| First Author | Shi X | Year | 2016 |
| Journal | PLoS One | Volume | 11 |
| Issue | 11 | Pages | e0166136 |
| PubMed ID | 27851790 | Mgi Jnum | J:250784 |
| Mgi Id | MGI:6099146 | Doi | 10.1371/journal.pone.0166136 |
| Citation | Shi X, et al. (2016) Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin beta3 Cytoplasmic Tail when Expressed in Mouse Platelets. PLoS One 11(11):e0166136 |
| abstractText | Previous studies in Chinese hamster ovary cells showed that truncational mutations of beta3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing beta3 with F754 and Y759 truncations, or NITY deletion (beta3-DeltaTNITYRGT, beta3-DeltaRGT, or beta3-DeltaNITY) were established through transplanting the homozygous beta3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different beta3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the beta3-DeltaTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the beta3-DeltaNITY platelets. Behaviors of the beta3-DeltaRGT platelets were consistent with the previous observations in the beta3-DeltaRGT knock-in platelets. The adhesion impairment of platelets with the beta3 mutants under flow was in different orders of magnitude shown as: beta3-DeltaTNITYRGT>beta3-DeltaRGT>beta3-DeltaNITY to fibrinogen-coated surface, and beta3-DeltaTNITYRGT>beta3-DeltaNITY>beta3-DeltaRGT to collagen-coated surface. To evaluate the interaction of the beta3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that beta3-DeltaRGT interacted with kindlin but not c-Src, beta3-DeltaNITY interacted with c-Src but not kindlin, while beta3-DeltaTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin beta3, i.e. T755NITYRGT762, R760GT762, and N756ITY759 participate in bidirectional, outside-in, and inside-out signaling, respectively and the association of c-Src or kindlin with beta3 integrin may regulate these processes. |
