Dynamic regulation of fibrinogen: integrin αIIbβ3 binding.

This study demonstrates that two orthogonal events regulate integrin alpha IIb beta 3's interactions with fibrinogen, its primary physiological ligand: (1) conformational changes at the alpha IIb-beta 3 interface and (2) flexibility in the carboxy terminus of fibrinogen's gamma-module. The first postulate was tested by capturing alpha IIb beta 3 on a biosensor and measuring binding by surface plasmon resonance. Binding of fibrinogen to eptifibatide-primed alpha IIb beta 3 was characterized by a k(on) of similar to 2 x 10(4) L mol(-1) s(-1) and a k(off) of similar to 8 x 10(-5) s(-1) at 37 degrees C. In contrast, even at 150 nM fibrinogen, no binding was detected with resting alpha IIb beta 3. Eptifibatide competitively inhibited fibrinogen's interactions with primed alpha IIb beta 3 (K-i similar to 0.4 nM), while a synthetic gamma-module peptide (HHLGGAKQAGDV) was only weakly inhibitory (K-i > 10 mu M). The second postulate was tested by measuring alpha IIb beta 3's interactions with recombinant fibrinogen, both normal (rFgn) and a deletion mutant lacking the gamma-chain AGDV sites (rFgn gamma Delta 408-411). Normal rFgn bound rapidly, tightly, and specifically to primed alpha IIb beta 3; no interaction was detected with rFgn gamma Delta 408-411. Equilibrium and transition-state thermodynamic data indicated that binding of fibrinogen to primed alpha IIb beta 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 gamma-C peptide and eptifibatide exhibit comparable electrostatic contacts with alpha IIb beta 3's ectodomain. The concept that fibrinogen's alpha IIb beta 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.