Four-Dimensional Characterization of Thrombosis in a Live-Cell, Shear-Flow Assay: Development and Application to Xenotransplantation

<div><p>Background</p><p>Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications.</p><p>Methodology/Principal Findings</p><p>Confluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm² and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001).</p><p>Conclusions/Significance</p><p>This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions.</p></div

A—C. Representative volumetric thrombus models.

<p>3D surface renderings of end-perfusion images (<i>t</i> = 50 minutes) for WT and GalTKO endothelia perfused with heparinized human blood (A & B, respectively) and WT endothelium perfused with heparinized human blood treated with 0.5 μg/mL abciximab (C). Panel A demonstrates significant adhesion and aggregation. In Panel B adhesion is reduced but aggregation is mostly intact, while in Panel C aggregation is ablated. Source images were taken at 100x magnification using a 100-millisecond exposure and rendered using ImageJ.</p

Results from perfusing porcine endothelia with syngeneic and allo-incompatible porcine blood.

<p>*All experiments were performed with heparinized blood. a.u., arbitrary units; TV, thrombus volume; Δ, relative change; SA, percent surface area coverage; FR, fluorescence ratio; T₅₀, time to 50% maximal surface area coverage. All Δ & <i>P</i> values are versus WT controls, and are expressed as mean ± SEM.</p><p>Results from perfusing porcine endothelia with syngeneic and allo-incompatible porcine blood.</p

Results from xenoperfusion of porcine endothelia with human blood.

<p>*All experiments were performed with heparinized blood. a.u., arbitrary units; TV, thrombus volume; Δ, relative change; SA, percent surface area coverage; FR, fluorescence ratio; T₅₀, time to 50% maximal surface area coverage. All Δ & <i>P</i> values are versus WT controls, and are expressed as mean ± SEM.</p><p>Results from xenoperfusion of porcine endothelia with human blood.</p

A & B. Representative xenothrombosis on wild type and GalTKO endothelia.

<p>Sequential source images of a single A) WT and B) GalTKO cell line endothelia perfused with heparinized human blood taken at 0, 10, 20, 30, 40 and 50-minute intervals (left-to-right by row). Panel A demonstrates the typical high-grade thrombosis stimulated by WT endothelium, with early and diffuse adhesion and aggregation. In Panel B, the GalTKO genotype reduces and delays adhesion, resulting in less overall thrombosis. However, relative aggregation remains intact where thrombus is present. Images are at 100x magnification using a 100-millisecond exposure. Scale bars: 100μm.</p

Thrombus generation curves for primary wild type and GalTKO endothelia.

<p>Summary of thrombus volume for primary WT versus GalTKO.hCD46 endothelia perfused with heparinized human blood. a.u., arbitrary units. Values are corrected for streak artifact (presumed embolization events) and photo-bleaching, and are expressed as mean ± SEM.</p