Mechanism of Soluble Laminin-Mediated Enhancement of Sickle Red Cell Adhesion to Endothelial Cells.
Authors: Ko Myung S. , Xu Kate K. , Telen Marilyn J. 
Published: April 2020

Abstract
We have previously shown that erythrocytes from patients homozygous for hemoglobin S (SS RBC) adhere to immobilized laminins −10,11 (α5 LAM) with high affinity through the B-CAM/LU receptor. SS RBC also avidly bind soluble LAM via B-CAM/LU. In addition, the interaction between endothelial cell (EC) integrin αVβ3 and erythrocyte ICAM4 is a significant contributor to the adhesion of SS RBC to EC. We have recently shown that soluble LAM, either purified or in plasma, enhances SS RBC adhesion to EC. We have now studied the mechanism by which soluble LAM enhances RBC adhesion to EC and the possible relationship between LAM-enhanced and ICAM-4 mediated adhesion of SS RBC. We tested the adhesion of SS RBC to EC under various potentially adhesion-blocking conditions to determine possible modes of interaction between SS RBC, LAM, SS RBC pre-incubated in LAM (SS RBC+LAM), and EC. We first studied whether blocking RBC-bound LAM with an anti-LAM antibody (Ab) could reduce the adhesion-enhancing effects of LAM. SS RBC were pre-incubated in 10 μg/mL soluble LAM for 1 hr at 37°C. After LAM incubation, SS RBC+LAM were washed and re-incubated for 1 hr in 4C7 mAb, an anti-LAM Ab, followed by washing. Adhesion of SS RBC+LAM incubated in 4C7 was compared to adhesion of SS RBC+LAM incubated with an irrelevant Ab and SS RBC not first incubated in LAM. SS RBC+LAM showed on average more than a two-fold increase in adhesion compared to SS RBC not incubated with LAM, at shear stresses of both 2 and 5 dynes/cm2 (n=3). In contrast, SS RBC+LAM incubated in Ab 4C7 showed &lt; a 50% increase in adhesion compared to SS RBC not pre-incubated in LAM. However, due to variability in adhesion among patient samples, these differences were not significant. In order to determine the EC structure to which SS RBC+LAM might bind, we then incubated EC for 1 hr in an IgM Ab to dystroglycan, a putative LAM receptor on EC. SS RBC+LAM were tested for adhesion to untreated EC, EC treated with anti-dystroglycan, and EC treated with a control IgM. Treatment of EC with anti-dystroglycan decreased the adhesion of SS RBC+LAM by 39.1% and 50.1% at shear stresses of 2 and 5 dynes/cm2, respectively, while EC treated with control IgM showed insignificant decreases in adhesion of 3.3% and 6.6% at shear stresses of 2 and 5 dynes/cm2, respectively (n=3; p=.0013 at 2 dynes/cm2; p=.0175 at 5 dynes/cm2). These results suggest that enhancement of SS RBC adhesion in the presence of LAM is due to LAM interaction with an EC receptor, possibly dystroglycan. We then explored whether the adhesion enhancing effects of LAM could still be observed when the interaction between erythrocyte ICAM4 and EC αVβ3 was blocked. EC were pre-treated for 1 hr with 25 μg/mL of soluble recombinant (sr) ICAM4, in order to block αvβ3 binding sites. We found that pre-incubation of SS RBC in 10 μg/mL LAM failed to enhance RBC adhesion to sr-ICAM4 treated EC (p=.0276 at 2 dynes/cm2, n=3). Similar results were obtained with the 7E3 mAb directed against αVβ3. This suggests that blocking the interaction between ICAM4 and αVβ3 abolishes adhesion despite the presence of RBC-bound LAM. Therefore, we conclude that soluble LAM enhances SS RBC adhesion to EC, probably by providing secondary adhesive interactions that can be abrogated by anti-LAM as well as anti-dystroglycan antibodies. However, the contribution of LAM to SS RBC adhesion was negligible in the absence of interaction between ICAM4 and αVβ3. Thus, the mechanism by which LAM enhances SS RBC adhesion to EC appears to be a complex process, dependent on both the binding of RBC-bound LAM to EC as well as ICAM4 interaction with EC αVβ3.