Background: Thrombomodulin (TBM) is an anticoagulant and anti-inflammatory protein expressed on endothelial cells (ECs). Mutations of TBM are seen in thrombotic microangiopathies due to aHUS. DSA, particularly those against HLA-II, are involved in endothelial allograft damage during ABMR, but mechanistic knowledge is still lacking. Thus far, the effects of anti-HLA I vs II antibodies on TBM have not been characterized.

Methods:We used human glomerular microvascular ECs to examine TBM expression by fluorescence microscopy and western blot on anti-HLA-treated cells. We then tested sera from 55 kidney recipients for soluble TBM by ELISA.

Results: Treatment of ECs with anti-HLA-I led to a dose-dependent increase in membrane TBM expression, whereas treatment with anti-HLA-II and TNF-α led to minimal levels of the protein. Western Blot on extracted membrane proteins confirmed these results and showed a different molecular weight for TBM in the anti-HLA-II well. Neither stimulation with anti-HLA-I nor II increased TBM levels in cell culture supernatants, but TNF-α stimulation induced high levels, suggesting different pathways between TNF-α and anti-HLA-II. We next measured cytosolic TBM and observed an accumulation with anti-HLA-II, but not with TNF-α. In patients, we found a significant association between the presence of circulating anti-HLA-II DSA and low serum levels of TBM.

Conclusions:Ligation of anti-HLA-I and II produces different effects on the endothelial surface expression of TBM and on serum levels in kidney recipients. Anti-HLA-II stimulation leads to a cytosolic accumulation of TBM with little membrane expression, suggesting a modification of the protein. This phenotype may be associated with a prothrombotic state, which could explain the higher occurrence of TG and the poor outcomes observed in patients with these antibodies.

CITATION INFORMATION: Beland S, Desy O, Valin P, De Serres S. Effects of Anti-HLA Antibodies on Endothelial Expression and Serum Levels of Thrombomodulin in Kidney Recipients. Am J Transplant. 2017;17 (suppl 3).

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