Functionally distinct anticoagulant mechanisms of endothelial cells

Abstract

Antithrombin and tissue factor pathway inhibitor (TFPI) provide different anticoagulant mechanisms. Having established a potent anticoagulant role of cultured human umbilical vein endothelial cells in vessel-on-a-chip microfluidic models, we now investigated how these cells modulated thrombin generation under stasis through antithrombin and TFPI pathways. We observed that endothelial monolayers in 96 well-plates strongly delayed and suppressed the thrombin generation process induced by tissue factor, regardless of the presence of whole blood, platelet-rich plasma or platelet-free plasma. Intervention studies indicated that the blocking of heparin-like proteoglycans with polybrene or protamine sulphate, similarly as the absence of antithrombin in plasma, reverted the endothelial anticoagulant activity. Heparinase treatment of the cells also reduced the anticoagulant potential. Furthermore, the presence of andexanet-alpha (inactivated factor Xa) and an anti-TFPI antibody were able to revert the endothelial effects. Jointly, these data point to additive anticoagulant mechanisms of endothelial cells through surface-expressed heparin-like proteoglycans and TFPI, both contributing to thrombin inhibition.