title: Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.
abstract: Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets.
authors: Immunology and Infectious Diseases Laboratory, GIGA-Signal Transduction Unit, University of Liège, B34, 1 Avenue de l'Hôpital, 4000, Liège, Belgium. srahmouni@ulg.ac.be.
authors: Souad
authors: S
authors: Rahmouni
authors: Laboratory of Thrombosis and Haemostasis, GIGA-Cardiovascular Sciences Unit, University of Liège, Liège, Belgium.
authors: Alexandre
authors: A
authors: Hego
authors: Laboratory of Thrombosis and Haemostasis, GIGA-Cardiovascular Sciences Unit, University of Liège, Liège, Belgium.
authors: Céline
authors: C
authors: Delierneux
authors: Laboratory of Thrombosis and Haemostasis, GIGA-Cardiovascular Sciences Unit, University of Liège, Liège, Belgium.
authors: Odile
authors: O
authors: Wéra
authors: Immunology and Infectious Diseases Laboratory, GIGA-Signal Transduction Unit, University of Liège, B34, 1 Avenue de l'Hôpital, 4000, Liège, Belgium.|Laboratory of Thrombosis and Haemostasis, GIGA-Cardiovascular Sciences Unit, University of Liège, Liège, Belgium.
authors: Lucia
authors: L
authors: Musumeci
authors: NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
authors: Lutz
authors: L
authors: Tautz
authors: Laboratory of Thrombosis and Haemostasis, GIGA-Cardiovascular Sciences Unit, University of Liège, Liège, Belgium.
authors: Cécile
authors: C
authors: Oury
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004195:Disease Models, Animal; D054640:Dual Specificity Phosphatase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D006487:Hemostasis / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D051379:Mice; D015539:Platelet Activation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010979:Platelet Function Tests / Q000379:methods; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000494:pharmacology; D013927:Thrombosis / Q000097:blood / Q000201:enzymology* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D010975:Platelet Aggregation Inhibitors; D054852:Small Molecule Libraries; C517025:DUSP3 protein, human; D054640:Dual Specificity Phosphatase 3; C517026:Dusp3 protein, mouse; D017027:Protein Tyrosine Phosphatases
keywords: ADP; Aggregation; Aggregation under flow; Bleeding time; CLEC-2; Calcium; Collagen; DSPs; DUSP3; Dual-specificity phosphatases; Ferric chloride; Flow cytometry; GPVI; Immunoprecipitation; Intravital microscopy; JON/A; PTPs; Platelets; Protein tyrosine phosphatases; Secretion; Selectin; Signaling; Small molecule inhibitors; Thrombin; Thromboembolism; VHR; Western blots