The Relationship of Plasma Laminin Levels to Anemia and the Effect of Soluble Laminin on Sickle Red Cell Adhesion in SCD.
Authors: Ko Myung , De Castro Laura M. , Jonassaint Jude C. , Batchvarova Milena , Zennadi Rahima , Telen Marilyn J. 
Published: April 2020

Abstract
Sickle red cell (SS RBC) adhesion is believed to be a major factor in vaso-occlusion. SS RBCs adhere especially avidly to the extracellular matrix protein laminin (LAM), which is found in the subendothelial basement membrane as well as soluble in plasma. We asked (1) Whether the level of soluble LAM in plasma is related to chronic complications of SCD; and (2) Whether soluble LAM enhances the ability of SS RBCs to adhere to endothelial cells (ECs). We first established an ELISA assay to quantitate plasma LAM and found that plasma from all normal subjects tested had LAM levels ≤0.5 μg/mL, which was comparable to the lower limit of detection for our assay. However, plasma from HbSS patients in steady state had levels ranging from ≤0.5 to &gt;8.1 μg/mL. Of 52 HbSS patients studied while in steady state, 5 had plasma LAM levels ≤0.5 μg/mL, 22 had levels &gt;0.5 and &lt;2.0 μg/mL, and 25 had levels between 2 and 8.1 μg/mL. We then compared these levels to a variety of clinical and laboratory endpoints. While patients with higher LAM levels (&gt;3.0 μg/mL) did not have a higher frequency of stroke, acute chest syndrome, priapism, leg ulcers, or aseptic necrosis than patients with levels &lt;1 μg/mL, there was an inverse relationship between plasma LAM level and hemoglobin (Hb) (p=0.004 by linear regression analysis). Patients with baseline (without hydroxyurea) Hb ≤8.5 g/dL (n=28) had mean plasma LAM levels of 3.44±0.42 (SEM) μg/mL, while patients with Hb &gt;8.5 g/dL had LAM levels of 1.72±0.41 μg/mL (n=18, p=0.008). To evaluate whether soluble LAM contributed to SS RBC adhesion, we assayed adhesion using TNF-activated human umbilical vein endothelial cells in a graduated height flow chamber. When SS RBCs were first preincubated with physiologic amounts of LAM, we saw a marked increase in SS RBC adhesion to ECs, and this effect was related to the amount of LAM added. With addition of 5 μg/mL LAM, adhesion to ECs at 2 and 5 dynes/cm2 was increased by 20% and 46% over baseline, respectively. Addition of 10 μg/mL LAM enhanced adhesion at these shear stresses by a mean of 154% and 249%, respectively (n=4). Furthermore, SS RBCs from patients with lower plasma LAM levels had lower baseline adhesion in the absence of added LAM. We then incubated SS RBCs with ABO-compatible SCD plasmas with either high or low LAM levels, washed these cells, and tested them for their ability to adhere to ECs. Plasmas with high LAM (n=3, LAM 4.5–8.1 μg/mL) strongly enhanced adhesion by a mean of 71.9% over baseline at 2 dynes/cm2, while those with low LAM levels (0.4–0.6 μg/mL) enhanced adhesion by only 21.1%. Finally, in order to show that it was the LAM in these plasmas that enhanced SS RBC adhesion, rather than another factor elevated in parallel with LAM, we precleared high LAM plasma with monoclonal antibody reactive with LAMs-10,-11 and anti-murine Ig-Sepharose beads and then compared the activity of this plasma to the same plasma sham precleared with only Sepharose beads. Plasma LAM levels were also assayed to confirm removal of LAM by this procedure. Plasmas from which LAMs-10,-11 had been removed failed to enhance SS RBC adhesion, while the same plasmas subjected to sham preclears increased SS RBC adhesion to ECs by 76.9% at 2 dyne/cm2 (n=2). Thus, we conclude that plasma LAM can enhance SS RBC adhesion to ECs, and elevated levels of plasma LAM may contribute to vaso-occlusion. In addition, the inverse correlation of plasma LAM levels to Hb levels further suggests a relationship between SS RBC adhesion and the severity of anemia in SCD.