Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin β3 Cytoplasmic Tail

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Xiaofeng Shi1,2, Jichun Yang1, Xiongying Cui1

  • 1State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.

Plos One
|November 17, 2016
PubMed

Insights

This study reveals how specific mutations in integrin beta 3 affect platelet function. Key sequences are crucial for fibrinogen binding, spreading, and adhesion under flow, impacting signaling pathways.

Area of Science:

  • Hematology
  • Cell Biology
  • Biochemistry

Background:

  • Previous studies identified truncational mutations of integrin beta 3 (β3) at F754 and Y759 sites, mimicking calpain cleavage, as regulators of integrin signaling in Chinese hamster ovary cells.
  • The functional significance of the β3 sequence from F754 to the C-terminus, particularly the N756ITY759 motif, in platelet biology remained largely unexplored.

Purpose of the Study:

  • To elucidate the roles of specific calpain cleavage-related sequences within integrin β3 in platelet function under static and flow conditions.
  • To investigate the impact of β3 truncations and deletions (β3-ΔTNITYRGT, β3-ΔRGT, β3-ΔNITY) on platelet adhesion, fibrinogen binding, and interactions with signaling molecules like kindlin and c-Src.

Main Methods:

  • Generation of mutant mice expressing β3 with defined truncations or deletions via bone marrow transplantation.
  • Assessment of platelet function including soluble fibrinogen binding, platelet spreading on immobilized fibrinogen, and adhesion under flow on fibrinogen- and collagen-coated surfaces.
  • Evaluation of protein interactions using GST pull-down and immunofluorescent assays to determine the binding of β3 mutants to kindlin and c-Src.

Main Results:

  • The β3-ΔTNITYRGT mutant showed drastic inhibition in fibrinogen binding and spreading, while β3-ΔNITY exhibited partially impaired binding and unaffected spreading.
  • Platelet adhesion under flow was impaired differently: β3-ΔTNITYRGT > β3-ΔRGT > β3-ΔNITY on fibrinogen, and β3-ΔTNITYRGT > β3-ΔNITY > β3-ΔRGT on collagen.
  • β3-ΔRGT interacted with kindlin but not c-Src; β3-ΔNITY interacted with c-Src but not kindlin; β3-ΔTNITYRGT interacted with neither.

Conclusions:

  • The calpain cleavage-related sequences T755NITYRGT762, R760GT762, and N756ITY759 in integrin β3 are critical for bidirectional, outside-in, and inside-out signaling in platelets, respectively.
  • The association of c-Src or kindlin with β3 integrin plays a regulatory role in these signaling processes, influencing platelet adhesion and aggregation.
  • This study provides in vivo evidence for the distinct functional roles of specific β3 integrin domains in platelet biomechanics and signaling.

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