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Biomimetic Thrombomodulin Conjugates and their Biological Roles

Gruzdys, Valentinas
http://rave.ohiolink.edu/etdc/view?acc_num=csu1462318995

Abstract Details

2016, Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, College of Sciences and Health Professions.
Hemostatic balance is maintained by a complex series of procoagulant and anticoagulant reactions involving systemic and local factors. Thrombomodulin (TM) that is expressed on endothelium contributes to hemostatic balance by serving as an essential cofactor for activation of both procoagulant and anticoagulant protein. Therefore, TM is an attractive molecule for studying procoagulant and anticoagulant mechanisms and for developing novel procoagulant and anticoagulant agents. In order to study TM function and develop TM-based anticoagulant agents, three biomimetic TM conjugates were investigated in this dissertation study, (i) liposomal TM conjugate, (ii) immobilized membrane system with TM and (iii) TM-glycopolymer conjugate. TM activates both protein C (PC) and thrombin activatable fibrinolysis inhibitor (TAFI). Lipid membrane association is involved in both activation processes, however, molecular details remain uninvestigated. Therefore, initial investigations focused on the lipid effects on the cofactor activities of TM via liposomal constructs with different lipids: phosphatidylcholine (PtCho), phosphatidylserine (PtSer) and phoshatidylethanolamine (PtEtn). Liposomal TM was assayed for PC and TAFI activation, respectively. Subsequent studies measured fibrinolysis with increased fibrin clot dissolution rates observed in high PtSer lipid environments due to lower TAFI activation. Subsequently, immobilized membrane system with TM was proposed to generate a biomimetic TM-based antithrombotic membrane system, which may be applied for biomedical device surface coating applications. A chemoselectively surface functionalizable tethered bilayer lipid membrane (tBLM) was developed via liposome immobilization, rupture and fusion processes. Introduction of triphenylphosphine (TP)- PEG-lipid into a liposome resulted in immobilization onto an azide surface via Staudinger ligation. Subsequent rupture of the immobilized liposome followed by a second TM- containing liposome fusion lead to the tBLM formation, which allowed for incorporation of membrane TM. The tBLM containing TM was assayed for PC activation, confirming successful incorporation of the membrane protein. Lastly, glyco-modification of recombinant TM with chain-end functionalized glycopolymers utilizing isourea bond and copper-free click chemistries was achieved. The protein glycoconjugates were confirmed by SDS-PAGE, western blot, and MALDI-TOF Mass Spectrometry. PC activation contribution by the glyco-modified recombinant TM was confirmed, showing no reduction in cofactor role of the glycol-modified TM. Overall, this work demonstrates methodologies for activity assessment, incorporation into biomimetic structures, as well as successful glycopolymer modification of TM.
Xue-Long Sun, PhD (Advisor)
David Anderson, PhD (Committee Member)
Michael Kalafatis, PhD (Committee Member)
Chandrasekhar Kothapalli, PhD (Committee Member)
Sihe Wang, PhD (Committee Member)
Aimin Zhou, PhD (Committee Member)
160 p.
Biochemistry; Chemistry
Thrombomodulin, glycopolymer, tethered bilayer lipid membrane, hemostasis

Recommended Citations

Citations

  • Gruzdys, V. (2016). Biomimetic Thrombomodulin Conjugates and their Biological Roles [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1462318995

    APA Style (7th edition)

  • Gruzdys, Valentinas. Biomimetic Thrombomodulin Conjugates and their Biological Roles. 2016. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1462318995.

    MLA Style (8th edition)

  • Gruzdys, Valentinas. "Biomimetic Thrombomodulin Conjugates and their Biological Roles." Doctoral dissertation, Cleveland State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1462318995

    Chicago Manual of Style (17th edition)

csu1462318995
237
© 2016, all rights reserved.
This open access ETD is published by Cleveland State University and OhioLINK.