1
Department of Internal Medicine, The University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
,
Neha Bhasin
2
Department of Pediatrics, The University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
,
Edward F. Plow
3
Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Cleveland Clinic, Cleveland, Ohio, USA
,
Pier Paolo Pandolfi
4
Cancer Research Institute, Beth Israel Deaconess Cancer Center, Departments of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
,
Paul B. Rothman
5
Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
,
Anil K. Chauhan
1
Department of Internal Medicine, The University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
,
Steven R. Lentz
1
Department of Internal Medicine, The University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
› Author AffiliationsFinancial Support: This work was supported by grants from the National Cancer Institute (CA134671) to PR and MN and the National Heart, Lung, and Blood Institute (HL118246 and HL118742 to AKC, HL062984 to SRL, and HL080070), a grant from the American Society of Hematology to SRL, and an American Heart Association postdoctoral award to HJ.
Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin β3, but the underlying effects of Dok-1 on αIIbβ3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in αIIbβ3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of αIIβ3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect “inside-out” platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p < 0.05 vs WT), increased PLCγ2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation (p < 0.01 vs WT), demonstrating that Dok-1 negatively regulates αIIbβ3 “outside-in” signalling. Finally, we found that Dok-1-/- mice exhibited significantly shortened bleeding times and accelerated carotid artery thrombosis in response to photochemical injury (p < 0.05 vs WT mice). We conclude that Dok-1 modulates thrombosis and haemostasis by negatively regulating αIIbβ3 outside-in signalling.
Supplementary Material to this article is available online at www.thrombosis-online.com.
7
Moser M,
Bauer M,
Schmid S.
et al. Kindlin-3 is required for beta2 integrin-mediated leukocyte adhesion to endothelial cells. Nature Med 2009; 15: 300-305.
9
Clark EA,
Shattil SJ,
Ginsberg MH.
et al. Regulation of the protein tyrosine kinase pp72syk by platelet agonists and the integrin alpha IIb beta 3. J Biol Chem 1994; 269: 28859-28864.
10
Lipfert L,
Haimovich B,
Schaller MD.
et al. Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. J Cell Biol 1992; 119: 905-912.
14
Obergfell A,
Eto K,
Mocsai A.
et al. Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signalling to the cytoskeleton. J Cell Biol 2002; 157: 265-275.
16
Gong H,
Shen B,
Flevaris P.
et al. G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin “outside-in” signalling. Science 2010; 327: 340-343.
17
Garcia A,
Prabhakar S,
Hughan S.
et al. Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins. Blood 2004; 103: 2088-2095.
18
Hughan SC,
Watson SP.
Differential regulation of adapter proteins Dok2 and Dok1 in platelets, leading to an association of Dok2 with integrin alphaIIbbeta3. J Thromb Haemost 2007; 5: 387-394.
19
Senis YA,
Antrobus R,
Severin S.
et al. Proteomic analysis of integrin alphaIIbbeta3 outside-in signalling reveals Src-kinase-independent phosphorylation of Dok-1 and Dok-3 leading to SHIP-1 interactions. J Thromb Haemost 2009; 7: 1718-1726.
20
Calderwood DA,
Fujioka Y,
de Pereda JM.
et al. Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signalling. Proc Natl Acad Sci USA 2003; 100: 2272-2277.
22
Law DA,
DeGuzman FR,
Heiser P.
et al. Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function. Nature 1999; 401: 808-811.
24
Di Cristofano A,
Niki M,
Zhao M.
et al. p62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl). J Exp Med 2001; 194: 275-284.
26
Wilson KM,
Lynch CM,
Faraci FM.
et al. Effect of mechanical ventilation on carotid artery thrombosis induced by photochemical injury in mice. J Thromb Haemost 2003; 1: 2669-2674.
29
McCarty OJ,
Larson MK,
Auger JM.
et al. Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow. J Biol Chem 2005; 280: 39474-39484.
30
Allen RD,
Zacharski LR,
Widirstky ST.
et al. Transformation and motility of human platelets: details of the shape change and release reaction observed by optical and electron microscopy. J Cell Biol 1979; 83: 126-142.
31
Wonerow P,
Pearce AC,
Vaux DJ.
et al. A critical role for phospholipase Cgamma2 in alphaIIbbeta3-mediated platelet spreading. J Biol Chem 2003; 278: 37520-37529.
33
Elvers M,
Pozgaj R,
Pleines I.
et al. Platelet hyperreactivity and a prothrombotic phenotype in mice with a gain-of-function mutation in phospholipase Cgamma2. J Thromb Haemost 2010; 8: 1353-1363.
34
Takizawa H,
Nishimura S,
Takayama N.
et al. Lnk regulates integrin alphaIIbbeta3 outside-in signalling in mouse platelets, leading to stabilisation of thrombus development in vivo. J Clin Invest 2010; 120: 179-190.
35
Oxley CL,
Anthis NJ,
Lowe ED.
et al. An integrin phosphorylation switch: the effect of beta3 integrin tail phosphorylation on Dok1 and talin binding. J Biol Chem 2008; 283: 5420-5426.
37
Collier NC,
Wang K.
Purification and properties of human platelet P235. A high molecular weight protein substrate of endogenous calcium-activated protease( s). J Biol Chem 1982; 257: 6937-6943.
41
van Dijk TB,
van Den Akker E,
Amelsvoort MP.
et al. Stem cell factor induces phosphatidylinositol 3’-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells. Blood 2000; 96: 3406-3413.
