Prevention of Thrombogenesis from Whole Human Blood on Plastic Polymer by Ultrathin Monoethylene Glycol Silane Adlayer

In contemporary society, a large percentage of medical equipment coming in contact with blood is manufactured from plastic polymers. Unfortunately, exposure may result in undesirable protein–material interactions that can potentially trigger deleterious biological processes such as thrombosis. To address this problem, we have developed an ultrathin antithrombogenic coating based on monoethylene glycol silane surface chemistry. The strategy is exemplified with polycarbonate–a plastic polymer increasingly employed in the biomedical industry. The various straightforward steps of surface modification were characterized with X-ray photoelectron spectroscopy supplemented by contact angle goniometry. Antithrombogenicity was assessed after 5 min exposure to whole human blood dispensed at a shear rate of 1000 s^–1. Remarkably, platelet adhesion, aggregation, and thrombus formation on the coated surface was greatly inhibited (>97% decrease in surface coverage) compared to the bare substrate and, most importantly, nearly nonexistent

Effects of Dp-3-g on human platelet aggregation.

<p>Human PRP and gel-filtered platelets were pre-incubated with control buffer (black), 0.5 µM Dp-3-g (blue), 5 µM (green) or 50 µM Dp-3-g (red) for 40 min at 37°C. Platelet aggregation with human PRP or gel-filtered platelets was performed at 37°C with a stir speed of 1000 rpm using an aggregometer. A) Human PRP. B) Human gel-filtered platelets. Values are mean ± SD, n = 3 per group. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001, as compared to control buffer.</p

Effects of Dp-3-g on human platelet activation.

<p>Human PRP or gel-filtered platelets were incubated with control buffer, 0.5 µM Dp-3-g, 5 µM Dp-3-g or 50 µM Dp-3-g for 40 min at 37°C. Platelet activation markers were analyzed via flow cytometry after stimulation by ADP, collagen, TRAP or thrombin. A) P-selectin expression on human PRP. B) P-selectin expression on human gel-filtered platelets. C) CD63 expression on human gel-filtered platelets. D) CD40L expression on human gel-filtered platelets. Values are mean ± SEM, n = 3 per group. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001, as compared to control buffer.</p

Effects of Dp-3-g on FeCl₃-induced thrombosis <i>in vivo</i>.

<p>A) Thrombus formation was initiated by topical application of FeCl₃ on mesenteric arterioles in C57BL/6 male mice, which were injected with fluorescently-labeled platelets and different concentration of Dp-3-g or control buffer. Thrombus formation was compared between groups based on the time to complete vessel occlusion. Values are mean ± SD, n = 6–10 per group. *** <i>P</i><0.001, as compared to control buffer. B) C57BL/6 mice were injected with 50 µM Dp-3-g or control buffer. Blood flow in the carotid artery following FeCl₃-induced injury was monitored until complete vessel occlusion was observed. Values are mean ± SD, n = 14 per group. * <i>P</i><0.05.</p

Effects of Dp-3-g on human platelet αIIbβ3 activation and fibrinogen binding.

<p>Human PRP or gel-filtered platelets were incubated with control buffer, 0.5 µM Dp-3-g, 5 µM Dp-3-g or 50 µM Dp-3-g for 40 min at 37°C. Platelet activation markers were analyzed via flow cytometry after stimulation by ADP, collagen, TRAP or thrombin. A) Activated integrin αIIbβ3 expression on platelets in human PRP. B) Activated integrin αIIbβ3 expression on human gel-filtered platelets. C) Platelet-bound fibrinogen in human PRP. D) Platelet-bound fibrinogen on human gel-filtered platelets. Values are mean ± SEM, n = 3 per group. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001, as compared to control buffer.</p

Effects of Dp-3-g on bleeding times in mice.

<p>Tail-vein bleeding times were examined in C57BL/6 mice. Either PBS (control) or different concentrations of Dp-3-g were administered via the tail vein 40 min before the bleeding time was determined. Values are mean ± SD, n = 8–10 per group. No significant differences in bleeding times were observed between treated and untreated mice.</p