasoocclusion. The known effects of thrombin on endothelial cell function and the increased activity of thrombin in sickle cell disease led us to examine the effect of thrombin on the adhesivity of cultured endothelial cells for sickle erythrocytes. In particular, we studied whether the effect of thrombin on interendothelial cell gap formation (ICGF) was involved in endothelial cell adhesivity for sickle erythrocytes. Those endothelial cell monolayers stimulated by thrombin to maximal levels of static sickle erythrocyte adherence also underwent striking cell contraction and enlargement of interendothelial cell gaps. Adhesivity also increased when gaps were induced with antilaminin antibodies or EDTA. Maximally adhesogenic thrombin conditions failed to increase adhesivity when gap formation was prevented by pretreatment of the monolayers with 8-bromo-cyclic adenosine monophosphate (bromo-cAMP) or glutaraldehyde, agents that respectively inhibit actin-myosin-dependent cell contraction or cross-link adjacent cells in the monolayer. The influence of these two agents on EDTA-enhanced adhesivity was linked to their ability to prevent gap formation. Glutaraldehyde prevented both increased adherence and gap formation; bromo-cAMP prevented neither. Interendothelial cell gap formation may contribute to vasoocclusion by facilitating sickle erythrocyte adherence.