Functional Reconstitution of Thrombomodulin within a Substrate-Supported Membrane-Mimetic Polymer Film

Abstract

A stable, protein C activating membrane-mimetic film was produced on a polyelectrolyte multilayer (PEM) by in-situ photopolymerization of a phospholipid assembly containing thrombomodulin (TM). The monoacrylated phospholipid monomer was initially synthesized and prepared as unilamellar vesicles with varying molar concentrations of TM. Notably, in mixed-lipid systems, K m values for protein C activation increased in direct proportion to the mole fraction of polymerizable lipid, which was likely due to reduced membrane mobility after photopolymerization. Membrane-mimetic films were also constructed on planar substrates with predictable surface concentrations of catalytically active TM. Significantly, at a TM surface concentration of 170 fmol/cm 2, the rate of protein C activation was comparable to that measured for a variety of confluent endothelial cell monolayers. Serial measurements of contact angles and protein C activation confirmed short-term film stability under a variety of in vitro conditions. Moreover, 125 I labeling of TM demonstrated little change in TM surface concentration over periods of up to 28 days. Significantly, polymeric lipid membranes functionalized with thrombomodulin efficiently inhibited thrombin generation. We believe that the design of membrane-mimetic films that have the capacity to activate the protein C pathway will provide a useful strategy for generating “actively” antithrombogenic surfaces.