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 6

Lnk deficiency in platelets leads to reduced binding of Fyn to αIIbβ3 and reduced tyrosine phosphorylation of the cytoplasmic domain of the β3 integrin subunit.

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Lnk deficiency in platelets leads to reduced binding of Fyn to αIIbβ3 an...
(A) WT and Lnk–/– platelets plated or maintained in suspension as in Figure 5A were lysed and analyzed by immunoblotting using anti–β3 integrin or anti–β3/p-Tyr747. In other sets, immunoprecipitation was elicited with anti-Fyn or anti-αIIb Ab. Each immunoblot panel is representative of 3 or 4 independent experiments, and estimated band densities are shown in the graphs. Band densities for WT samples from adherent platelets on fibrinogen were defined as 100%. Lys, lysates. (B) Washed platelets from WT and Fyn–/– mice were plated on fibrinogen for 45 minutes or maintained in suspension in a BSA-coated dish, lysed, and analyzed. The error bars in A and B indicate mean ± SD. (C) Left panels show the features of WT or Fyn–/– platelets allowed to spread on fibrinogen-coated coverslips for 45 minutes in the absence of agonist. The cells were fixed, permeabilized, and stained with Alexa 488 Fluor–phalloidin to visualize F-actin. Scale bars: 10 μm. The graph shows spreading quantified by computer analysis of their surface areas (mean ± SD).