Analysis of Fyn function in hemostasis and αIIbβ3-integrin signaling

Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signalling, and the beta 3 subunit of alpha IIb beta 3 integrin has been shown to interact with multiple SFK members. Here, we analyzed the interactions and functional consequences of Fyn and Src binding to alpha IIb beta 3. Fyn associated with the beta 3 subunit in resting and thrombin-aggregated platelets, whereas interaction between Src and alpha IIb beta 3 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 alpha IIb beta 3. On the basis of these differences, we wanted to determine the sequence requirements for the interaction of Fyn and Src within the beta 3-cytoplasmic domain. Whereas Src association required the C-terminal region of beta 3, 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 beta 3-cytoplasmic tail. Using deletion mutants of beta 3-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 beta 3-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 alpha IIb beta 3-dependent cell spreading. Our results suggest that Fyn and Src have distinct requirements for interaction with alpha IIb beta 3; and, consequently, the two SFK can mediate different functional responses.