Blood-contacting medical devices, such as vascular grafts, stents, heart valves, and catheters, are often used to treat cardiovascular diseases. These implantable medical devices, even if labeled as biocompatible, can cause serious complications in patients. Thrombus formation and infection are the main causes of failure of these devices. In contrast to the healthy endothelium, which actively resists thrombosis, artificial surfaces promote clotting through a complex series of interconnected processes that include protein adsorption, adhesion of platelet, leukocytes and red blood cells, ending with thrombosis.

Nitric oxide is an important intercellular and intracellular signal molecule that regulates the cardiovascular, nervous, and immune systems. It is generated from L-arginine by a family of enzymes, called nitric oxide synthases. Under physiological conditions, a constitutive Ca2+-dependent NOS in endothelial cells (eNOS) is responsible for the continuous release of NO. In addition to its role in vasodilation and regulation of vascular tone, continuous release of NO by the endothelium has also been shown to counteract the adhesion of platelets to the inner walls of blood vessels. From this standpoint, NO is a natural antithrombotic agent in blood vessels. In this regard, NO-releasing films as coatings on blood-contacting devices have shown the potential to be effective in preventing platelet adhesion, activation, and aggregation, therefore reducing the risk of thrombus formation on the treated surfaces.

In this project, we use layer-by-layer thin film building strategy to form layers of polyethyleneimine (PEI) and recombinant NOS as NO-releasing coatings. Charge-based Layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/PEI films. When surfaces coated with PEI/NOS multilayered films are exposed to substrate arginine, a source of reducing equivalents, and other ingredients of the NOS reaction, nitric oxide is formed and released. In this work, we characterize the PEI/NOS thin films in terms of structure of NOS within the films as well as the amount of active NOS. Fourier transform infrared (FTIR) spectroscopic analysis was used to characterize structure-activity relationships of these NOS-containing thin films. We used cyclic voltammetry to determine the active enzyme concentration on the modified surfaces, and how this relates to enzymatic activity in terms of NO released fluxes from the thin films. Finally, we conduct platelet adhesion assays to determine if the amount of platelets adsorbed on the PEI/NOS films is affected by the amounts of NO released from these coatings.