Fig 1.
Retroviral β3 expression enables bidirectional signaling.
(A) The expression of β3 and GFP in the platelets of vector and β3 transplanted mice, as well as β3 deficient homozygote (β3-/-), heterozygote (β3+/-), and wild-type (β3+/+) mice. (B) Alexa-Fluor 647-conjugated fibrinogen binding was measured in β3-/-, β3+/-, β3+/+ platelets and in GFP-positive platelets gated by flow cytometry from transplanted animals upon Mn2+ (blue lines), ADP/Epi (orange lines) and PAR4 peptide (green lines) stimulation or no agonist (red lines for control). The detailed scatter diagram was shown in S1 Fig (C) Spreading of β3-/-, β3+/-, β3+/+ platelets and GFP-positive platelets of transplanted mice (The GFP and β3 expressions in the platelets are shown in A.) on immobilized fibrinogen in presence of PAR4 peptide. Platelets were visualized using tetramethyl rhodamine isothiocynate-conjugated phalloidin. The scale bar represents 40 μm.
Fig 2.
β3 expression of GFP-positive platelets in transfected platelets.
(A) The amino acid sequences of the cytoplasmic tail of wild-type and mutated β3. DNA fragment was also sequenced (S2 Fig). (B) Integrin β3 and GFP expression in platelets of representative transplanted mice. (C) Statistical histogram of mean fluorescence intensity (mean±SEM) of β3 expression in GFP-positive platelets from at least three individual animals for each type of mutants. *P<0.05, compared to the wild-type β3 expressing platelets.
Fig 3.
Loss of the β3 cytoplasmic NITY motif, rather than RGT motif, impairs inside-out αIIbβ3 signaling.
Alexa-Fluor 647-conjugated fibrinogen binding was measured in GFP-positive platelets gated by flow cytometry from vector, β3, β3-ΔRGT, β3-ΔTNITYRGT and β3-ΔNITY transplanted mice upon Mn2+ (blue lines), ADP/Epi (orange lines) and PAR4 peptide (green lines) stimulation, or no agonist (red lines for control). (A) The representative images of fibrinogen binding. (B) The mean fluorescent intensity (MFI) of fibrinogen binding in the present of agonists (Mn2+, ADP/Epi, or PAR4 peptide) or antagonists (RGDS) was calculated based on the basal level fibrinogen binding platelets without treatment by agonists or antagonists. (C) The mean fluorescent intensity of fibrinogen binding with agonists (ADP/Epi, or PAR4 peptide) or antagonists (RGDS) was calibrated with that stimulated by Mn2+. Statistical chart is from at least three individual animals so performed (mean±SEM). *P<0.05, compared to the wild-type β3 transfected platelets.
Fig 4.
Outside-in αIIbβ3 signaling requires the β3 C-terminal RGT motif, but not NITY motif.
Platelet of transplanted mice spreading on immobilized fibrinogen only (Fg), immobilized fibrinogen accompanied with ADP (Fg+ADP), or PAR4 peptide (Fg+PAR4 peptide). (A) The representative images of actin staining and GFP expression under fluorescence microscopy. The scale bar represents 40 μm. The β3 and GFP expressions of the representative transfected platelets are shown in Panel B. The green fluorescence of β3 wild-type and β3-ΔRGT transfected platelets looks dim (A), because the mean green fluorescence intensities of them are relatively low in comparison to other kinds of transfected platelets (B). Several GFP-negative platelets are observed to spread well in response to stimulation in β3-ΔRGT and β3-ΔTNITYRGT platelet groups, but not in the vector ones (A). That is because there is almost no recipient platelet left in vector transfected mice (B). The GFP-positive ratio of the wild-type β3 group (A) looks higher than that shown in flow cytometry (B), probably resulting from a loss of GFP-β3- platelets by washing during the process of slide preparation. (C) The area occupied by GFP-positive adherent platelets was measured using the Image J program. The results are the mean ± SEM from 20–60 GFP-positive individual platelets of at least three animals analyzed for each type of mutants. *P<0.05 and **P<0.01, compared to the wild-type β3 transfected platelets.
Fig 5.
Effects of different β3 mutations on platelet function under flow.
Whole blood from transplanted mice was perfused through the capillary tubes coated with fibrinogen or collagen at 125 s-1 for 12 minutes or 1,500 s-1 for 5 minutes, respectively. The adherent GFP-positive platelets were recorded in real-time under fluorescence microscopy. (A) The representative images of adherent GFP-positive platelets on fibrinogen or collagen-coated surface. The scale bar represents 40 μm. (B) The number of adherent GFP-positive platelets on fibrinogen. (C) The adhesion ratio on fibrinogen (relative value). (D) The coverage area of adherent and aggregated GFP-positive platelets on collagen. (E) The adhesion ratio on collagen (relative value). Results are the mean ± SEM from 10–15 randomly selected visual fields for each type of mutants. *P<0.05 compared to the wild-type β3 transfected platelets.
Fig 6.
Interaction of different β3 (WT and mutants) with signaling molecules.
(A) Expression of correct truncational mutants in each of the GST-β3 cytoplasmic tail fusion proteins was verified with antibodies specifically recognizing calpain cleaved forms of β3 (Ab 759 and Ab 754) and an antibody recognizing the COOH terminus of (Ab 762). (B) Glutathione-Sepharose 4B beads coated with GST-β3 cytoplasmic tail fusion proteins were incubated overnight with platelet lysates at 4°C. After washing the special antibodies were used to detect talin, kindlin-3, and c-Src binding. Anti-GST antibody was used to verify the loading of the β3 cytoplasmic tail fusion proteins.
Fig 7.
Co-localization of different β3 (WT and mutants) with signaling molecules.
Stably transfected cells (As characterized in S6 Fig) were allowed to spread on fibrinogen-coated slides for 120 min, fixed, and permeabilized. (A) The slides were stained with a mouse anti-β3 monoclonal antibody, SZ21 (green), and rabbit anti-kindlin-2 antibodies (red), followed by fluorescence-labeled secondary antibodies, as well as Dapi (blue). (B) Methods applied were similar to Panel A, except the use of rabbit anti-Src antibodies (red). Data were collected with a Leica laser confocal microscope. The scale bar represents 25 μm.