A Monoclonal Antibody to Lactadherin Inhibits Sickle Red Blood Cell Adhesion to Vascular Endothelial Cells in a Plasma Milieu.

Adherence of red blood cells to the endothelium initiates vaso-occlusion in sickle cell anemia. The increased adhesiveness of sickle erythrocytes is accompanied by several changes in the lipids of the erythrocyte membrane, including increased expression of phosphatidylserine (PS). One important PS-binding protein is lactadherin (also known as milk fat globule-EGF factor 8), a 45-kDa glycoprotein containing an Arg-Gly-Asp (RGD) sequence. It is secreted by macrophages and is present in normal plasma. Lactadherin promotes phagocytosis of PS-expressing apoptotic lymphocytes and sickle red blood cells by anchoring them to integrins on macrophages. Here, we investigated the role of endogenous lactadherin in adhesion of sickle erythrocytes to the endothelium. We developed a murine monoclonal antibody to human lactadherin, called L688, and investigated its effect on the adhesion of sickle red blood cells to histamine-stimulated human umbilical vein endothelial cells under hydrodynamic flow. In three experiments using washed erythrocytes resuspended in autologous plasma from three different patients with sickle cell anemia, L688 (20 μg/ml) inhibited adhesion by 24–30% (p<0.01). Further evidence for an important role for lactadherin in sickle erythrocyte adhesion to endothelial cells was provided by the observation that exogenous lactadherin enhanced adhesion in a concentration-dependent manner. Lactadherin-mediated adhesion was also inhibited by monoclonal antibody abciximab, (c7E3, 10 μg/ml) which targets the β3 integrin subunit common to both αIIbβ3 and αVβ3. Control antibodies had no effect. Finally, the lactadherin-dependent adhesion of sickle erythrocytes to activated endothelium was inhibited by PS vesicles but not by phosphatidylcholine vesicles, confirming an important role for PS in sickle cell adhesion. Consistent with this, normal erythrocytes can be induced to adhere to stimulated HUVEC in a lactadherin-dependent manner by treatment with N-ethylmaleimide (10 mM) and calcium ionophore A23187 (4 μM) — treatment that exposes PS on the outer leaflet of the red cell membrane. Together, these results indicate that lactadherin mediates sickle cell adhesion to the endothelium by bridging PS on the erythrocytes with αVβ3 integrin on the endothelium. We propose that anemia in sickle cell disease is at least partially due to phagocytosis of lactadherin-coated sickle erythrocytes in the spleen, liver, and lymph nodes. Those erythrocytes that are not ingested immediately by macrophages will become more adhesive for endothelium. Thus, lactadherin appears to be involved both in sickle cell clearance from the circulation and in adhesion to endothelium.