Page 125 - 2020_11-Haematologica-web

In vivo role and mechanism of platelet migfilin
Migfilin positively regulates early aIIbβ3 outside-in signaling in platelets
aggregation. Both spread areas on fibrinogen and the aggregation rates by manganese were largely reduced in migfilin-/- platelets (Figure 4A-B). WT-migfilin-CCR7, but not MT-migfilin-CCR7 peptide, fully rescued platelet spreading on fibrinogen and Mn2+ induced platelet aggre- gation (Figure 4A-B). The late outside-in signaling was assessed by a clot retraction experiment.21-23 Contrary to
Figure 3. WT-migfilin-CCR7 (5 mM) rescued the impaired aggregation and ATP release of washed migfilin-/- platelet in response to thrombin or collagen. (A) Platelets were stimulated with collagen (0.4 mg/mL, 0.8 mg/mL), thrombin (0.018 U/mL, 0.025 U/mL) in the presence of WT-migfilin-CCR7 (5 mM) peptide or MT-migfilin-CCR7 (5 mM) peptide. Platelet aggregation and ATP release were assessed with a Chrono-log lumi-aggregometer under stirring at 1,200 rpm. Traces were representative of at least three independent experiments. (B) WT (□) and migfilin-/- (■) platelets treated with WT-migfilin-CCR7 and MT-migfilin-CCR7 respectively were stimulated by collagen and thrombin. Results are expressed as mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical significance was evaluated with paired Student t test (*P<0.05, **P<0.01, ns: no significant difference).
Since the effects of migfilin in platelets are aIIbβ3- dependent, outside-in signaling mediated by aIIbβ3 was evaluated. First, early outside-in signaling was assessed by two functional assays, i.e., spreading of platelets on immobilized fibrinogen and Mn2+ (0.5mM)-caused platelet
A
B
0.4 (mg/mL)
0.018 (U/mL)
0.8 (mg/mL)
0.025 (U/mL)
0.4 (mg/mL)
0.018 (U/mL)
0.8 (mg/mL)
0.025 (U/mL)
Collagen (0.4 mg/mL)
Collagen (0.8 mg/mL)
Thrombin (0.018 U/mL)
Thrombin (0.025 U/mL)
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