(118a) Modification of Polyethylene Terephthalate Surfaces and Analysis of Immobilized Ntpdase Kinetics

When blood comes in contact with foreign materials, platelets become activated and secrete ADP molecules, leading to platelet aggregation in a concentration-dependent manner. In nature, ADP-induced platelet activation is counteracted by nucleoside triphosphate diphosphohydrolase (NTPDase). NTPDase rapidly metabolizes ADP to AMP and inorganic phosphate. In this study, NTPDase was immobilized on polyethylene terephthalate (PET) surfaces to inhibit ADP-induced platelet adhesion.

Initially, a primary amine group was attached to PET by aminolysis using ethylenediamine. NTPDase was then immobilized via an imine bond between the primary amine on PET and the aldehyde group on the enzyme. NTPDase was also immobilized on aminated PET by carbodiimide coupling, which resulted in an amide bond between the primary amine on PET and the carboxylic acid group on the enzyme. Kinetic studies showed significant NTPDase activity using both processes. However, studies showed that the imine bonds are unstable and the reaction kinetics are mass transfer limited. Due to cracks forming on PET following exposure to ethylenediamine, it was postulated that the mass transfer problems were due to transport limitations of ADP within the cracks.

To avoid cracks on polymer surface, PET surfaces were modified by introducing carboxylic acid groups. For this purpose, benzylic hydroxyl groups were first introduced to PET to form carboxyl groups on the surface. NTPDase was immobilized on carboxylated PET (PET-COOH) surfaces by carbodiimide coupling, which results in an amide bond between the carboxylic acid groups on PET and the ε-aminolysine groups on the enzyme. SEM pictures showed that polymer surface had no cracks?which is important for any biomedical applications. Kinetic studies showed significant NTPDase activity and the amide bond used for immobilization was stable.

After successfully immobilizing NTPDase, kinetic studies were performed in a batch system by exposing an ADP solution to the NTPDase modified PET-COOH in the presence of 5 mM CaCl2 and 0.1 M Tris buffer (pH 7.4). The rate of hydrolysis of ADP at physiological conditions (37ºC and pH 7.4) was studied by measuring the release of inorganic phosphate. Based on these results, the kinetic parameters for immobilized NTPDase kinetics were calculated. For understanding the kinetics in a continuous flow system, NTPDase kinetic experiments were performed using a slit flow chamber.

Future studies will evaluate in-vitro studies of NTPDase modified polymers using platelet suspensions, platelet rich plasma and whole blood to establish the effectiveness of the polymers in inhibiting platelet adhesion.