REGULAR ARTICLE| Volume 104, ISSUE 4, P239-247, November 15, 2001

Synergistic Outside–In Regulation of Platelet Activation by GPIIb/IIIa Ligand-Induced Conformation and Oligomerization


      Full platelet activation with serotonin secretion and thromboxane A2 (TxA2) formation induced by a low dose of thrombin receptor agonist peptide (TRAP) or high dose ADP requires platelet aggregation. This requirement can be replaced by pretreatment of platelets with a combination of reagents including: GPIIb/IIIa inhibitors yielding ligand-induced binding sites (LIBS), either arginine–glycine–aspartate–serine (RGDS) peptide or Ro 43-5054, cytochalasin to disrupt actin filaments and crosslinking by a GPIIb/IIIa mAb (pl-62). Crosslinking is required since Fab fragments of pl-62 do not support activation. Engagement of the Fc receptor by the mAb Fc domain is not required for pl-62 augmentation, since it is not blocked by the anti-Fc receptor mAb, IV-3. Another GPIIb/IIIa inhibitor, Ro 44-9883, not yielding LIBS epitopes, serves as a negative control and shows a requirement for LIBS in addition to crosslinking. Focal adhesion kinase tyrosine phosphorylation induced by TRAP is blocked by these GPIIb/IIIa antagonists, but restored by pl-62 crosslinking independent of LIBS induction. Tyrosine phosphorylation of a peptide comigrating with p38 MAP kinase is also inhibited by these antagonists and restored by pl-62 crosslinking. However, p38 MAP kinase activation by low dose TRAP is not affected by these aggregation inhibitors. Tyrosine phosphorylation of a 34-kDa phosphoprotein in the absence of aggregation or TxA2 formation was uniquely augmented by Ro 43-5054 but not Ro 44-9883 under the above activation conditions.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Thrombosis Research
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Carroll RC
        • Wang XF
        • Lanza F
        • Steiner B
        • Kouns WC
        Blocking platelet aggregation inhibits thromboxane A(2) formation by low dose agonists but does not inhibit phosphorylation and activation of cytosolic phospholipase A(2).
        Thromb Res. 1997; 88: 109-125
        • Dickfeld T
        • Ruf A
        • PogatsaMurray G
        • Muller I
        • Engelmann B
        • Taubitz W
        • Fischer J
        • Meier O
        • Gawaz M
        Differential antiplatelet effects of various glycoprotein IIb–IIIa antagonists.
        Thromb Res. 2001; 101: 53-64
        • Hato T
        • Pampori N
        • Shattil SJ
        Complementary roles for receptor clustering and conformational change in the adhesive and signaling functions of integrin alpha(IIb)beta(3).
        J Cell Biol. 1998; 141: 1685-1695
        • Isenberg WM
        • McEver RP
        • Phillips DR
        • Shuman MA
        • Bainton DF
        Immunogold-surface replica study of ADP-induced ligand binding and fibrinogen receptor clustering in human platelets.
        Am J Anat. 1989; 185: 142-148
        • Kieffer N
        • Guichard J
        • Breton-Gorius J
        Dynamic redistribution of platelet surface receptors after contact induced platelet activation and spreading.
        Am J Pathol. 1992; 140: 57-73
        • Carroll RC
        • Butler RG
        • Morris PA
        • Gerrard JM
        Separable assembly of platelet pseudopodal and contractile cytoskeletons.
        Cell. 1982; 30: 385-393
        • Wheeler ME
        • Cox AC
        • Carroll RC
        Retention of the glycoprotein IIb–IIIa complex in the isolated platelet cytoskeleton. Effects of separable assembly of platelet pseudopodal and contractile cytoskeletons.
        J Clin Invest. 1984; 74: 1080-1089
        • Wheeler ME
        • Gerrard JM
        • Carroll RC
        Reciprocal transmembranous receptor–cytoskeleton interactions in concanavalin A-activated platelets.
        J Cell Biol. 1985; 101: 993-1000
        • Clark EA
        • Shattil SJ
        • Ginsberg MH
        • Bolen J
        • Brugge JS
        Regulation of the protein tyrosine kinase pp72syk by platelet agonists and the integrin αIIbβ3.
        J Biol Chem. 1994; 269: 28859-28864
        • Law DA
        • NannizziAlaimo L
        • Ministri K
        • Hughes PE
        • Forsyth J
        • Turner M
        • Shattil SJ
        • Ginsberg MH
        • Tybulewicz VLJ
        • Phillips DR
        Genetic and pharmacological analyses of Syk function in alpha(IIb)beta(3) signaling in platelets.
        Blood. 1999; 93: 2645-2652
        • Sarkar S
        • Rooney MM
        • Lord ST
        Activation of integrin-beta(3)-associated syk in platelets.
        Biochem J. 1999; 338: 677-680
        • Saci A
        • Rendu F
        • BachelotLoza C
        Platelet alpha IIb-ss 3 integrin engagement induces the tyrosine phosphorylation of Cbl and its association with phosphoinositide 3-kinase and Syk.
        Biochem J. 2000; 351: 669-676
        • Kouns WC
        • Kirchhofer D
        • Hadvary P
        • Edenhofer A
        • Weller T
        • Pfenninger G
        • Baumgartner HR
        • Jennings LK
        • Steiner B
        Reversible conformational changes induced in glycoprotein IIb–IIIa by a potent and selective peptidomimetic inhibitor.
        Blood. 1992; 80: 2539-2547
        • Steiner B
        • Haring P
        • Jennings LK
        • Kouns WC
        Five independent neo-epitopes on GPIIb–IIIa are differentially exposed by two potent peptidomimetic platelet inhibitors.
        Thromb Haemostasis. 1993; 69 (Abstract): 860
        • Kouns WC
        • Steiner B
        • Kunicki TJ
        • Moog S
        • Jutzi J
        • Jennings LK
        • Cazenave J-P
        • Lanza F
        Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia.
        Blood. 1994; 84: 1108-1115
        • Wang TH
        • Wang HS
        • Ichijo H
        • Giannakakou P
        • Foster JS
        • Fojo T
        • Wimalasena J
        Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways.
        J Biol Chem. 1998; 273: 4928-4936
        • Worthington RE
        • Carroll RC
        • Boucheix C
        Platelet activation by CD9 monoclonal antibodies is mediated by the Fc-gamma-II receptor.
        Br J Haematol. 1990; 74: 216-222
        • Rubinstein E
        • Boucheix C
        • Urso I
        • Carroll RC
        Fc gamma receptor mediated interplatelet activation by a monoclonal antibody against beta2 microglobulin.
        J Immunol. 1991; 147: 3040-3046
        • Woods VLJ
        • Wolff LE
        • Keller DM
        Resting platelets contain a substantial centrally located pool of glycoprotein IIb–IIIa complex which may be accessible to some but not other extracellular proteins.
        J Biol Chem. 1986; 261: 15242-15251
        • Kramer RM
        • Roberts EF
        • Um SL
        • Borsch-Haubold AG
        • Watson SP
        • Fisher MJ
        • Jakubowski JA
        p38 Mitogen-activated protein kinase phosphorylates cytosolic phospholipase A(2) (cPLA(2)) in thrombin-stimulated platelets—evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA(2).
        J Biol Chem. 1996; 271: 27723-27729
        • Hunt TW
        • Carroll RC
        • Peralta EG
        Heterotrimeric G proteins containing Gαi3 regulate multiple effector enzymes in the same cell. Activation of phospholipases C and A2 and inhibition of adenylyl cyclase.
        J Biol Chem. 1994; 269: 29565-29570
        • Law DA
        • Nannizzi-Alaimo L
        • Phillips DR
        Outside–in integrin signal transduction—alpha(IIb)beta(3)-(GP IIb–IIIa) tyrosine phosphorylation induced by platelet aggregation.
        J Biol Chem. 1996; 271: 10811-10815
        • Cazes E
        • Nurden P
        • Nurden AT
        Abciximab binding to glycoprotein IIb–IIa and protein tyrosine phosphorylation in human platelets.
        Blood. 1999; 93 (letter, comment): 4019-4020