Targeting the RT loop of Src SH3 in Platelets Prevents Thrombosis without Compromising Hemostasis

Abstract

Conventional antiplatelet agents indiscriminately inhibit both thrombosis and hemostasis, and the increased bleeding risk thus hampers their use at more aggressive dosages to achieve adequate effect. Blocking integrin 𝜶IIb𝜷3 outside-in signaling by separating the 𝜷3/Src interaction, yet to be proven in vivo, may nonetheless resolve this dilemma. Identification of a specific druggable target for this strategy remains a fundamental challenge as Src SH3 is known to be responsible for binding to not only integrin 𝜷3 but also the proteins containing the PXXP motif. In vitro and in vivo mutational analyses show that the residues, especially E97, in the RT loop of Src SH3 are critical for interacting with 𝜷3. DCDBS84, a small molecule resulting from structure-based virtual screening, is structurally validated to be directed toward the projected target. It specifically disrupts 𝜷3/Src interaction without affecting canonical PXXP binding and thus inhibits the outside-in signaling-regulated platelet functions. Treatment of mice with DCDBS84 causes a profound inhibition of thrombosis, equivalent to that induced by extremely high doses of 𝜶IIb𝜷3 antagonist, but does not compromise primary hemostasis. Specific targets are revealed for a preferential inhibition of thrombosis that may lead to new classes of potent antithrombotics without hemorrhagic side effects.

Structure, signal transduction, activation, and inhibition of integrin αIIbβ3

“…Src contains the following domains: an N-terminal 14-carbon myristoyl group, two highly conserved Src homology domains (SH2 and SH3), a catalytic domain, and a C-terminal tail [57]. Recently our group found that the residues, especially E 97 , in the RT loop of Src SH3 are critical for interacting with β3 [58]. DCDBS84, a small molecule disrupting β3/Src interaction, inhibits the outside-in signaling-regulated platelet functions [58].…”

“…Recently our group found that the residues, especially E 97 , in the RT loop of Src SH3 are critical for interacting with β3 [58]. DCDBS84, a small molecule disrupting β3/Src interaction, inhibits the outside-in signaling-regulated platelet functions [58]. Src myristoylation promotes cell membrane binding and is imperative for function in vivo.…”

“…While, αIIbβ3 is responsible for platelet stable adhesion and required to resist the washing or shedding by shear stress [56,[126][127][128], contributing to maintain the attachment-detachment balance of thrombus formation under flow [129]. β3-knockout or β3-blocked platelets hardly adhere and aggregate to the wound or wound-mimicking surface at shear stress in vivo [58] or in vitro [129,130]. Under flow in vivo, the thrombus formed in the mice with β3 double mutations in Y 747 and Y 759 , which have a defect in αIIbβ3 outside-in signaling, have a loosely-packed and less-activated core, compared that in wild-type mice [131].…”