Protamine modifications and their new biomedical applications.

Several new biomedical applications based on varied patterns of protein modifications of protamine, a polycationic protein from fish, were developed. These modification patterns, including non-limited proteolysis, limited proteolysis, and polyethylene glycol modification (PEGylation), were used to tailor protamine to accede specific biomedical applications such as in monitoring thrombolytic therapy and in regulating heparin anticoagulant therapy with improved efficacy and safety. In the first application, novel electrochemical assays suitable to monitor the activities of plasminogen activators (PAs) and plasminogen (PLG) in plasma/blood were designed. These assays were based on the properties of the degradation products produced by the non-limited proteolysis of protamine. They were achieved by using protamine as the substrate for plasmin, the ultimate protease generated in the thrombolytic cascade in plasminogen activation, to measure the activity of the generated plasmin activity during thrombolytic therapy by using a novel protamine-sensitive membrane electrode detection method. The features of detecting both the exogenous PAs and the endogenous PLG activities in plasma prompted a great usefulness of these assays for patients undergoing thrombolytic therapy. In the second application, a detailed description of the preparation, characterization, and in vitro testing of low molecular weight protamine (LMWP) fragments as a potential non-toxic heparin/LMWH antidote were presented. These LMWP fragments were prepared using the limited proteolysis of protamine by thermolysin digestion. By maintaining a similar charge density of protamine, it was found that these LMWP fragments were as effective and, indeed, fully capable of neutralizing a broad spectrum of heparin-induced anticoagulant activity. When comparing with protamine, however, these LMWP fragments exhibited a significantly reduced toxicity and thereby yielding an improved safety profiles. Such properties were reflected by a markedly reduced ability in activating the complement system and in cross-reacting with the anti-protamine antibodies by LMWP; two primary events contributing to protamine-induced toxicity. In the last but not least application, chemical modification of protamine with PEG polymers was used to provide beneficial effects for protamine to serve as a clinical heparin antidote. Preliminary results showed that the PEG-protamine conjugates retained nearly 100% of the heparin-neutralizing function of protamine but yielded a significantly reduced complement-activating ability of protamine. Overall, this dissertation research may shed the light of developing many useful and important biomedical applications for proteins using innovative modification methods.Ph.D.Applied SciencesBiomedical engineeringPharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124957/2/3016818.pd