protease activated receptor 4 par4  (Proteintech)

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: Platelet aggregation is impaired in S 485 A kindlin-3 platelets. (A) Comparison of kindlin-3 phosphorylation in wild-type (WT) and S 485 A mouse platelets. Platelet suspensions were treated with protease-activated receptor 4 (PAR4) agonist peptide for the times indicated, and incubations were terminated by the addition of 2× Laemmli sample buffer. Phosphorylated kindlin-3, total kindlin-3, β 3 integrin, talin, and actin loading control were detected on Western blots. (B) Representative traces showing the aggregation kinetics of WT and S 485 A K3 mouse platelets stimulated with 0.1 U/mL or 0.2 U/mL of thrombin. (C) Quantification of the thrombin-induced aggregation. Results are representative of 3 independent experiments, ∗ P < .001. (D) Representative trace showing the aggregation kinetics of WT and S 485 A K3 mouse platelets stimulated with a high dose of thrombin (0.5 U/mL). (E) Representative trace showing the aggregation kinetics of WT and S 485 A K3 mouse platelets stimulated with 1 mM PAR4 agonist peptide. (F) Quantification of the 1 mM PAR4 agonist-induced aggregation. Results are representative of 2 independent experiments, ∗ P < .001. (G and H) Representative traces showing the aggregation kinetics of WT and S 485 A K3 mouse platelets stimulated with U46619 and collagen. (I) Quantification of 4 μg/mL collagen-induced aggregation. Results are representative of 2 independent experiments, ∗ P < .001.

Article Snippet: Collagen was from Chrono-Log (P/N 385), human thrombin was from Sigma Aldrich (T6884), mouse protease-activated receptor 4 (PAR4) agonist peptide (1-6) amide trifluoroacetate was from Bachem (H-6054.0005BA), (5 Z )-7-[(1 R ,4 S ,5 S ,6 R )-6-[(1 E ,3 S )-3-Hydroxy-1-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid (U46619) was from Tocris (1932), prostaglandin E1 was from Sigma Aldrich (P5515), and human fibrinogen was from Enzyme Research Laboratories.

Techniques: Comparison, Phospho-proteomics, Control, Western Blot

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: Collagen was from Chrono-Log (P/N 385), human thrombin was from Sigma Aldrich (T6884), mouse protease-activated receptor 4 (PAR4) agonist peptide (1-6) amide trifluoroacetate was from Bachem (H-6054.0005BA), (5 Z )-7-[(1 R ,4 S ,5 S ,6 R )-6-[(1 E ,3 S )-3-Hydroxy-1-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid (U46619) was from Tocris (1932), prostaglandin E1 was from Sigma Aldrich (P5515), and human fibrinogen was from Enzyme Research Laboratories.

Techniques: Phospho-proteomics, Activation Assay, Staining, Comparison, Expressing, Labeling, Binding Assay