Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo
Hitoshi Takizawa, Satoshi Nishimura, Naoya Takayama, Atsushi Oda, Hidekazu Nishikii, Yohei Morita, Sei Kakinuma, Satoshi Yamazaki, Satoshi Okamura, Noriko Tamura, Shinya Goto, Akira Sawaguchi, Ichiro Manabe, Kiyoshi Takatsu, Hiromitsu Nakauchi, Satoshi Takaki, Koji Eto
Hitoshi Takizawa, Satoshi Nishimura, Naoya Takayama, Atsushi Oda, Hidekazu Nishikii, Yohei Morita, Sei Kakinuma, Satoshi Yamazaki, Satoshi Okamura, Noriko Tamura, Shinya Goto, Akira Sawaguchi, Ichiro Manabe, Kiyoshi Takatsu, Hiromitsu Nakauchi, Satoshi Takaki, Koji Eto
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Research Article Hematology

Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo

Abstract

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk–/– mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin αIIbβ3–mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk–/– mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk–/– platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the β3 integrin subunit, and reduced binding of Fyn to integrin αIIbβ3. These results provide new insight into the mechanism of αIIbβ3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.

Authors

Hitoshi Takizawa, Satoshi Nishimura, Naoya Takayama, Atsushi Oda, Hidekazu Nishikii, Yohei Morita, Sei Kakinuma, Satoshi Yamazaki, Satoshi Okamura, Noriko Tamura, Shinya Goto, Akira Sawaguchi, Ichiro Manabe, Kiyoshi Takatsu, Hiromitsu Nakauchi, Satoshi Takaki, Koji Eto

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Figure 5

Lnk associates with c-Src, Fyn, and Fyb in a manner dependent on outside-in signaling.

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Lnk associates with c-Src, Fyn, and Fyb in a manner dependent on outside...
(A) Washed platelets from WT and Lnk–/– mice were plated on fibrinogen (Fbg) for 45 minutes or maintained in suspension in a BSA-coated dish. Platelet lysate (left) or proteins immunoprecipitated using anti-Lnk Abs (right, IP: Lnk) were separated and probed with anti-phosphotyrosine (pY) mAb. (B) Lysates from WT platelets prepared as in A and immunoprecipitates obtained using irrelevant control rabbit sera (IP: Ctrl) or anti-Lnk (IP: Lnk) were probed by immunoblotting for the indicated proteins. (C) WT platelets were incubated with 5 μM PP2 to block Src kinase activity or with PP3, an inactive congener of PP2. PP2 but not PP3 diminished tyrosine phosphorylation of cellular proteins and the association of c-Src and Fyb, with Lnk in platelets adhering to fibrinogen. The observation was confirmed using 20 μM SU6656, an unrelated selective c-Src inhibitor (data not shown). Arrows indicate bands for the expected phosphoproteins corresponding to Fyb, Lnk, and c-Src. Results shown are representative of 3 independent experiments. The 2 panels on the right show the quantification of Western blot bands from 3 experiments (mean ± SD). Maximal band density was defined as 100%.