Download PDF
Thromb Haemost 2007; 98(05): 1072-1080
DOI: 10.1160/TH07-02-0089
Platelets and Blood Cells
Schattauer GmbH

Role of murine integrin α2β1 in thrombus stabilization and embolization: Contribution of thromboxane A2

Marijke J. E. Kuijpers
1   Departments of Biochemistry, University of Maastricht, Maastricht, The Netherlands
,
Miroslava Pozgajova
3   Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany
,
Judith M. E. M. Cosemans
1   Departments of Biochemistry, University of Maastricht, Maastricht, The Netherlands
2   Departments of Human Biology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands
,
Imke C. A. Munnix
1   Departments of Biochemistry, University of Maastricht, Maastricht, The Netherlands
,
Beate Eckes
4   Department of Dermatology, University of Cologne, Cologne, Germany
,
Bernhard Nieswandt
3   Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany
,
Johan W. M. Heemskerk
1   Departments of Biochemistry, University of Maastricht, Maastricht, The Netherlands
2   Departments of Human Biology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands
› Author Affiliations Financial support: This work was supported by grants from the Netherlands Heart Foundation 2002-B014 and 2005-B079, the Netherlands Organization for Scientific Research 902–16–276 and the Deutsche Forschungsgemeinschaft through SFB 589 (to B.E.).
Further Information

Publication History

Received 05 February 2007

Accepted after resubmission 15 August 2007

Publication Date:
30 November 2017 (online)

    Permissions and Reprints

Summary

Platelets stably interact with collagen via glycoprotein (GP)VI and α2β1 integrin. With α2-null mice, we investigated the role of α2β1 in thrombus formation and stability in vivo and in vitro. Using a FeCl3-induced thrombosis model, in arteries from α2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects, while the thromboxane A2 analogue U46619 was borderline effective in suppressing the embolisation in α2-null mice. In vitro, perfusion of α2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from α2-null mice. We conclude that integrin α2β1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A2 release. The increased embolization in α2-null mice may argue against the use of α2β1 integrin inhibitors for antithrombotic therapy.

Keywords

Collagen receptors - integrins - mice - platelets - thrombus
 

  • References

  • 1 Nieswandt B, Brakebusch C, Bergmeier W. et al. Glycoprotein VI but not α2β1 integrin is essential for platelet interaction with collagen. EMBO J 2001; 20: 2120-2130.
    CrossrefPubMedGoogle Scholar
  • 2 Nieswandt B, Schulte V, Bergmeier W. et al. Longterm antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice. J Exp Med 2001; 193: 459-469.
    CrossrefPubMedGoogle Scholar
  • 3 Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling, the platelet paradigm. Blood 1998; 91: 2645-2657.
    PubMedGoogle Scholar
  • 4 Jung SM, Moroi M. Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin α2β1. J Biol Chem 2000; 275: 8016-8026.
    CrossrefPubMedGoogle Scholar
  • 5 Schoolmeester A, Vanhoorelbeke K, Katsutani S. et al. Monoclonal antibody IAC-1 is specific for activated α2β1 and binds to amino acids 199 to 201 of the integrin α2β1-domain. Blood 2004; 104: 390-396.
    CrossrefPubMedGoogle Scholar
  • 6 Nieuwenhuis HK, Akkerman JWN, Houdijk WP. et al. Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia. Nature 1985; 318: 470-472.
    CrossrefPubMedGoogle Scholar
  • 7 Nieuwenhuis HK, Sakariassen KS, Houdijk WP. et al. Deficiency of platelet membrane glycoprotein Ia associated with a decreased platelet adhesion to subendothelium: a defect in platelet spreading. Blood 1986; 68: 692-695.
    PubMedGoogle Scholar
  • 8 Kehrel B, Balleisen L, Kokott R. et al. Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder. Blood 1988; 71: 1074-1078.
    PubMedGoogle Scholar
  • 9 Nieswandt B, Watson SP. Platelet collagen interaction: is GPVI the central receptor?. Blood 2003; 102: 449-461.
    CrossrefPubMedGoogle Scholar
  • 10 Kunicki TJ, Kritzik M, Annis DS. et al. Hereditary variation in platelet integrinα2β1 density is associated with two silent polymorphisms in the α2 gene coding sequence. Blood 1997; 89: 1939-1943.
    PubMedGoogle Scholar
  • 11 Cuisset T, Frere C, Quilici J. et al. Lack of association between the 807 C/T polymorphism of glycoprotein Ia gene and post-treatment platelet reactivity after aspirin and clopidogrel in patients with acute coronary syndrome. Thromb Haemost 2007; 97: 212-217.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 12 Holtkotter O, Nieswandt B, Smyth N. et al. Integrin α2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen. J Biol Chem 2002; 277: 10789-10794.
    CrossrefPubMedGoogle Scholar
  • 13 Grüner S, Prostredna M, Schulte V. et al. Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo. Blood 2003; 102: 4021-4027.
    CrossrefPubMedGoogle Scholar
  • 14 Chen J, Diacovo TG, Grenache DG. et al. The α2 integrin subunit-deficient mouse: a multifaceted phenotype including defects of branching morphogenesis and hemostasis. Am J Pathol 2002; 161: 337-344.
    CrossrefPubMedGoogle Scholar
  • 15 He L, Pappan LK, Grenache DG. et al. The contributions of the α2β1 integrin to vascular thrombosis in vivo. Blood 2003; 102: 3652-3657.
    CrossrefPubMedGoogle Scholar
  • 16 Siljander PRM, Munnix ICA, Smethurst PA. et al. Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood. Blood 2004; 103: 1333-1341.
    PubMedGoogle Scholar
  • 17 Chen H, Kahn ML. Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets. Mol Cell Biol 2003; 23: 4764-4777.
    CrossrefPubMedGoogle Scholar
  • 18 Heemskerk JWM, Bevers EM, Lindhout T. Platelet activation and blood coagulation. Thromb Haemost 2002; 88: 186-193.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Kuijpers MJE, Schulte V, Bergmeier W. et al. Complementary roles of glycoprotein VI and α2β1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo. Faseb J 2003; 17: 685-867.
    CrossrefPubMedGoogle Scholar
  • 20 Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 1998; 94: 657-666.
    CrossrefPubMedGoogle Scholar
  • 21 Auger JM, Kuijpers MJE, Senis YA. et al. Adhesion of human and mouse platelets to collagen under shear: a unifying model. Faseb J 2005; 19: 825-827.
    CrossrefPubMedGoogle Scholar
  • 22 Sarratt KL, Chen H, Zutter MM. et al. GPVI and α2β1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow. Blood 2005; 106: 1268-1277.
    CrossrefPubMedGoogle Scholar
  • 23 Munnix ICA, Strehl A, Kuijpers MJE. et al. The glycoprotein VI-phospholipase Cγ2 signaling pathway controls collagen- and tissue factor-induced thrombus formation in vitro and in vivo. Atheroscler Thromb Vasc Biol 2005; 25: 2673-2678.
    CrossrefPubMedGoogle Scholar
  • 24 Nakamura T, Jamieson GA, Okuma M. et al. Platelet adhesion to native type I collagen fibrils. Role of GPVI in divalent cation-dependent and -independent adhesion and thromboxane A2 generation. J Biol Chem 1998; 273: 4338-4344.
    CrossrefPubMedGoogle Scholar
  • 25 Lecut C, Feijge MAH, Cosemans JME. et al. Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of α2β1 but not αIIbβ3 integrin. Thromb Haemost 2005; 94: 107-114.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 26 Denis C, Methia N, Frenette PS. et al. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc Natl Acad Sci USA 1998; 95: 9524-9529.
    CrossrefPubMedGoogle Scholar
  • 27 Van Gestel MA, Heemskerk JWM, Slaaf DW. et al. In vivo blockade of platelet ADP receptor P2Y12 reduces embolus and thrombus formation but not thrombus stability. Arterioscler Thromb Vasc Biol 2003; 23: 518-523.
    CrossrefPubMedGoogle Scholar
  • 28 Kuijpers MJE, Nieuwenhuys CMA, Feijge MAH. et al. Regulation of tissue factor-induced coagulation and platelet aggregation in flowing whole blood. Thromb Haemost 2005; 93: 97-105.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 29 Cosemans JME, Munnix ICA, Wetzker R. et al. Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization. Blood 2006; 108: 3045-3052.
    CrossrefPubMedGoogle Scholar
  • 30 Ni H, Ramakrishnan V, Ruggeri ZM. et al. Increased thrombogenesis and embolus formation in mice lacking glycoprotein V. Blood 2001; 98: 368-373.
    CrossrefPubMedGoogle Scholar
  • 31 Ni H, Denis CV, Subbarao S. et al. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 2000; 106: 385-392.
    CrossrefPubMedGoogle Scholar
  • 32 Lecut C, Schoolmeester A, Kuijpers MJE. et al. Principal role of glycoprotein VI in α2β1 and αIIbβ3 activation during collagen-induced thrombus formation. Arterioscler Thromb Vasc Biol 2004; 24: 1727-1733.
    CrossrefPubMedGoogle Scholar
  • 33 Samaha FF, Kahn ML. Novel platelet and vascular roles for immunoreceptor signaling. Arterioscler Thromb Vasc Biol 2006; 26: 2588-2593.
    CrossrefPubMedGoogle Scholar
  • 34 Atkinson BT, Jarvis GE, Watson SP. Activation of GPVI by collagen is regulated by α2β1 and secondary mediators. J Thromb Haemost 2003; 1: 1278-1287.
    CrossrefPubMedGoogle Scholar
  • 35 Falati S, Gross P, Merrill-Skoloff G. et al. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med 2002; 8: 1175-1181.
    CrossrefPubMedGoogle Scholar
  • 36 Mackman N. Mouse models in haemostasis and thrombosis. Thromb Haemost 2004; 92: 440-443.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Dubois C, Panicot-Dubois L, Merrill-Skoloff G. et al. Glycoprotein VI-dependent and -independent pathways of thrombus formation in vivo. Blood 2006; 107: 3902-3906.
    CrossrefPubMedGoogle Scholar
  • 38 Mangin P, Yap CL, Nonne C. et al. Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRγ receptor deficiency. Blood 2006; 107: 4346-4353.
    CrossrefPubMedGoogle Scholar
  • 39 Offermanns S, Toombs CF, Hu YH. et al. Defective platelet activation in Gαq-deficient mice. Nature 1997; 389: 183-186.
    CrossrefPubMedGoogle Scholar
  • 40 Moers A, Nieswandt B, Massberg S. et al. G13 is an essential mediator of platelet activation in hemostasis and thrombosis. Nat Med 2003; 9: 1418-1422.
    CrossrefPubMedGoogle Scholar
  • 41 Woulfe D, Jiang H, Morgans A. et al. Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2. J Clin Invest 2004; 113: 441-450.
    CrossrefPubMedGoogle Scholar
  • 42 Nieswandt B, Schulte V, Zywietz A. et al. Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin αIIbβ3 activation in platelets. J Biol Chem 2002; 277: 39493-39498.
    CrossrefPubMedGoogle Scholar
  • 43 Grüner S, Prostredna M, Aktas B. et al. Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced α2β1 levels or concomitant aspirin therapy. Circulation 2004; 110: 2946-2951.
    CrossrefPubMedGoogle Scholar
  • 44 André P, Delaney SM, LaRocca T. et al. P2Y12 regulates platelet adhesion/ activation, thrombus growth, and thrombus stability in injured arteries. J Clin Invest 2003; 112: 398-406.
    CrossrefPubMedGoogle Scholar
  • 45 Paul BZ, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A2-induced platelet aggregation. Essential role for P2tac and α2a receptors. J Biol Chem 1999; 274: 29108-29114.
    CrossrefPubMedGoogle Scholar
  • 46 André P, Prasad KS, Denis CV. et al. CD40L stabilizes arterial thrombi by a β3 integrin-dependent mechanism. Nat Med 2002; 8: 247-252.
    CrossrefPubMedGoogle Scholar
  • 47 Prevost N, Woulfe DS, Jiang H. et al. Eph kinases and ephrins support thrombus growth and stability by regulating integrin outside-in signaling in platelets. Proc Natl Acad Sci USA 2005; 102: 9820-9825.
    CrossrefPubMedGoogle Scholar
  • 48 Brass LF, Zhu L, Stalker TJ. Minding the gaps to promote thrombus growth and stability. J Clin Invest 2005; 115: 3385-3392.
    CrossrefPubMedGoogle Scholar
  • 49 Nanda N, Andre P, Bao M. et al. Platelet aggregation induces platelet aggregate stability via SLAM family receptor signaling. Blood 2005; 106: 3028-3034.
    CrossrefPubMedGoogle Scholar
  • 50 Goschnick MW, Lau LM, Wee JL. et al. Impaired "outside-in" integrin αIIbβ3 signaling and thrombus stability in TSSC6-deficient mice. Blood 2006; 108: 1911-1918.
    CrossrefPubMedGoogle Scholar