title: Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions.
abstract: BACKGROUND
  Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions.
  
  
  METHODS
  Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion.
  
  
  RESULTS
  No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions.
  
  
  CONCLUSIONS
  In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.
authors.affiliation: 1 Department of Clinical Research, University of Bern, Bern, Switzerland. 2 Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland. 3 Clinic of Plastic and Hand Surgery, University Hospital, Bern, Switzerland. 4 Clinic of Cardiovascular Surgery, University Hospital, Bern, Switzerland. 5 Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany. 6 Revivicor, Inc., Blacksburg, VA. 7 Division of Immunology and Allergology, University Hospital and Medical Faculty, Geneva, Switzerland.
authors.forename: Anjan K
authors.initials: AK
authors.lastname: Bongoni
authors.forename: David
authors.initials: D
authors.lastname: Kiermeir
authors.forename: Jonas
authors.initials: J
authors.lastname: Schnider
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Jenni
authors.forename: Pavan
authors.initials: P
authors.lastname: Garimella
authors.forename: Andrea
authors.initials: A
authors.lastname: Bähr
authors.forename: Nikolai
authors.initials: N
authors.lastname: Klymiuk
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
authors.forename: David
authors.initials: D
authors.lastname: Ayares
authors.forename: Esther
authors.initials: E
authors.lastname: Voegelin
authors.forename: Mihai A
authors.initials: MA
authors.lastname: Constantinescu
authors.forename: Jörg D
authors.initials: JD
authors.lastname: Seebach
authors.forename: Robert
authors.initials: R
authors.lastname: Rieben
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001706:Biopsy; D001777:Blood Coagulation; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D005342:Fibrinolysis; D005552:Forelimb; D005690:Galactose / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D008297:Male; D051916:Membrane Cofactor Protein / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000473:pathology; D010477:Perfusion; D010958:Plasminogen / Q000737:chemistry; D013552:Swine; D010959:Tissue Plasminogen Activator / Q000737:chemistry; D019076:Transgenes; D014183:Transplantation, Heterologous
chemical_list: D007074:Immunoglobulin G; D007075:Immunoglobulin M; D051916:Membrane Cofactor Protein; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D005690:Galactose