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First published online 22 April 2008
doi: 10.1242/jcs.014076

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Analysis of Fyn function in hemostasis and IIbβ3-integrin signaling

Department of Molecular Cardiology and Joseph J. Jacobs Center for Thrombosis & Vascular Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA

^* Author for correspondence (e-mail: reddyk{at}ccf.org)

Accepted 12 February 2008

Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signalling, and the β3 subunit of IIbβ3 integrin has been shown to interact with multiple SFK members. Here, we analyzed the interactions and functional consequences of Fyn and Src binding to IIbβ3. Fyn associated with the β3 subunit in resting and thrombin-aggregated platelets, whereas interaction between Src and IIbβ3 was seen predominantly in resting but not in thrombin-aggregated platelets. We have also observed that Fyn but not Src localized to focal adhesions in CHO cells adherent to fibrinogen through IIbβ3. On the basis of these differences, we wanted to determine the sequence requirements for the interaction of Fyn and Src within the β3-cytoplasmic domain. Whereas Src association required the C-terminal region of β3, Fyn continued to interact with mutants that could no longer associate with Src and that contained as few as 13 membrane-proximal amino acids of the β3-cytoplasmic tail. Using deletion mutants of β3-cytoplasmic tails expressed as GST-fusion proteins, we narrowed down the Fyn-binding site even further to the amino acid residues 721-725 (IHDRK) of the β3-cytoplasmic domain. On the basis of these observations, we explored whether Fyn^–/– mice exhibited any abnormalities in hemostasis and platelet function. We found that Fyn^–/– mice significantly differed in their second bleeding times compared with wild-type mice, and platelets from Fyn^–/– mice exhibited delayed spreading on fibrinogen-coated surfaces. Using mutant forms of Fyn, it appears that its kinase activity is required for its localization to focal adhesions and to mediate IIbβ3-dependent cell spreading. Our results suggest that Fyn and Src have distinct requirements for interaction with IIbβ3; and, consequently, the two SFK can mediate different functional responses.

Key words: Integrin, IIbβ3, Src-family kinases

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