In this proposal, we hypothesize that engineered thin films designed with the capacity for rapid and repeatable covalent recharging of selective molecular constituents, which block thrombin and purinergic pathways will display sustained resistance to thrombus formation in vivo. In the process, the lifetime of bioactive """"""""anti- thrombogenic"""""""" films will be extended with enhanced patency of synthetic small diameter arterial substitutes. Specifically, we intend to: (1) Synthesize and characterize heterofunctional surfaces with """"""""covalently rechargeable"""""""" non-fouling and anti-thrombogenic properties. In the first phase of these investigations, we plan to define the capacity of evolved mutant sortases to facilitate the fabrication of rechargeable, bioactive thin films comprised of species that block thrombin (e.g. TM, heparin) or purinergic (e.g. CD39) pathways. Synthetic heparin oligosaccharides will be used in the course of these studies;enhancing our ability to design glycocalyx-like films. The capacity of these films to block platelet thrombus formation will be determined in vitro and in vivo. During the second phase, rechargeable architectures that contain both non-fouling (e.g. polyethylene glycol) and anti-thrombogenic components will be assessed. (2) Evolve sortase-coupling systems to afford sequence specific transpeptidases with enhanced catalytic activity that facilitate in situ regeneration of biologically active surfaces. In the first phase, directed evolution will be applied to develop new orthogonal sortase variants that display sequence specificity to new peptide motifs. In order to enrich mutants for maximal activity, mutant sortase libraries cloned in yeast cells will be subjected to evolutionary pressure to enhance the affinity for LPESG, LAETG, or LAESG substrates. In the second phase, surviving sortase genes from the first phase will be diversified by gene shuffling and subjected to evolutionary pressure to further enhance catalytic activity by increasing selection stringency, reducing substrate concentration, and shortening reaction time with each successive round. (3) Determine the capacity of blood contacting devices with rechargeable anti-thrombogenic and non- fouling properties to display sustained resistance to thrombus formation in vivo. Film architectures and orthogonal sortase variants that afford rapidly rechargeable anti-thrombogenic films, which effectively inhibit platelet thrombus growth will be used to coat the luminal surface of small diameter ePTFE vascular grafts. Implant studies will be performed using a rabbit carotid interposition model to assess short- and long-term film bioactivity, stability, and rechargeability resistance to platelet deposition, as well as 30-day and 6-month patency and graft healing.

Public Health Relevance

The fabrication of a small diameter vascular prosthesis (<6 mm) remains unsolved due to the absence of a surface coating that reliably resists thrombus formation over clinically relevant time scales. We hypothesize that engineered thin films designed with the capacity for rapid and repeatable covalent recharging of selective molecular constituents, which block thrombin and purinergic pathways will display sustained resistance to thrombus formation in vivo. In the process, the lifetime of bioactive anti-thrombogenic films wil be extended with enhanced patency of synthetic small diameter arterial substitutes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056819-15
Application #
8705566
Study Section
Bioengineering, Technology and Surgical Sciences Study Section (BTSS)
Program Officer
Kindzelski, Andrei L
Project Start
1996-07-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
15
Fiscal Year
2014
Total Cost
$413,308
Indirect Cost
$108,780
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Dydek, E Victoria; Chaikof, Elliot L (2016) Simulated thrombin responses in venous valves. J Vasc Surg Venous Lymphat Disord 4:329-35
Dydek, E Victoria; Chaikof, Elliot L (2016) Simulated Thrombin Generation in the Presence of Surface-Bound Heparin and Circulating Tissue Factor. Ann Biomed Eng 44:1072-84
Ham, Hyun Ok; Qu, Zheng; Haller, Carolyn A et al. (2016) In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A. Nat Commun 7:11140
Kim, Wookhyun; Haller, Carolyn; Dai, Erbin et al. (2015) Targeted antithrombotic protein micelles. Angew Chem Int Ed Engl 54:1461-5
Qu, Zheng; Krishnamurthy, Venkat; Haller, Carolyn A et al. (2014) Immobilization of actively thromboresistant assemblies on sterile blood-contacting surfaces. Adv Healthc Mater 3:30-5
Dorr, Brent M; Ham, Hyun Ok; An, Chihui et al. (2014) Reprogramming the specificity of sortase enzymes. Proc Natl Acad Sci U S A 111:13343-8
Jordan, Sumanas W; Corriere, Matthew A; Vossen, Carla Y et al. (2012) Flow-simulated thrombin generation profiles as a predictor of thrombotic risk among pre-menopausal women. Thromb Haemost 108:258-65
Kim, Wookhyun; Xiao, Jiantao; Chaikof, Elliot L (2011) Recombinant amphiphilic protein micelles for drug delivery. Langmuir 27:14329-34
Jordan, S W; Chaikof, E L (2011) Simulated surface-induced thrombin generation in a flow field. Biophys J 101:276-86
Qu, Zheng; Muthukrishnan, Sharmila; Urlam, Murali K et al. (2011) A biologically active surface enzyme assembly that attenuates thrombus formation. Adv Funct Mater 21:4736-4743

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