Recombinant and Chemo-/Bio-Orthogonal Synthesis of Liposomal Thrombomodulin

Summary

Principal Investigator: Xue Long Sun
Abstract: DESCRIPTION (provided by applicant): Thrombotic diseases such as myocardial infarction, stroke, and thromboembolism are severe with significant mortality and morbidity in the United States. Antithrombotic agents can be used for both prevention and treatment of active vascular thrombosis. However, current antithrombotic therapy is limited by the risk of bleeding, hemorrhagic complication. Recent advances in molecular bases of haemostasis have highlighted new targets for novel antithrombotic agent design. Endothelial thrombomodulin (TM) plays a critical role in local haemostasis by binding thrombin and subsequently converting protein C to its active form (APC), which is an anticoagulant protease that selectivelyinactivates coagulation factors VA and VIIIa. In addition, the binding of thrombin to TM drastically alters the thrombin's procoagulant activities to anticoagulant activities. Importantly, TM expression, however, decreases in perturbed endothelial cells, predisposing to thrombotic occlusion and particularly in response to a variety of inflammatory stimuli, direct vessel wall injury, and oxidant stress. TM is a type I membrane protein. The lipid bilayer in which it resides serves as an essential 'cofactor', locally concentrating and coordinating the appropriate alignment of reacting cofactors and substrates for protein C activation. Liposomes, in which lipid composition closely resembles that of cell membranes, have been extensively studied as cell membrane model as well as carrier for delivering certain vaccines, enzymes, drugs, or genes to their active sites. Therefore, we propose a TM-liposome conjugate to mimic the native endothelial antithrombotic mechanism of both TM and lipid components and thus will provide a more forceful than current antithrombotic agent. Using membrane protein as a drug presents special challenges since it is difficult to purify and manipulate an amphiphilic membrane protein and difficult to maintain the active form of a membrane protein as in cell membrane. In this proposal, we want to test a central hypothesis that recombinant and chemo- and bio-orthogonal membrane-mimetic assembling membrane protein thrombomodulin (recombinant TM-liposome conjugate) provides a potent antithrombotic agent and a rational design strategy for generating a membrane mimetic drug. The Specific Aims are the following: (1)Synthesize and characterize recombinant TM (rTM)- liposome conjugates in chemo-/bio-orthogonal approach;(2) Evaluate in vitro antithrombotic activity of the rTM-liposome conjugates;(3) Define the capacity of rTM-liposome conjugates to limit coagulation events as well as their pharmacokinetics in vivo. PUBLIC HEALTH RELEVANCE: Thrombotic disorders continue to represent a major cause of morbidity and mortality in the United States despite available methods of diagnosis and treatment. Currently available anticoagulants share the common property of disrupting normal hemostatic pathways. Anticoagulation is often accompanied by hemorrhagic or other side effects, which necessitate interruption of therapy. Furthermore, no beneficial effects in preventing restenosis after revascularization procedures have yet been obtained with the established antithrombotic agents. Thus, an antithrombotic agent that is safer and more effective than currently available is highly demanded. Recent understanding of haemostasis in the molecular bases has highlighted new targets for novel antithrombotic agent design. Physiologically, endothelial thrombomodulin (TM) plays a critical role in local haemostasis. However, TM expression decreases in perturbed endothelial cells, predisposing to thrombotic occlusion and particularly in response to a variety of inflammatory stimuli, direct vessel wall injury, and oxidant stress. In this proposal, we want to develop a recombinant and chemo-/bio-orthogonal approach to synthesize liposomal TM conjugate that mimics the native endothelial antithrombotic mechanism of both TM and lipid components and thus would be a novel and more potent antithrombotic agent.
Funding Period: 2010-03-09 - 2015-02-28
more information: NIH RePORT

Top Publications

  1. pmc Surface-bound cytomimetic assembly based on chemoselective and biocompatible immobilization and further modification of intact liposome
    Yong Ma
    Department of Chemistry, Cleveland State University, Ohio 44115, United States
    Bioconjug Chem 21:1994-9. 2010
  2. pmc Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin
    Rui Jiang
    Department of Chemistry, Chemical and Biomedical Engineering, Cleveland State University, 2121 Euclid Ave, Cleveland, OH 44115 USA
    Chembiochem 15:42-6. 2014
  3. ncbi Recombinant thrombomodulin of different domains for pharmaceutical, biomedical, and cell transplantation applications
    Lin Wang
    Department of Chemistry, Chemical and Biomedical Engineering, Cleveland State University, Cleveland, Ohio 44115
    Med Res Rev 34:479-502. 2014
  4. pmc Membrane mimetic surface functionalization of nanoparticles: methods and applications
    Jacob Weingart
    Department of Chemistry, Cleveland State University, Cleveland, OH 44115, United States
    Adv Colloid Interface Sci 197:68-84. 2013
  5. pmc Bio-inspired liposomal thrombomodulin conjugate through bio-orthogonal chemistry
    Hailong Zhang
    Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA
    Bioconjug Chem 24:550-9. 2013
  6. ncbi Liposomal glyco-microarray for studying glycolipid-protein interactions
    Yong Ma
    Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA
    Anal Bioanal Chem 404:51-8. 2012
  7. pmc Recent advances in sialic acid-focused glycomics
    Huan Nie
    School of Life Science and Technology, Harbin Institute of Technology, Harbin, Heilongjiang, China
    J Proteomics 75:3098-112. 2012
  8. pmc End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation
    Rui Jiang
    Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, USA
    Bioconjug Chem 23:643-9. 2012
  9. pmc Azide-reactive liposome for chemoselective and biocompatible liposomal surface functionalization and glyco-liposomal microarray fabrication
    Yong Ma
    Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, United States
    Langmuir 27:13097-103. 2011
  10. pmc Liposome surface functionalization based on different anchoring lipids via Staudinger ligation
    Pratima Vabbilisetty
    Department of Chemistry, Chemical and Biomedical Engineering, Cleveland State University, Cleveland, Ohio 44115, USA
    Org Biomol Chem 12:1237-44. 2014

Detail Information

Publications11

  1. pmc Surface-bound cytomimetic assembly based on chemoselective and biocompatible immobilization and further modification of intact liposome
    Yong Ma
    Department of Chemistry, Cleveland State University, Ohio 44115, United States
    Bioconjug Chem 21:1994-9. 2010
    ....
  2. pmc Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin
    Rui Jiang
    Department of Chemistry, Chemical and Biomedical Engineering, Cleveland State University, 2121 Euclid Ave, Cleveland, OH 44115 USA
    Chembiochem 15:42-6. 2014
    ....
  3. ncbi Recombinant thrombomodulin of different domains for pharmaceutical, biomedical, and cell transplantation applications
    Lin Wang
    Department of Chemistry, Chemical and Biomedical Engineering, Cleveland State University, Cleveland, Ohio 44115
    Med Res Rev 34:479-502. 2014
    ..Particularly, recent advances in exploring recombinant TM of different domains for pharmaceutical, biomedical, and cell transplantation applications are summarized. ..
  4. pmc Membrane mimetic surface functionalization of nanoparticles: methods and applications
    Jacob Weingart
    Department of Chemistry, Cleveland State University, Cleveland, OH 44115, United States
    Adv Colloid Interface Sci 197:68-84. 2013
    ..This review herein describes recent advances in the preparations and characterization of integrated functional NPs covered by artificial cell membrane structures and their use in various biomedical applications. ..
  5. pmc Bio-inspired liposomal thrombomodulin conjugate through bio-orthogonal chemistry
    Hailong Zhang
    Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA
    Bioconjug Chem 24:550-9. 2013
    ..The reported liposomal protein conjugate approach provides a rational design strategy for both studying membrane protein TM's functions and generating a membrane protein TM-based anticoagulant agent...
  6. ncbi Liposomal glyco-microarray for studying glycolipid-protein interactions
    Yong Ma
    Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA
    Anal Bioanal Chem 404:51-8. 2012
    ..This liposomal glyco-microarray is simple and broadly applicable and thus will find important biomedical applications, such as studying glycolipid-protein interactions and toxin screening applications...
  7. pmc Recent advances in sialic acid-focused glycomics
    Huan Nie
    School of Life Science and Technology, Harbin Institute of Technology, Harbin, Heilongjiang, China
    J Proteomics 75:3098-112. 2012
    ..This review summarizes these recent advances in glycomic studies on SAs and sialylglycans. Specially, derivatization and capturing of SAs and sialylglycans combined with mass spectrometry analysis are highlighted...
  8. pmc End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation
    Rui Jiang
    Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, USA
    Bioconjug Chem 23:643-9. 2012
    ..This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biological activity...
  9. pmc Azide-reactive liposome for chemoselective and biocompatible liposomal surface functionalization and glyco-liposomal microarray fabrication
    Yong Ma
    Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, United States
    Langmuir 27:13097-103. 2011
    ....
  10. pmc Liposome surface functionalization based on different anchoring lipids via Staudinger ligation
    Pratima Vabbilisetty
    Department of Chemistry, Chemical and Biomedical Engineering, Cleveland State University, Cleveland, Ohio 44115, USA
    Org Biomol Chem 12:1237-44. 2014
    ..Furthermore, the density and accessibility of grafted carbohydrate residues on the liposome surface were evaluated for the two anchoring lipid-derived liposomes with lectin binding, respectively. ..

Research Grants30

  1. Dopamine and Angiotensin Receptor Interactions in Genetic Hypertension
    Robin A Felder; Fiscal Year: 2013
    ..abstract_text> ..
  2. Hemostatic factors and sickle cell disease
    Punam Malik; Fiscal Year: 2013
    ..abstract_text> ..
  3. Factor VIIa Interaction with Endothelial Cell Protein C Receptor
    VIJAYA MOHAN RAO LELLA; Fiscal Year: 2013
    ..The proposed studies will have major and important implications for future treatment strategies of bleeding disorders and sepsis. ..
  4. Endothelial cell phosphatidylserine, topography, and procoagulant activity
    Gary E Gilbert; Fiscal Year: 2013
    ..It is also related to the role of the blood coagulation mechanism in preventing or limiting bacterial infections. Finally, it may also help to explain the relationship of blood coagulation to inflammatory diseases. ..
  5. Structure-function of cytoprotective coagulation proteases and their receptors
    LAURENT OLIVIER MOSNIER; Fiscal Year: 2013
    ..abstract_text> ..