Tuesday, November 9, 2010: 8:30 AM
Grand Ballroom J (Salt Palace Convention Center)
The natural response of the body to a foreign material entering the blood stream is swift adsorption of an array of plasma proteins followed by a chain of biochemical responses that often lead to thrombosis at the surface of the material. Although this process serves as a part of the body's innate immune system it presents an obstacle in the development of effective blood contacting medical devices. In particular it is a concern with devices like vascular grafts and artificial heart values where such a reaction could lead to a number of dangerous outcomes such as a heart attack or stroke. New methods to better enhance material hemocompatibility are required to lessen the danger of such an event. The technique proposed here is the use of antigenic disguise proteins found in various microorganisms to accomplish this goal. Antigenic disguise is a term that can be used to describe any of a number of methods microorganisms employ to shield themselves from being targeted for elimination. This work focuses on the spirochete bacteria, Treponema pallidum, which is believed to bind plasma fibronectin (FN) and use it to prevent detection by the host's immune system. This ability is thought to originate in a number of surface proteins, one of which is labeled Tp0483. The focus of this study was to investigate the binding between Tp0483 and human plasma FN as well as to demonstrate the potential application of this protein in enhancing biomaterial hemocompatibility. Tp0483 was adsorbed to COO- functionalized self-assembled monolayers on gold surfaces and the binding of FN to this protein was investigated using surface plasmon resonance (SPR). It is hypothesized that FN changes its conformation or orientation when binding to Tp0483, hence, contributing to the observed antigenic disguise. FN possesses a number of binding domains for an array of different molecules and if the configuration of FN is altered when binding to Tp0483, it is likely that the availability of these binding domains will also change. The accessibility of the FN-binding domains for RGD and heparin were examined following binding of the FN to Tp0483 and compared to FN binding to the COO- functionalized SAM. The results showed that anti-RGD bound significantly more to FN when the FN was adsorbed onto the unmodified COO- SAM as compared to the Tp0483 surface (205 pg/mm2 versus 63 pg/mm2) indicating that FN binds to Tp0483 through the RGD site making it unavailable for binding to the anti-RGD. Heparin did not bind to FN adsorbed on either Tp0483 or the COO- SAMs indicating that the heparin binding domain on the FN was not accessible in either case. The charge and position of the heparin-binding domain likely accounts for the general lack of heparin binding. The heparin-binding domain possesses a positive charge so it interacts strongly with the negatively charged COO- SAM meaning that it is not available to bind heparin from solution. Additionally, binding to FN on a Tp0483 coated surface is likely inhibited because the primary heparin-binding domain is in close proximity to the RGD site. Studies of the collagen-binding domain on FN are currently ongoing. Hemocompatibilty enhancement was studied by observing the effect of surface adsorbed Tp0483 and/or FN on plasma protein binding. Specifically fibrinogen (Fg), which is a necessary component of inflammatory cell recruitment, was added to a COO- surface coated with Tp0483, FN, or a combination of the two at physiological concentration and the surface concentration determined using SPR. These results demonstrated that the combination of Tp0483 and FN on the surface decreased Fg binding by 79% compared to binding directly on to the COO- SAMS. Fg binding was also decreased when either Tp0483 or FN was absorbed on the surface alone but only by 42% and 45%, respectively. These results support the potential of Tp0483/FN in improving hemocompatibility through inhibition of Fg binding. Current studies are focusing on the effect of Tp0483 and FN on binding of other plasma proteins along with their effects on platelet activation.