Fig 1.
Interaction of CIB1 with αIIbβ3 requires intracellular calcium rise.
(A) DIC images of human platelets treated as indicated. (B) Confocal images of platelets in suspension pre-treated with or without BAPTA-AM (50 μM) and then allowed to spread on immobilized Fg-coated coverglass for 45 min. Accumulation of CIB1 at the filopodia and the membrane periphery are indicated by arrows; original magnification, X 1600. (C) Western blot analysis of cIgG or anti-CIB1 immunoprecipitates of lysates of platelets treated as in B. Input represents sample of platelet lysate before immunoprecipitation. αIIb bands are shown in upper blot. Same blot was reprobed with anti-CIB1 to ensure an equal amount of protein in the immunoprecipitates (lower blot). (D) Densitometric analysis of coimmunoprecipitated αIIb bands normalized to corresponding CIB1 band (**P<0.01). Error bars indicate mean ± SEM of at least three independent experiments.
Fig 2.
Association of CIB1 with αIIb is needed for platelet spreading but not filopodia formation.
(A) Western blot analysis of anti-CIB1 immunoprecipitates of lysates from platelets incorporated with either αIIb peptide (2.5 μM) or anti-CIB1 antibody (0.1 mg/ml) as indicated. DMSO, scrambled αIIb peptide (2.5 μM) and irrelevant isotype specific antibody (cIgG, 0.1 mg/ml) were used as control. Upper blot shows the co-immunoprecipitation of αIIb. Same blot was reprobed with anti-CIB1 to ensure equal amount of protein in the immunoprecipitates (lower blot). Input represents sample of platelet lysate prior to immunoprecipitation. (B) Quantitation of αIIb bands from (A) normalized to corresponding CIB1 band. (*P<0.05) (C) Confocal images of platelets incorporated with αIIb peptide, anti-CIB1 or corresponding controls DMSO, scrambled αIIb peptide, or cIgG and were allowed to spread on Fg for 45 min and stained for F-actin; boxed area is enlarged to visualize colocalization (arrows); non-specific red speckles are shown by arrowheads; original magnification, X 1600. (D) Quantitation of platelets from C showing CIB1 staining at the filopodia (***P<0.001). Error bars indicate mean ± SEM of at least three independent experiments.
Fig 3.
Dynamic cytoskeletal rearrangement is required for activation of FAK.
(A) Confocal images of vehicle treated (DMSO) platelets, of platelets pre-treated with cytochalasin D (10 μM) for 15 min (Pre-CD) and then allowed to spread on Fg for 30 min, of platelets allowed to spread for 30 min prior to treatment with cytochalasin D for additional 15 min (Post-CD). Arrow indicates CIB1 staining at the tip of the filopodia. Arrowhead indicates filopodia lacking CIB1 staining; original magnification, X 1600. (B) Western blot analysis of anti-CIB1 immunoprecipitates of lysates from platelets treated as in A. Top blot shows phosphorylated Y397FAK. The blot was reprobed for total FAK co-immunoprecipitated with CIB1 (upper middle), and for CIB1 to ensure equal amount of protein in the immunoprecipitates (bottom). Samples from B were also blotted for αIIb (lower middle). Shown are the representative blots from three separate experiments performed independently.
Fig 4.
Association of CIB1 with αIIb is essential for the recruitment and activation of FAK.
(A) Western blot analysis of anti-CIB1 immunoprecipitates of lysates of platelets incorporated with anti-CIB1 or αIIb peptide and corresponding scrambled peptide or DMSO and allowed to spread on Fg. Input represents sample of platelet lysates prior to immunoprecipitation. FAK bands are shown in upper blot. Same blot was reprobed with anti-CIB1 to ensure an equal amount of protein in the precipitates (lower blot). (B) Western blot analysis of anti-FAK immunoprecipitates of lysates of platelets treated as above. cIgG was used as control. Activated FAK bands identified using anti-pY397 are shown in upper blot. The same blot was reprobed for total FAK to ensure an equal amount of protein in the precipitates. (C) Quantitation of optical density of phosphorylated FAK bands from B, normalized to corresponding total FAK band (**P<0.01). Error bars indicate mean ± SEM of at least three independent experiments. (D) Western blot analysis of anti-FAK immunoprecipitate of lysates of Cib1+/+ and Cib1-/- mouse platelets exposed to BSA or immobilized Fg. Activated FAK bands identified using anti-pY397 are shown in upper blot. The same blot was reprobed for total FAK to ensure equal protein in the precipitates. (E) Quantitation of optical density of phosphorylated FAK bands from D, normalized to corresponding total FAK band. (**P<0.01).
Fig 5.
Association of c-Src with CIB1-FAK complex augments its activity.
(A) Washed platelets were exposed to BSA or adhered to Fg for 45 min. The pre-cleared detergent lysates of these platelets were immunoprecipitated with anti-CIB1 or cIgG. Autoradiogram (AR) of MBP phosphorylation (upper panel). Western blot of immunoprecipitates probed with anti-Src (upper middle), and with anti-FAK (lower middle). The membrane was reprobed with anti-CIB1 to ensure equal amount of protein in the precipitates (lower panel). (B) Densitometric analysis of co-immunoprecipitated c-Src (upper middle blot in (A) (**P < 0.01). Error bars indicate mean ± SEM of at least three independent experiments. (C) Src kinase activity in CIB1 immunoprecipitates from platelets attached to Fg compared to BSA (***P<0.001).
Fig 6.
CIB1 association with αIIb is required for c-Src kinase activity.
(A) Confocal images of washed platelets pretreated in suspension with DMSO or Src kinase inhibitor PP2 (10 μM) for 20 min and allowed to spread on Fg for 45 min. Arrows indicate the localization of CIB1. (B) Western blot analysis of lysates from platelets incorporated with either αIIb peptide or anti-CIB1 antibody; DMSO, scrambled αIIb peptide, and cIgG as control probed with anti-Src pY418. The same blot was reprobed with anti-Src antibody to show the equal protein loading. (C) Densitometric analysis of (B). ***P< 0.001 and **P<0.01. (D) Western blot analysis of lysates prepared from Cib1+/+ and Cib1-/- mouse platelets. Activated c-Src bands identified using anti-c-Src-pY418 are shown in upper blot. The same blot was reprobed for total c-Src to ensure equal amount of protein loaded. (E) Densitometric analysis of c-Src-pY418 bands from D; normalized to corresponding total c-Src band (**P<0.01) from three independent experiments.
Fig 7.
Schematic representation of the initial steps of outside-in signaling through αIIbβ3.
Inactive αIIbβ3 in resting platelets complexed with JAM-A (left). Initial steps of outside-in signaling initiated by Fg binding to αIIbβ3 prior to the association of CIB1 leading to filopodia formation (middle). Signaling complex recruited by CIB1 to the αIIb tail leading to Src activation and platelet spreading (right).