title: Four-dimensional characterization of thrombosis in a live-cell, shear-flow assay: development and application to xenotransplantation.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of General Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Donald G
authors.initials: DG
authors.lastname: Harris
authors.affiliation: Division of Cardiac Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Prabhjot K
authors.initials: PK
authors.lastname: Benipal
authors.affiliation: Division of Cardiac Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Xiangfei
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Division of Cardiac Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Lars
authors.initials: L
authors.lastname: Burdorf
authors.affiliation: Division of Cardiac Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Agnes M
authors.initials: AM
authors.lastname: Azimzadeh
authors.affiliation: Division of Cardiac Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America; Surgical Care Clinical Center, VA Maryland Health Care System, Baltimore, Maryland, United States of America.
authors.forename: Richard N
authors.initials: RN
authors.lastname: Pierson
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D001681:Biological Assay; D001696:Biomechanical Phenomena; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical; D042783:Endothelial Cells / Q000276:immunology; D004730:Endothelium, Vascular / Q000276:immunology; D064593:Heterografts; D006648:Histocompatibility; D006801:Humans; D007700:Kinetics; D034421:Sus scrofa; D013927:Thrombosis / Q000276:immunology*
chemical_list: D000925:Anticoagulants
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