Background A limitation of current antiplatelet therapies is their inability to

Background A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. This phosphatase is usually highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated through the collagen receptor glycoprotein VI (GPVI) and the C-type lectin-like receptor 2 (CLEC-2). DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared to wild-type mice and showed severely impaired thrombus formation upon ferric chloride-induced carotid artery injury. Intriguingly bleeding times were not altered in DUSP3-deficient mice. At the molecular level DUSP3 deficiency impaired Syk tyrosine phosphorylation subsequently reducing phosphorylation of PLCγ2 and calcium fluxes. To investigate DUSP3 function in human platelets a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen and T-5224 CLEC-2-induced human platelet aggregation thereby phenocopying the effect of DUSP3 deficiency in murine cells. Conclusions DUSP3 plays a selective and essential role in collagen- and CLEC-2-mediated platelet activation and thrombus formation H1-related (VHR) phosphatase is usually a DSP encoded by the gene. DUSP3 (185 amino acids; Mr 21 kDa) which only contains a catalytic (PTP) domain name 6 has been reported to dephosphorylate the mitogen-activated protein kinases (MAPKs) ERK1/2 and JNK1/2.7 Additional reported substrates include EGFR and ErbB2.8 DUSP3 is implicated in cell cycle regulation and its expression is altered in human cancer.9-11 However since all of these studies were performed either in vitro using recombinant proteins or in cell lines using transient overexpression or siRNA knockdown the true physiological function of DUSP3 has remained elusive. We recently generated a full Dusp3-knockout (Dusp3-KO) mouse.12 Dusp3-KO mice were healthy fertile T-5224 T-5224 and showed no JNKK1 spontaneous T-5224 phenotypic abnormality. However DUSP3 deficiency prevented neo-angiogenesis and bFGF-induced microvessel outgrowth.12 In the present study we identified DUSP3 as a key and nonredundant player in GPVI- and CLEC-2-mediated signaling pathways in mouse and human platelets. We show that DUSP3 deficiency limits platelet activation and arterial thrombosis. Moreover we developed a specific small-molecule inhibitor of DUSP3 which was able to phenocopy DUSP3 deficiency in platelets. Methods Platelet RNA sampling and Microarray Platelets from 256 healthy volunteers were isolated from citrate-anticoagulated blood. Donors were informed about the objectives of the study and signed an informed consent. The study was approved by the ethical committee review board of the Liège University Hospital. RNA extraction and microarray procedures are described in the Supplementary Material. Mice C57BL/6-Dusp3-KO were generated by homologous recombination.12 Heterozygous mice were mated to generate +/+ and ?/? littermates used for experimentation (8-12 weeks old male mice). All experiments were approved by the local ethics committee. Isolation of human and mouse platelets Human platelets were prepared from peripheral blood freshly drawn from healthy donors as previously described.13 Mouse washed platelets (WPs) were prepared as previously described.14 Isolation of human and murine B and T cells Human B and T cells were sorted from freshly collected blood using EasySep B and T cell-negative selection kits (Stemcell Technologies). Mouse B and T cells were sorted from spleens. Platelet aggregation analyses Light T-5224 transmission was recorded during platelet aggregation induced by collagen convulxin (CVX) collagen related peptide (CRP) rhodocytin thrombin U46619 or ADP in the presence of 2 mM CaCl2 on a Lumi-Aggregometer (Chrono-log). Flow cytometry WPs were stimulated for 15 min with different concentrations of collagen CRP thrombin or ADP under non-stirring conditions. Saturating concentrations of FITC-conjugated P-selectin and PE-conjugated JON/A antibodies were added. Samples were analyzed on a FACSCantoII flow cytometer (BD Biosciences). Electron microscopy Platelet pellets were fixed for 60 min in 2.5% glutaraldehyde in S?rensen’s buffer (0.1 M pH 7.4) post-fixed for 30 min with 1% osmium tetroxide dehydrated in a series of ethanol concentrations and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined on a Jeol-CX100II transmission electron.