title: Enhanced effect of inhibition of thrombin on endothelium in murine endotoxaemia: specific inhibition of thrombocytopenia.
abstract: BACKGROUND
  In systemic endotoxaemia, bacterial lipopolysaccharide causes the rapid expression of tissue factor (TF) and disseminated intravascular coagulation and in animal models, anticoagulants limit pathology and promote survival. Recent studies have emphasised the importance of TF expressed by mononuclear cells for initiating thrombin generation during endotoxaemia and suggested that endothelial cell TF is of little relevance. However, the precise importance of endothelium for intravascular thrombin generation has not been established. In this study, we compared the effect of equivalent levels of hirudin tethered to either endothelium or platelets and monocytes.
  
  
  METHODS
  CD31-Hir-Tg mice express a vesicle-targeted, membrane-tethered hirudin fusion protein on endothelium, platelets and monocytes. Bone marrow chimeras between these mice and C57BL/6 were generated The level of intravascular hirudin expressed during endotoxaemia was quantified by inhibition studies using an anti-hirudin antibody and reference to the circulating thrombin anti-thrombin complexes generated in control mice given soluble hirudin.
  
  
  CONCLUSIONS
  Antibody inhibition studies indicated that individual chimeras expressed similar levels of hirudin fusion protein on endothelium alone as on platelets and leukocytes combined and accordingly, the levels of thrombin anti-thrombin complexes and fibrinogen in each chimera were similar, indicating equivalent inhibition of thrombin generation. However, mice with hirudin on endothelium alone developed significantly less thrombocytopenia. These results suggest a hitherto unrecognized role of endothelium in thrombin-dependent platelet sequestration during endotoxaemia. The data have implications for the development of therapeutic strategies based on targeted anticoagulation to limit disseminated intravascular coagulation.
authors.affiliation: MRC Centre for Transplantation, King's College London, Innate Immunity Section, Division of Transplantation Immunology and Mucosal Biology, Guy's Hospital, Great Maze Pond, United Kingdom SE1 9RT.
authors.forename: Daxin
authors.initials: D
authors.lastname: Chen
authors.forename: John H
authors.initials: JH
authors.lastname: McVey
authors.forename: Anthony
authors.initials: A
authors.lastname: Dorling
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism*; D019446:Endotoxemia / Q000378:metabolism*; D006629:Hirudins / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000494:pharmacology; D013552:Swine; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013921:Thrombocytopenia / Q000097:blood / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D006629:Hirudins; D008070:Lipopolysaccharides; D011993:Recombinant Fusion Proteins; D013925:Thromboplastin; D013917:Thrombin
keywords: BL/6; C57BL/6; EC; Endothelial cell; Endothelial protein C receptor; Endothelium; Endotoxin; FIX; FX; Factor IX; Factor X; HEQ; Hirudin Equivalent”; LPS; Lipopolysaccharide; P-selectin glycoprotein ligand-1; PSGL-1; TAT; TF; Thrombin; Thrombin anti-thrombin; Thrombocytopenia; Tissue factor; WT; Wild-type; ePCR