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/cm2, 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, 125I 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.

In vivo biocompatibility and stability of a substrate-supported polymerizable membrane-mimetic film

“…Additionally, we have previously demonstrated the ability of membrane-mimetic films to serve as a versatile template for the assembly of membrane-bound macromolecules that may lead to improved hemocompatibility and biocompatibility [29][30][31][32]41]. In this report, we evaluate, and subsequently improve, the in vivo stability and biocompatibility of polymerized, self-assembled membrane-mimetic thin films assembled on an alginate/PLL polyelectrolyte multiplayer.…”

“…Moreover, we have demonstrated the ability to functionalize these surfaces by creating glycocalyx-mimetic surfaces [29] and protein C activating surfaces by the functional reconstitution of thrombomodulin [30][31][32]. Significantly, we have recently extended this approach to form membrane-mimetic films on the surface of implantable biomaterials, such as cell encapsulation devices [28] and the lumenal surface of a small diameter ePTFE vascular prosthesis [33].…”

Progress and challenges in macroencapsulation approaches for type 1 diabetes (T1D) treatment: Cells, biomaterials, and devices

“…Instant blood mediated inflammation reaction (IBMIR) is another mechanism for acute graft rejection that involves platelet consumption, complement activation and the initiation of the coagulation cascade (van der Windt et al, 2007). To prevent acute rejection as a result of direct contact with the blood, conjugation of thrombomodulin (Feng et al, 2002; Tseng et al, 2006a; Wilson et al, 2010) and anti-coagulation agents such as heparin and warfarin (Edens et al, 1994; Maillet et al, 1988; Tseng et al, 2006b, c; Wang et al, 2013), use of low-molecular weight dextran sulfate (Goto et al, 2004; van der Windt et al, 2007), and genetic modifications of islets such as adenoviral transduction of complement regulatory factors (CD55, CD59) (Schmidt et al, 2003; van der Windt et al, 2007) have been investigated.…”

Challenges and emerging technologies in the immunoisolation of cells and tissues

“…Moreover, membrane-mimetic films have been assembled on microcapsules containing CHO cells [212] and pancreatic islets [211] with minimal loss of cell viability or function. Importantly, we have previously demonstrated the ability of membrane-mimetic films to serve as a template for the assembly of membrane-bound macromolecules that may abrogate inflammatory responses to encapsulated cells, specifically heparin and the transmembrane enzyme thrombomodulin [213][214][215][216][217]. Though perhaps better known for its anticoagulant properties, immobilized heparin can inhibit the formation of NO through its capacity to bind superoxide dismutase [218], and has been shown to limit complement activity by inhibiting the formation of C3 convertase and the assembly of C5b-9 [219][220][221].…”