Rohun U. Palekar, BS, Chandu Vemuri, MD, Jon N

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Antithrombin nanoparticles inhibit stent thrombosis in ex vivo static and flow models Rohun U. Palekar, BS, Chandu Vemuri, MD, Jon N. Marsh, PhD, Batool Arif, MS, Samuel A. Wickline, MD Journal of Vascular Surgery Volume 64, Issue 5, Pages 1459-1467 (November 2016) DOI: 10.1016/j.jvs.2015.08.086 Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 1 A, Schematic of perfluorocarbon nanoparticles (PFC-NP) with surface-conjugated D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK-NP). B, After implantation, activated thrombin adsorbs to vascular stents forming a prothrombotic coating. NP-PPACK binds to and inactivates this adsorbed thrombin transforming the stent surface with a functionally antithrombotic coating. Journal of Vascular Surgery 2016 64, 1459-1467DOI: (10.1016/j.jvs.2015.08.086) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 2 A, S-2238 incubation confirms thrombin adsorption to stent surfaces. B, Incubation of treated stents with PPP results in significant clot deposition after 60 minutes of exposure with noninhibitor-treated stents compared with lack of clot growth on D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK-nanoparticle [NP])-treated stents. C, Rapid thrombus deposition after 5 minutes of platelet-rich plasma (PRP) exposure, compared with no growth on PPACK-NP-treated stents for the duration of the experiment. D, Pretreatment with vorapaxar demonstrates little benefit in preventing stent thrombosis alone, with significantly less clot deposition when used in combination with PPACK-NP. E, Ultrasound imaging of platelet-poor plasma-exposed stents confirms lack of clot deposition on PPACK-NP-treated stents compared with saline controls. In all panels, ∗P < .05, ∗∗P < .005, ∗∗∗P < .0005. Journal of Vascular Surgery 2016 64, 1459-1467DOI: (10.1016/j.jvs.2015.08.086) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 3 Scanning electron microscopy (SEM) of platelet-rich plasma (PRP)-exposed stents treated with (A) saline, (B) control nanoparticles (NP), (C) free D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK), and (D) NP conjugated with PPACK (PPACK-NP). Stents treated with saline or control NP show deposition of a dense fibrin mesh compared with PPACK or PPACK-NP-treated stents. SEM confirms the deposition of PPACK-NP on the stent surface with a measured diameter of 185.46 ± 2.92 nm. Journal of Vascular Surgery 2016 64, 1459-1467DOI: (10.1016/j.jvs.2015.08.086) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 4 Picrosirus red staining of thrombosed stents after 10 minutes of stent exposure to flowing plasma containing each treatment group and 10 units of thrombin. In saline and control nanoparticle (NP)-treated plasma, occlusive thrombi were deposited on the stents, with minimal deposition on stents in D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK) and PPACK-NP-treated plasma. Journal of Vascular Surgery 2016 64, 1459-1467DOI: (10.1016/j.jvs.2015.08.086) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 5 Thrombin inhibition allows for maintenance of normal cell viability and proliferation in human aortic endothelial cells (HAECs) compared with noninhibitor controls. In saline and control nanoparticle (NP)-treated groups, proliferation of endothelial cells was significantly decreased compared with baseline levels of proliferation after 4 days of thrombin exposure. PPACK, D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone. ∗P < .05. Journal of Vascular Surgery 2016 64, 1459-1467DOI: (10.1016/j.jvs.2015.08.086) Copyright © 2015 Society for Vascular Surgery Terms and Conditions