A directional switch of integrin signalling and a new anti-thrombotic strategy

Abstract

Integrins are critical in thrombosis and hemostasis1. Antagonists of the platelet integrin αIIbβ3 are potent anti-thrombotic drugs, but also have the life-threatening adverse effect of bleeding2,3. It is thus desirable to develop new antagonists that do not cause bleeding. Integrins transmit signals bidirectionally4,5. Inside-out signaling activates integrins via a talin-dependent mechanism6,7. Integrin ligation mediates thrombus formation and outside-in signaling8,9, which requires Gα13 and greatly expands thrombi. Here we show that Gα13 and talin bind to mutually exclusive, but distinct sites within the integrin β3 cytoplasmic domain in opposing waves. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for outside-in signaling. Integrin ligation induces transient talin dissociation and Gα13 binding to an ExE motif, which selectively mediates outside-in signaling and platelet spreading. The second talin binding wave is associated with clot retraction. An ExE motif-based inhibitor of Gα13-integrin interaction selectively abolishes outside-in signaling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without affecting bleeding time. Thus, we have discovered a novel mechanism for the directional switch of integrin signaling and, based on this mechanism, we designed a potent new anti-thrombotic that does not cause bleeding.

Gα13 Switch Region 2 Binds to the Talin Head Domain and Activates αIIbβ3 Integrin in Human Platelets

“…G 13 signal transduction (8). Our present and previous demonstration that G␣ 13 SR1 signaling is specific for PAR1-mediated activation is inconsistent with recent work (27,28) describing a binding interaction between G␣ 13 SR1 and platelet ␤3 integrin, as a basis for ␣llb␤3 integrin activation. If this binding interaction were indeed the mechanism of G␣ 13 -mediated integrin activation in platelets, one would expect inhibition of G 13 SR1 signaling to not only block platelet stimulation caused by PAR1, but platelet stimulation caused by all activating agents.…”

Current State and Novel Approaches of Antiplatelet Therapy

“…On the basis of recent research discoveries, there is far wider scope for therapeutic regulation of platelet targets, including αIIbβ3, 28,81 integrin α6β1, 82 GPIb-IX-V, and GPVI adhesive function and signaling. In addition to vWF-and GPIbα-targeting inhibitors in development (Table 2), alternative strategies could have advantages, for example, small molecules with increased scope for bioavailability.…”

“…81 Notably, it seems feasible to dissect out precise signaling functions of αIIbβ3 involving Gα13 or talin associations, which control platelet activation, adhesion, and aggregation/clot contraction, and to inhibit thrombus formation in mice comparable with current αIIbβ3 inhibitors, yet without adverse bleeding. 81 Platelet integrin α6β1 also represents a novel antiplatelet target.…”

“…81 Notably, it seems feasible to dissect out precise signaling functions of αIIbβ3 involving Gα13 or talin associations, which control platelet activation, adhesion, and aggregation/clot contraction, and to inhibit thrombus formation in mice comparable with current αIIbβ3 inhibitors, yet without adverse bleeding. 81 Platelet integrin α6β1 also represents a novel antiplatelet target. Recently, using platelet-specific knockout of integrin α6, the main vascular laminin receptor was indicated to play an important part in platelet adhesion, activation, and in arterial thrombosis without significant effect on bleeding risk.…”

The key position: influence of staple location on constrained peptide conformation and binding

“…This interaction is critical for integrin activation, and triggers platelet aggregation and thrombosis formation 7 . Disruption of this PPI therefore offers a novel approach to anti-platelet therapeutics for the treatment of heart disease 8,9 . Integrin binding to talin occurs over an extensive surface, comprised of two main regions, termed the membrane proximal (MP) and membrane distal (MD) interfaces 10 , with an overall Kd in the range of 270-600µM 11,12 .…”