Thrombin-cleaved syndecan-3/-4 ectodomain fragments mediate endothelial barrier dysfunction

OBJECTIVE: The endothelial glycocalyx constitutes part of the endothelial barrier but its degradation leaves endothelial cells exposed to transmigrating cells and circulating mediators that can damage the barrier or promote intercellular gaps. Syndecan proteins are key components of the endothelial glycocalyx and are shed during disease states where expression and activity of proteases such as thrombin are elevated. We tested the ability of thrombin to cleave the ectodomains of syndecans and whether the products could act directly on endothelial cells to alter barrier function. APPROACH AND RESULTS: Using transmission electron microscopy, we illustrated the presence of glycocalyx in human lung microvasculature. We confirmed expression of all syndecan subtypes on the endothelial surface of agarose-inflated human lungs. ELISA and western blot analysis suggested that thrombin can cleave syndecan-3/-4 ectodomains to produce fragments. In vivo, syndecan-3 ectodomain fragments increased extravasation of albumin-bound Evans blue in mouse lung, indicative of plasma protein leakage into the surrounding tissue. Syndecan-3/-4 ectodomain fragments decreased transendothelial electrical resistance, a measure of cell-cell adhesive barrier integrity, in a manner sensitive to a Rho kinase inhibitor. These effects were independent of glycosylation and thrombin receptor PAR1. Moreover, these cleavage products caused rapid VE-cadherin-based adherens junction disorganization and increased F-actin stress fibers, supporting their direct effect on endothelial paracellular permeability. CONCLUSIONS: We suggest that thrombin can cleave syndecan-3/4 ectodomain into fragments which interact with endothelial cells causing paracellular hyperpermeability. This may have important implications in the pathogenesis of vascular dysfunction during sepsis or thrombotic disease states where thrombin is activated.