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Blood, 7 May 2009, Vol. 113, No. 19, pp. 4754-4762.
Prepublished online as a Blood First Edition Paper on December 24, 2008; DOI 10.1182/blood-2008-06-162693.
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Submitted June 20, 2008
Accepted November 14, 2008
Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins
Marie N O'Connor, Isabelle I Salles, Ana Cvejic, Nicholas A Watkins, Adam Walker, Stephen F Garner, Chris I Jones, Iain C Macaulay, Michael Steward, Jaap-Jan Zwaginga, Sarah L Bray, Frank Dudbridge, Bernard de Bono, Alison H Goodall, Hans Deckmyn*, Derek L Stemple, and Willem H Ouwehand
Department of Haematology, University of Cambridge, Cambridge, United Kingdom
Laboratory for Thrombosis Research, Katholieke Universiteit Leuven, Campus Kortrijk, Leuven, Belgium
National Health Service Blood and Transplant, Cambridge, United Kingdom
Domantis Limited, Cambridge, United Kingdom
Department of Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom
Department of Experimental Immunohaematology, Sanquin Research, Amsterdam, Netherlands
Medical Research Council Biostatistics Unit, Institute of Public Health, Cambridge, United Kingdom
European Bioinformatics Institiute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
Wellcome Trust Sanger Institute, Hinxton, United Kingdom
* Corresponding author; email: hans.deckmyn{at}kuleuven-kortrijk.be.
In this study we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell the megakaryocyte, together with nucleated blood cell elements, endothelial cells and erythroblasts, identified novel platelet membrane proteins with hitherto unknown role in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of five of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet Glycoprotein (GP) IIb and the coagulation protein Factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of four of the five novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.
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