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. 2021 Aug 5;107(12):2846–2858. doi: 10.3324/haematol.2021.278537

Figure 5.

Figure 5.

Altered αIIbβ3 integrin activation, degranulation and aggregation of thymosin β4 knockout platelets. (A and B) Activation of platelet αIIbβ3 integrin (JON/A-PE) (A) and degranulation (α-P-selectin-FITC) (B) in wild-type (WT) and Tmsb4x knockout (KO) platelets upon stimulation with the indicated agonists was determined by flow cytometry (n=12). U46: U46619; CRP: collagen-related peptide; Rhd: rhodocytin. Unpaired, two-tailed Student’s t-test. *P<0.05, **P<0.005. (C) Dense granule secretion was assessed by luminometric measurement of released ATP of activated WT and Tmsb4x KO platelets. Results are given as mean ATP concentration [mM] ± standard deviation (SD) (n=12 per group). Unpaired, two-tailed Student’s t-test. *P<0.05, **P<0.005, ***P<0.001. (D) Aggregation responses of washed platelets or platelet-rich plasma (PRP) in turbidometric aggregometry (n=6). (E) Western blot analysis of phosphotyrosine levels in resting and CVX-stimulated WT and Tmsb4x KO platelets using the 4G10 antibody. GAPDH served as loading control. CVX: convulxin; K: Tb4-/-; W: WT. (F and G) Phosphorylation and total protein levels of Syk in resting and CVX-stimulated WT and Tmsb4x KO platelets were analyzed (E) and quantified (F) by an automated quantitative capillary-based immunoassay platform. Values are mean ± SD (n = 3). Unpaired, two-tailed Student’s t-test. *P<0.05, **P<0.005, ***P<0.001. MFI: mean fluorescence intensity.