Page 170 - Haematologica-5

P.H. Mangin et al.
mouse platelets on fibrinogen is blocked by the Src kinase inhibitor PP2 but not by the Syk kinase inhibitor PRT060318 (Figure 4B). Morphological changes of mouse platelets on fibrinogen are also not altered in platelets from irradiated mice transplanted with Syk-deficient fetal liver (Figure 4B) or from PF4.Cre-Sykfl/fl mice (Online Supplementary Figure S1). Western blotting for Syk con- firmed lack of expression of the tyrosine kinase in the two transgenic models (Figure 4B and not shown). Thrombin stimulated full spreading of wild-type and Syk-deficient platelets (Figure 4B). Formation of lamellipodia and stress
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fibers in human GPVI transgenic mouse platelets was blocked by PRT060318 (Figure 4C). The ability of Src and Syk inhibitors to block spreading of human platelets and human GPVI transgenic mouse platelets on fibrinogen is consistent with platelet activation by GPVI. This is sup- ported by demonstration of phosphorylation of the FcR γ- chain. The limited spreading of mouse platelets on fibrino- gen is mediated through a Src-dependent but Syk-inde- pendent pathway. Together, these results support a model in which immobilized fibrinogen activates human but not mouse platelets through GPVI.
Figure 2. Human but not mouse glyco- protein VI supports platelet adhesion and spreading on immobilized fibrino- gen. (A). Washed platelets from wild- type mice (WT mice), GPVI-deficient mice (GPVI-/- mice) or from healthy donors (Human) were allowed to adhere to human or mouse fibrinogen (FGN) for 30 or 45 min, respectively, and fixed with PFA and stained with Alex-488-
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phalloidin (4 μg/mL). Representative epifluorescence images of washed platelets adhering to fibrino- gen. Scale bars represent 10 μm. (A)(ii). Bar graph representing the number of platelets adhering to immobilized fib- rinogen per mm2. Adhesion is expressed as mean±SEM in five random fields, in three separate experiments (two-way ANOVA, Bonferroni post-hoc test: P>0.05). (A)(iii). Bar graph representing the percentage of platelets spreading on immobilized fibrinogen. Spreading is expressed as the mean±SEM in five ran- dom fields, in six separate experiments. Significance was attained using a two- way ANOVA, Bonferroni post-hoc test: ****P<0.001. (B). Washed control (WT) or β3-deficient (β3-/-) platelets were allowed to adhere to fibrinogen for 60 min, fixed with PFA and stained with TRITC-phalloidin (2 μg/mL). (B)(i). Representative epifluorescence images of washed mouse platelets adhering to fibrinogen. Scale bars represent 10 μm. (B)(ii). Bar graph representing the num- ber of platelets adhering to immobilized fibrinogen per mm2. Adhesion is expressed as the mean±SEM in eight random fields, in four separate experi- ments (Mann-Whitney test, **P<0.001). (C). Washed platelets from wild-type mice (WT mice) or mice expressing human GPVI (hGPVI mice) were allowed to adhere to fibrinogen for 60 min, fixed with PFA and stained with TRITC-phalloidin (2 μg/mL). (C)(i). Representative epifluorescence images of washed platelets adhering to fibrino- gen. Scale bars represent 10 μm. (C)(ii). Bar graph (left) representing the num- ber of platelets adhering to immobilized fibrinogen per mm2. Adhesion is expressed as the mean±SEM in eight random fields, in four separate experi- ments (Mann-Whitney test, P>0.05). Bar graph (right) representing the per- centage of platelets spreading on immo- bilized fibrinogen. Spreading is expressed as the mean±SEM in eight random fields, in four separate experi- ments (Mann-Whitney test, **P<0.001).
haematologica | 2018; 103(5)
(A)(i).