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Thromb Haemost 2013; 109(06): 1131-1140
DOI: 10.1160/TH12-09-0645
Platelets and Blood Cells
Schattauer GmbH

Dextran sulphate induces fibrinogen receptor activation through a novel Syk-independent PI-3 kinase-mediated tyrosine kinase pathway in platelets

Todd M. Getz
1   Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
3   Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
,
Bhanu Kanth Manne
1   Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
3   Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
,
Lorena Buitrago
1   Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
3   Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
,
Yingying Mao
1   Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
3   Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
,
Satya P. Kunapuli
1   Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
2   Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
3   Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
› Author Affiliations Financial support: This work is supported by HL93231 from National Institutes of Health to SPK. TG is supported by NIH training grant in Thrombosis (HL07777). YM is supported by AHA postdoctoral fellowship 10POST3730006.
Further Information

Publication History

Received: 17 September 2012

Accepted after major revision: 01 March 2013

Publication Date:
22 November 2017 (online)

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Summary

In our attempt to find a physiological agonist that activates PAR3 receptors, we screened several coagulation proteases using PAR4 null platelets. We observed that FXIIa and heat inactivated FXIIa, but not FXII, caused platelet aggregation. We have identified a contaminant activating factor in FXIIa preparation as dextran sulfate (DxS), which caused aggregation of both human and mouse platelets. DxS-induced platelet aggregation was unaffected by YM254890, a Gq inhibitor, but abolished by pan-Src family kinase (SFK) inhibitor PP2, suggesting a role for SFKs in this pathway. However, DxS-induced platelet aggregation was unaffected in FcRγ-chain null murine platelets, ruling out the possibility of glycoprotein VI-mediated events. More interesting, OXSI-2 and Go6976, two structurally unrelated inhibitors shown to affect Syk, had only a partial effect on DxS-induced PAC-1 binding. DxS-induced platelet aggregation and intracellular calcium increases were abolished by the pan PI-3 kinase inhibitor LY294002, or an isoform-specific PI-3 kinase β inhibitor TGX-221. Pretreatment of platelets with Syk inhibitors or ADP receptor antagonists had little effect on Akt phosphorylation following DxS stimulation. These results, for the first time, establish a novel tyrosine kinase pathway in platelets that causes fibrinogen receptor activation in a PI-3 kinase-dependent manner without a crucial role for Syk.

Keywords

Platelets - signalling - dextran-sulfate - Src-Family kinases - Syk kinase - PI-3 kinase
 

  • References

  • 1 Packham MA. Role of platelets in thrombosis and hemostasis. Can J Physiol Pharmacol 1994; 72: 278-284.
    CrossrefPubMedGoogle Scholar
  • 2 Shattil SJ, Kashiwagi H, Pampori N. Integrin signalling: the platelet paradigm. Blood 1998; 91: 2645-2657.
    PubMedGoogle Scholar
  • 3 Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998; 95: 8070-8074.
    CrossrefPubMedGoogle Scholar
  • 4 Offermanns S, Laugwitz KL, Spicher K. et al. G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci USA 1994; 91: 504-508.
    CrossrefPubMedGoogle Scholar
  • 5 Brass LF, Manning DR, Cichowski K. et al. Signalling through G proteins in platelets: to the integrins and beyond. Thromb Haemost 1997; 78: 581-589.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 6 Offermanns S. The role of heterotrimeric G proteins in platelet activation. Biol Chem 2000; 381: 389-396.
    PubMedGoogle Scholar
  • 7 Paul BZ, Daniel JL, Kunapuli SP. Platelet shape change is mediated by both calcium-dependent and -independent signalling pathways. Role of p160 Rho-as-sociated coiled-coil-containing protein kinase in platelet shape change. J Biol Chem 1999; 274: 28293-28300.
    PubMedGoogle Scholar
  • 8 Getz TM, Dangelmaier CA, Jin J. et al. Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets. J Thromb Haemost 08: 2283-2293.
    PubMedGoogle Scholar
  • 9 Jin J, Mao Y, Thomas D. et al. RhoA downstream of G(q) and G(12/13) pathways regulates protease-activated receptor-mediated dense granule release in platelets. Biochem Pharmacol 2009; 77: 835-844.
    CrossrefPubMedGoogle Scholar
  • 10 Dorsam RT, Kim S, Jin J. et al. Coordinated signalling through both G12/13 and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets. J Biol Chem 2002; 277: 47588-47595.
    CrossrefPubMedGoogle Scholar
  • 11 Watson SP, Auger JM, McCarty OJ. et al. GPVI and integrin alphaIIb beta3 signalling in platelets. J Thromb Haemost 2005; 03: 1752-1762.
    CrossrefPubMedGoogle Scholar
  • 12 Ezumi Y, Shindoh K, Tsuji M. et al. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets. J Exp Med 1998; 188: 267-276.
    CrossrefPubMedGoogle Scholar
  • 13 Spalton JC, Mori J, Pollitt AY. et al. The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signalling in platelets. J Thromb Haemost 2009; 07: 1192-1199.
    CrossrefPubMedGoogle Scholar
  • 14 Getz TM, Mayanglambam A, Daniel JL. et al. Go6976 abrogates GPVI-me-diated platelet functional responses in human platelets through inhibition of Syk. J Thromb Haemost 09: 608-610.
    PubMedGoogle Scholar
  • 15 Huang TF, Liu CZ, Yang SH. Aggretin, a novel platelet-aggregation inducer from snake (Calloselasma rhodostoma) venom, activates phospholipase C by acting as a glycoprotein Ia/IIa agonist. Biochem J 1995; 309: 1021-1027.
    CrossrefPubMedGoogle Scholar
  • 16 Shin Y, Morita T. Rhodocytin, a functional novel platelet agonist belonging to the heterodimeric C-type lectin family, induces platelet aggregation independently of glycoprotein Ib. Biochem Biophys Res Commun 1998; 245: 741-745.
    CrossrefPubMedGoogle Scholar
  • 17 Suzuki-Inoue K, Ozaki Y, Kainoh M. et al. Rhodocytin induces platelet aggregation by interacting with glycoprotein Ia/IIa (GPIa/IIa, Integrin alpha 2beta 1). Involvement of GPIa/IIa-associated src and protein tyrosine phosphorylation. J Biol Chem 2001; 276: 1643-1652.
    CrossrefPubMedGoogle Scholar
  • 18 Bergmeier W, Bouvard D, Eble JA. et al. Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha. J Biol Chem 2001; 276: 25121-25126.
    CrossrefPubMedGoogle Scholar
  • 19 Suzuki-Inoue K, Fuller GL, Garcia A. et al. A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2. Blood 2006; 107: 542-549.
    CrossrefPubMedGoogle Scholar
  • 20 Senis YA, Tomlinson MG, Garcia A. et al. A comprehensive proteomics and ge-nomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b–B, a novel immunoreceptor tyrosine-based inhibitory motif protein. Mol Cell Proteomics 2007; 06: 548-564.
    CrossrefPubMedGoogle Scholar
  • 21 Hughes CE, Pollitt AY, Mori J. et al. CLEC-2 activates Syk through dimerisation. Blood 115: 2947-2955.
    PubMedGoogle Scholar
  • 22 Suzuki-Inoue K, Kato Y, Inoue O. et al. Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells. J Biol Chem 2007; 282: 25993-26001.
    CrossrefPubMedGoogle Scholar
  • 23 Polgar J, Clemetson JM, Kehrel BE. et al. Platelet activation and signal transduc-tion by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. J Biol Chem 1997; 272: 13576-13583.
    CrossrefPubMedGoogle Scholar
  • 24 Daniel JL, Dangelmaier C, Strouse R. et al. Collagen induces normal signal transduction in platelets deficient in CD36 (platelet glycoprotein IV). Thromb Haemost 1994; 71: 353-356.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 25 Sambrano GR, Weiss EJ, Zheng YW. et al. Role of thrombin signalling in platelets in haemostasis and thrombosis. Nature 2001; 413: 74-78.
    CrossrefPubMedGoogle Scholar
  • 26 Nakanishi-Matsui M, Zheng YW, Sulciner DJ. et al. PAR3 is a cofactor for PAR4 activation by thrombin. Nature 2000; 404: 609-613.
    CrossrefPubMedGoogle Scholar
  • 27 van der Graaf F, Keus FJ, Vlooswijk RA. et al. The contact activation mechanism in human plasma: activation induced by dextran sulfate. Blood 1982; 59: 1225-1233.
    PubMedGoogle Scholar
  • 28 Brass L. Understanding and evaluating platelet function. Hematology Am Soc Hematol Educ Program 2010; 387-396.
    PubMedGoogle Scholar
  • 29 Jarvis GE, Atkinson BT, Snell DC. et al. Distinct roles of GPVI and integrin alpha(2)beta(1) in platelet shape change and aggregation induced by different collagens. Br J Pharmacol 2002; 137: 107-117.
    CrossrefPubMedGoogle Scholar
  • 30 Bhavaraju K, Kim S, Daniel JL. et al. Evaluation of [3-(1-methyl-1H–indol-3-yl-methylene)-2-oxo-2, 3-dihydro-1H–indole-5-sulfonamide] (OXSI-2), as a Syk-selective inhibitor in platelets. Eur J Pharmacol 2008; 580: 285-290.
    CrossrefPubMedGoogle Scholar
  • 31 Chari R, Kim S, Murugappan S. et al. Lyn, PKC-delta, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion. Blood 2009; 114: 3056-3063.
    CrossrefPubMedGoogle Scholar
  • 32 Schoenwaelder SM, Ono A, Nesbitt WS. et al. Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces. J Biol Chem 2010; 285: 2886-2896.
    CrossrefPubMedGoogle Scholar
  • 33 Walton KW. The biological behaviour of a new synthetic anticoagulant (dextran sulphate) possessing heparin-like properties. Br J Pharmacol Chemother 1952; 07: 370-391.
    CrossrefPubMedGoogle Scholar
  • 34 Gronowicz E, Coutinho A. Responsiveness of lymphoid precursors to polyclonal B-cell activators. Scand J Immunol 1975; 04: 429-437.
    CrossrefPubMedGoogle Scholar
  • 35 Watson SP, Herbert JM, Pollitt AY. GPVI and CLEC-2 in hemostasis and vascular integrity. J Thromb Haemost 08: 1456-1467.
    PubMedGoogle Scholar
  • 36 Barkalow KL, Falet H, Italiano Jr JE. et al. Role for phosphoinositide 3-kinase in Fc gamma RIIA-induced platelet shape change. Am J Physiol Cell Physiol 2003; 285: C797-C805.
    CrossrefPubMedGoogle Scholar
  • 37 Kato Y, Hori S, Fujita N. et al. A novel anti-platelet monoclonal antibody induces mouse platelet aggregation through an Fc receptor-independent mechanism. Biochem Biophys Res Commun 1998; 242: 250-255.
    CrossrefPubMedGoogle Scholar
  • 38 Watson AA, Christou CM, James JR. et al. The platelet receptor CLEC-2 is active as a dimer. Biochemistry 2009; 48: 10988-10996.
    CrossrefPubMedGoogle Scholar
  • 39 Garcia A, Quinton TM, Dorsam RT. et al. Src family kinase-mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets. Blood 2005; 106: 3410-3414.
    CrossrefPubMedGoogle Scholar
  • 40 Suzuki-Inoue K, Wilde JI, Andrews RK. et al. Glycoproteins VI and Ib-IX-V stimulate tyrosine phosphorylation of tyrosine kinase Syk and phospholipase Cgamma2 at distinct sites. Biochem J 2004; 378: 1023-1029.
    CrossrefPubMedGoogle Scholar
  • 41 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.
    CrossrefPubMedGoogle Scholar
  • 42 Trumel C, Payrastre B, Plantavid M. et al. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase. Blood 1999; 94: 4156-4165.
    PubMedGoogle Scholar
  • 43 Bowers RK, Marder P, Green LJ. et al. A platelet biomarker for assessing phosphoinositide 3-kinase inhibition during cancer chemotherapy. Mol Cancer Ther 2007; 06: 2600-2607.
    CrossrefPubMedGoogle Scholar
  • 44 Garcia A, Kim S, Bhavaraju K. et al. Role of phosphoinositide 3-kinase beta in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways. Biochem J 2010; 429: 369-377.
    CrossrefPubMedGoogle Scholar
  • 45 Kim S, Mangin P, Dangelmaier C. et al. Role of phosphoinositide 3-kinase beta in glycoprotein VI-mediated Akt activation in platelets. J Biol Chem 2009; 284: 33763-33772.
    CrossrefPubMedGoogle Scholar
  • 46 Kim S, Jin J, Kunapuli SP. Akt activation in platelets depends on Gi signalling pathways. J Biol Chem 2004; 279: 4186-4195.
    CrossrefPubMedGoogle Scholar
  • 47 Venkatesan N, Ouzzine M, Kolb M. et al. Increased deposition of chondroitin/ dermatan sulfate glycosaminoglycan and upregulation of beta1,3-glucuronosyl-transferase I in pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 300: L191-L203.
    PubMedGoogle Scholar
  • 48 Cavaco RA, Kaul S, Chapman T. et al. Idiopathic pulmonary fibrosis associated with pulmonary vein thrombosis: a case report. Cases J 2009; 02: 9156.
    CrossrefPubMedGoogle Scholar
  • 49 Morrissey JH, Choi SH, Smith SA. Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation. Blood l119: 5972-5979.
    PubMedGoogle Scholar
  • 50 Morrissey JH. Polyphosphate: a link between platelets, coagulation and inflammation. Int J Hematol 2012; 95: 346-352.
    CrossrefPubMedGoogle Scholar
  • 51 Smith SA, Morrissey JH. Polyphosphate as a general procoagulant agent. J Thromb Haemost 2008; 06: 1750-1756.
    CrossrefPubMedGoogle Scholar
  • 52 Gao C, Boylan B, Fang J. et al. Heparin promotes platelet responsiveness by potentiating {alpha}IIb{beta}3-mediated outside-in signalling. Blood 117: 4946-4952.
    PubMedGoogle Scholar