Viability of a Novel Peptide P12 as an Antithrombotic Agent

Medical devices and implants are an ongoing point of research due to their impressive capabilities to enhance and support human infrastructure, but pose major challenges as they must incorporate a biocompatible material that can support endothelialization and prevent thrombosis. Specifically, current cardiovascular devices like stents lack reliable endothelialization and because of exposure to constant contact with blood flow within vasculature, result in life-threatening blood clots. Fibrinogen is a plasma-soluble protein that, once cleaved, results in fibrin monomer polymerization into fibrous clots. Previous studies have demonstrated that most stent materials are subject to fibrinogen deposition and denaturation, which results in conversion into fibrin monomers and polymerization through αC domain connections [1]. A multitude of possible solutions have been studied by utilizing varying hydrophobic or hydrophilic surface properties, which feature different materials or coatings with limited success. Here, we will explore incorporation of a surface coating using P12, a peptide derived from fibronectin, to prevent thrombosis.<br/>Initial studies provided insight on how P12 is effective for inhibiting the fibrin polymerization process on polystyrene (PS) surfaces. We studied various fibrinogen and P12 concentrations, but ultimately we found that incorporation of P12 during the incubation of 4 mg/ml of fibrinogen, the concentration in blood, on PS surfaces prevented substantial fiber formations. We also analyzed this at the molecular level with low concentrations of fibrinogen and by using Atomic Force Microscopy and simulation technology, we concluded that the hydrophobic nature of P12 allowed it to bind to exposed αC domains of fibrin on the PS surface. This prevented aggregation of neighboring fibrin monomers and ultimately prevented polymerization into fibers.<br/>Further studies were conducted to overcome the possible negatives of using P12, which includes the uncertainty of P12 reaching the implant or cytotoxic considerations. In order to utilize P12 properly, we investigated using it as a material coating to enhance its effectiveness in a controlled location. We studied this by applying 100 µM of P12 solution onto PS surfaces until full coverage was achieved. Fibrinogen solutions of 4 mg/ml incubated the coated surface for one hour. The P12 coating resulted in &lt;1% of the surface containing fibers and adversely, without the coating resulted in &gt;50% of the surface covered in fibers. From this data, we concluded that a coating of P12 is very effective in thrombosis prevention.<br/>Cytotoxicity is another major problem with implementation of a biomaterial coating. We therefore plated fibroblasts on P12 coated tissue culture plastic (TCP) and added additional P12 to the media to directly test cell survivability. We found that there were no significant differences on P12 coating or adding P12 to cells. These results are indicative of no associated cytotoxicity of the P12 surface. We further investigated this by testing cell adhesion and proliferation on P12 coated polystyrene and found an increase in cell growth and adhesion by the addition of P12 on an otherwise non-biocompatible polystyrene surface. This data concluded that a P12 coating not only rendered surfaces non-thrombogenic and non-toxic, but also enhanced biocompatibility.<br/>Future research will look to investigate the effects of a P12 coating on its ability to support endothelialization and look to create a robust interface that incorporates P12 directly into the matrix of the material chemically so that the coating cannot be degraded or dislodged from the surface.<br/>[1] Zhang, L., Casey, B., Galanakis, D. K., Marmorat, C., Skoog, S., Vorvolakos, K., Simon, M., & Rafailovich, M. H. (2017). The influence of surface chemistry on adsorbed fibrinogen conformation, orientation, fiber formation and platelet adhesion. Acta Biomaterialia, 54, 164–174. https://doi.org/10.1016/j.actbio.2017.03.002