6‐O‐sulfation of endothelial glycocalyx heparan sulfates regulates systemic coagulation

Abstract

ObjectiveThe endothelial glycocalyx is a glycosaminoglycan‐rich layer that lines the surface of all vessels and is critical for vascular homeostasis, including the maintenance of an anticoagulant vascular surface. Heparan sulfate (HS), a linear polysaccharide composed of repeating disaccharide units of glucosamine and iduronic acid, is the major glycosaminoglycan comprising the endothelial glycocalyx. The mechanism by which glycocalyx HS regulates coagulation is unknown. As the biological function of HS derives from its unique pattern of sulfation (2‐O‐, 6‐O‐, and/or N‐sulfation) within constituent disaccharides, we hypothesized that the degree of heparan sulfate sulfation critically regulates systemic hemostasis.MethodsWe performed thromboelastography on whole blood collected from tamoxifen‐treated VE‐Cadherin‐Cre‐ERT2‐Sulf1/2 knockout mice, which have loss of endothelial sulf‐1 (a 6‐O specific HS sulfatase) and thus increased heparan sulfate 6‐O‐sulfation at the endothelial surface. Tamoxifen‐treated floxed mice (no Cre recombinase) served as controls. To determine the effects of HS in clot initiation and propagation, separate from platelet contributions, we treated platelet‐poor plasma from healthy donors (pooled and deidentified) with highly‐sulfated HS (HS09) and lower‐sulfated HS (HS01) and measured endogenous thrombin potential with calibrated automated thrombography. To explore the potential mechanism by which HS functions as anticoagulant, we repeated calibrated automated thrombography with antithrombin‐III‐, heparin cofactor‐II depleted plasma.ResultsMice lacking endothelial sulf‐1 demonstrated increased R‐time, decreased angle, and increased K‐time compared to tamoxifen‐treated controls, suggesting that increased endothelial‐surface 6‐O HS sulfation decreases coagulation. In platelet poor plasma, highly‐sulfated HS09 attenuated thrombin generation in dose‐dependent manner while such effect was not observed with the same dose of less‐sulfated HS01. Repeated analysis using platelet poor plasma depleted of antithrombin‐III and of heparin cofactor‐II showed dose‐dependent decrease of thrombin generation with HS09, suggesting that HS exerts this anticoagulant effect independently of heparin‐like activation of antithrombin‐III.ConclusionThe anticoagulant effect of endothelial‐surface HS is dependent on 6‐O sulfation of its constituent disaccharides, and this effect is mediated by the mechanisms distinct from well‐known heparin anticoagulation mechanisms.