Journal: Research and Practice in Thrombosis and Haemostasis
Article Title: Kindlin-3 phosphorylation is crucial for thrombosis and hemostasis in vivo
doi: 10.1016/j.rpth.2025.102863
Figure Lengend Snippet: Kindlin-3 S 485 phosphorylation is required for α IIb β 3 integrin activation in platelets. (A–D) Overlays of representative histograms of wild-type (WT; cyan) and S 485 A kindlin-3 (pink) platelets (A) unstained and (B) stained with JON/A monoclonal antibody at resting state or upon stimulation with (C) thrombin (Thr; 0.1 U/mL), (D) protease-activated receptor 4 (PAR4) agonist peptide (2.4 mM), or (E) U46619 (0.2 μg/mL). (F) Statistical comparison of data is shown in B–E. ∗ P < .001; S 485 A kindlin-3 vs WT, n = 4. (G) For total surface expression of α II β 3 integrin, platelets were stained with monoclonal antibody Leo.H4. (H) Alexa Fluor 647-labeled fibrinogen fragment D binding to S 485 A kindlin-3 platelets is reduced compared with WT platelets in response to Thr (0.1 U/mL), U46619 (0.2 μg/mL), PAR4 agonist peptide (2.4 mM), and resting platelets. (∗ P < .05; n = 6). (I) Platelet degranulation measured by the surface expression of P-selectin is the same in WT and S 485 A kindlin-3 platelets ( n = 2).
Article Snippet: Rabbit polyclonal antibodies (Abs) against mouse kindlin-3 were from Invitrogen (PA5-116402), rabbit polyclonal Abs against β 3 integrin were from Cell Signaling Technology (#4702), mouse mAb against talin was from Sigma Aldrich (T3287, clone 8d4), rabbit mAb against actin was from Cell Signaling Technology (#8456), rat mAb against mouse active α IIb β 3 integrin was from Emfret Analytics (M023-2, clone JON/A), and rat mAb against total α II β 3 integrin was from Emfret Analytics (M021-1, Leo.H4).
Techniques: Phospho-proteomics, Activation Assay, Staining, Comparison, Expressing, Labeling, Binding Assay