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Dynamic regulation of fibrinogen: integrin ?IIb?3 binding.

ABSTRACT: This study demonstrates that two orthogonal events regulate integrin ?IIb?3's interactions with fibrinogen, its primary physiological ligand: (1) conformational changes at the ?IIb-?3 interface and (2) flexibility in the carboxy terminus of fibrinogen's ?-module. The first postulate was tested by capturing ?IIb?3 on a biosensor and measuring binding by surface plasmon resonance. Binding of fibrinogen to eptifibatide-primed ?IIb?3 was characterized by a k(on) of ~2 × 10(4) L mol(-1) s(-1) and a k(off) of ~8 × 10(-5) s(-1) at 37 °C. In contrast, even at 150 nM fibrinogen, no binding was detected with resting ?IIb?3. Eptifibatide competitively inhibited fibrinogen's interactions with primed ?IIb?3 (K(i) ~0.4 nM), while a synthetic ?-module peptide (HHLGGAKQAGDV) was only weakly inhibitory (K(i) > 10 ?M). The second postulate was tested by measuring ?IIb?3's interactions with recombinant fibrinogen, both normal (rFgn) and a deletion mutant lacking the ?-chain AGDV sites (rFgn ??408-411). Normal rFgn bound rapidly, tightly, and specifically to primed ?IIb?3; no interaction was detected with rFgn ??408-411. Equilibrium and transition-state thermodynamic data indicated that binding of fibrinogen to primed ?IIb?3, while enthalpy-favorable, must overcome an entropy-dominated activation energy barrier. The hypothesis that fibrinogen binding is enthalpy-driven fits with structural data showing that its ?-C peptide and eptifibatide exhibit comparable electrostatic contacts with ?IIb?3's ectodomain. The concept that fibrinogen's ?IIb?3 targeting sequence is intrinsically disordered may explain the entropy penalty that limits its binding rate. In the hemostatic milieu, platelet-platelet interactions may be localized to vascular injury sites because integrins must be activated before they can bind their most abundant ligand.

SUBMITTER: Hantgan RR

PROVIDER: S-EPMC3210020 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Dynamic regulation of fibrinogen: integrin αIIbβ3 binding.

Hantgan Roy R RR Stahle Mary C MC Lord Susan T ST

Biochemistry 20101101 43

This study demonstrates that two orthogonal events regulate integrin αIIbβ3's interactions with fibrinogen, its primary physiological ligand: (1) conformational changes at the αIIb-β3 interface and (2) flexibility in the carboxy terminus of fibrinogen's γ-module. The first postulate was tested by capturing αIIbβ3 on a biosensor and measuring binding by surface plasmon resonance. Binding of fibrinogen to eptifibatide-primed αIIbβ3 was characterized by a k(on) of ~2 × 10(4) L mol(-1) s(-1) and a k ...[more]

PMID: 20828133

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Project description:Essentials peroxisome proliferator-activated receptor γ (PPARγ) agonists inhibit platelet function. PPARγ agonists negatively regulate outside-in signaling via integrin αIIbβ3. PPARγ agonists disrupt the interaction of Gα13 with integrin β3. This is attributed to an upregulation of protein kinase A activity.SummaryBackground Agonists for the peroxisome proliferator-activated receptor (PPARγ) have been shown to have inhibitory effects on platelet activity following stimulation by GPVI and GPCR agonists. Objectives Profound effects on thrombus formation led us to suspect a role for PPARγ agonists in the regulation of integrin αIIbβ3 mediated signaling. Both GPVI and GPCR signaling pathways lead to αIIbβ3 activation, and signaling through αIIbβ3 plays a critical role in platelet function and normal hemostasis. Methods The effects of PPARγ agonists on the regulation of αIIbβ3 outside-in signaling was determined by monitoring the ability of platelets to adhere and spread on fibrinogen and undergo clot retraction. Effects on signaling components downstream of αIIbβ3 activation were also determined following adhesion to fibrinogen by Western blotting. Results Treatment of platelets with PPARγ agonists inhibited platelet adhesion and spreading on fibrinogen and diminished clot retraction. A reduction in phosphorylation of several components of αIIbβ3 signaling, including the integrin β3 subunit, Syk, PLCγ2, focal adhesion kinase (FAK) and Akt, was also observed as a result of reduced interaction of the integrin β3 subunit with Gα13. Studies of VASP phosphorylation revealed that this was because of an increase in PKA activity following treatment with PPARγ receptor agonists. Conclusions This study provides further evidence for antiplatelet actions of PPARγ agonists, identifies a negative regulatory role for PPARγ agonists in the control of integrin αIIbβ3 outside-in signaling, and provides a molecular basis by which the PPARγ agonists negatively regulate platelet activation and thrombus formation.

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Dynamic regulation of fibrinogen: integrin ?IIb?3 binding.

Publications

Dynamic regulation of fibrinogen: integrin αIIbβ3 binding.

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