Background: Targeting platelet outside-in signaling inhibits thrombosis without compromising hemostasis. We previously showed that blockade of outside-in signaling was achieved by targeting the interaction of integrin β3 with c-Src. The c-Src SH3 is known to bind the canonical proline-rich motif and thereby regulates many biological processes. Therefore, it is essential to identify the sequences within SH3 responsible for the β3/SH3 interaction so that one may tell it apart from the canonical binding in the context of targeting strategy.

Aims: To identify the targets of integrin β3 in c-Src SH3 and evaluate their roles in anti-thrombosis and hemostasis in transgenic mouse model.

Methods: A synthetic small molecule known to interrupt the β3/SH3 interaction was used to mimic the β3 cytoplasmic domain. NMR, chemical shift perturbation and SPR were applied to identify the targets. Co-IP was assayed for β3/Src interaction and platelet aggregation, adhesion and spreading on immobilized fibrinogen for platelet function. The transgenic mouse model was generated and the FeCl₃-induced carotid arterial thrombus formation and tail bleeding time assay were performed.

Results: The RT-loop of SH3, especially E97, was critically required for binding the small molecule and the β3 cytoplasmic domain but not necessarily for binding the canonical PXXP-containing RLP1 peptide and for the c-Src kinase activity that were primarily dependent upon the amino acids in the n-Src loop (Fig.1). The c-Src^E97A mouse model was established. The β3/Src interaction in c-Src^E97A mice was interrupted and the platelet function was significantly diminished. Notably, c-Src^E97A mice exhibited a reduced thrombotic potential in the FeCl₃-induced thrombosis model with a normal hemostasis in the tail-bleeding assay (Fig.2).

Conclusions: We identified that E97 in the RT-loop of c-Src SH3 was critical for β3 binding but was unlikely to contribute significantly to the canonical binding and kinase activity. The sequences around E97 may thus serve as potential anti-thrombotic targets.

[Identification of the target sites of integrin β3 in c-Src SH3]

[Effects of the c-SrcE97A mutation on the β3/Src interaction, platelet function, thrombus formation and tail bleeding in transgenic mice]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/identification-of-the-sequence-specificity-in-c-src-sh3-required-for-binding-to-integrin-%ce%b23-as-a-novel-anti-thrombotic-target-without-compromising-primary-hemostasis/