Background: Immune thrombocytopenia (ITP) is an autoimmune disease in which anti-platelet antibody (APA) is produced. APA-coated platelets are captured and phagocytized by macrophages in the spleen. Recent study revealed that spleen tyrosine kinase (Syk) inhibitor is effective in the treatment of ITP, because Syk phosphorylation is the key step of the phagocytosis by macrophages. Activated Fc receptor signal transduction is initiated by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) tyrosine residues by SRC family kinases. Recruitment of Syk to dually phosphorylated ITAMs triggers the activation of Syk. To prove the hypothesis that the inhibition of SRC family kinase induces the decreased phosphorylation of Syk, resulting in decreased phagocytosis by macrophages, a SRC family kinase inhibitor, dasatinib, was used in the experiments.
Methods, Results and Discussion:
In vitro study; Murine macrophage cell line, RAW, was incubated with APA coated murine platelets for 30 minutes. Phagocytosis by RAW was significantly decreased with dasatinib (100nM, p<0.01), indicating SRC family kinase activity is required for efficient phagocytosis. Phosphorylated Syk was decreased in RAW, incubated with anti-Fc receptor antibody (rat IgG) and anti-rat IgG antibody with dasatinib (100nM), shown in the Western blot analysis (Figure 1). These results suggest that Syk phosphorylation is the key step in phagocytosis.
In vivo study; (1) Three hours before APA intra-peritoneal injection, dasatinib (2.5mg/kg) was oral-administrated. Six hours after APA injection, platelet counts were measured. The platelet counts were 366 ± 164 x109/L with dasatinib (n=4, mean ± SD) and 114 ± 51 x109/L without dasatinib (n=4)(P=0.026)(Figure 2). (2) Osmotic pump, filled with APA, were inserted in murine intra-peritoneal cavity and dasatinib (2.5mg/kg) was oral-administered once daily for 7 days. The platelet counts were 499 ± 98 x109/L with dasatinib (n=4, mean ± SD) and 82 ± 131 x109/L without dasatinib (n=4) at day 7 (p<0.0022) (Figure 3). These results strongly suggest that dasatinib inhibit the phagocytosis in vivo.
Conclusion: Dasatinib inhibits phosphorylation of Syk, inducing decreased phagocytosis of APA-coated platelets via decreased SRC family kinase activity. These findings reveal that SRC family kinase controls the efficiency of phagocytosis in part through the regulation of Syk function. Dasatinib might be effective in the treatment of ITP.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.