Synthesis of Biomimetic Proteoglycan and Its Antithrombotic Activity

Cleveland State University, Cleveland OH

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Abstract

Project Summary/Abstract The objective of this proposal is to develop novel method to synthesize recombinant thrombomodulin (TM)-glycosaminoglycan (GAG) conjugates and investigate the significance of GAG on the antithrombotic activity and pharmacokinetic properties of recombinant TM. TM is an endothelial cell membrane proteoglycan that plays essential roles in controlling vascular haemostatic balance by activating anticoagulant and anti-inflammatory proteins and simultaneously inactivating procoagulant and proinflammatory proteins. Pathological activation of the endothelium results in TM deficiency leading to uncontrolled thrombosis. Therefore, recombinant TM or its active fragments seems to represent an effective approach for both treatment and prevention of thrombotic diseases. TM serves a receptor for thrombin and directly inhibits thrombin?s procoagulant activity. Furthermore, TM-thrombin complex accelerates the conversion of protein C to its active form (APC), which is an anticoagulant protease that selectively inactivates coagulation factors Va and VIIIa. The EGF-like domains 4,5,6 of TM has cofactor activity for thrombin binding and subsequently protein C activation. Thus, recombinant TM of the EGF- like domains 4,5,6 (rTM456) is a potent anticoagulant candidate. TM is a proteoglycan and its GAG contributes TM?s additional thrombin binding and overall protein stability. In this proposal, we hypothesize that chemical glyco-engineering of rTM456 with defined GAG or GAG mimetics will enhance the rTM456?s anticoagulant activity and improve the rTM456?s stability and solubility in aqueous solutions and biological fluids. Three specific aims will be investigated to test the hypothesis, which are: (i) chemical and enzymatic synthesis and characterization of rTM456-GAG conjugates; (ii) Evaluation of in vitro anticoagulant activity, toxicity, stability, and solubility of rTM456-GAG conjugates; and (iii) evaluation of in vivo antithrombotic activity of rTM456-GAG conjugates by using endotoxin-induced disseminated intravascular coagulation (DIC) model in rats and its pharmacokinetics in rats. Overall, these studies will establish effective method for synthesizing proteoglycans and add to our understanding of the biological functions of EGF-like domains 4,5,6 and GAG of TM. Furthermore, this research will lay the foundation for developing TM-based antithrombotic agent with enhanced antithrombotic activity and pharmacokinetic properties.