The Effects of the Spleen Tyrosine Kinase Inhibitor, Fostamatinib, on an Immune Thrombocytopenia Mouse Model

Background: Immune thrombocytopenia (ITP) is the most common autoimmune bleeding disorder. The major mechanism involves the phagocytosis of antibody-coated platelets by Fc-gamma receptor (FcγR)-bearing macrophages in the reticuloendothelial system. Many of the FcγR-mediated activities are dependent on the spleen tyrosine kinase (Syk) pathway: and we hypothesized that Syk could be one of the intracellular pathways which could be targeted for the treatment of ITP.

Objectives: The novel Syk inhibitor drug, fostamatinib, was used effectively for the treatment of ITP; but, the mechanism of action of fostamatinib in ITP has not been studied. The aims of the present study were to test the hypotheses of whether (1) The Syk pathway is involved in both the pathogenesis of ITP and in the treatment response to ITP, (2) Fostamatinib, a novel Syk inhibitor drug, has any effects on the complement system, lymphocytes, and macrophages in ITP, and whether (3) Fostamatinib decreases the migration of T lymphocytes to the bone marrow and the spleen in ITP.

Methods: Six to 8 week-old BALB/c mice were divided into eight groups: I) Sham-operated; II) isolated ITP; III) Fostamatinib (3 gr/kg, p.o.); IV) Fostamatinib + Liposomal clodronate (LC) (0.2 ml/U/mouse, i.p.); V) Fostamatinib + cobra venom factor (CVF) (25 U/mouse, i.p.); VI) Intravenous immunoglobulin (IVIG) (2 gr/kg, i.m.); VII) IVIG + LC; and VIII) IVIG + CVF. ITP model was formed by the i.v. injection of 4 μg of anti-CD41/Integrin alpha-2b antibody. Platelets were counted using a hemocytometer. The femurs of mice were dissected, their ends were cut off, and bone marrows were gently sprayed out. Flow cytometry was used for determining VLA-4 expression of CD3-positive T-cells, and fluorescein intensity of CD3(+)/VLA-4(+) cells. Spleen tissue sections were cut and stained with Syk, phospho (p)-Syk, platelet/endothelial cell adhesion molecule (PECAM), and CX3CR1.

Results: Administration of IVIG (p<0.001) and fostamatinib (p<0.001) prevented the fall in platelet counts in an ITP mouse model. Macrophage depletion by LC, and complement depletion by CVF did not provide any extra increments in the platelet counts. The intensity of Syk (p=0.0001) and p-Syk (p=0.0001) stainings were significantly higher in the isolated-ITP group than in the sham-operated group. Groups treated with fostamatinib demonstrated less Syk and p-Syk staining scores than ITP group (p<0.001 for fostamatinib and fostamatinib + LC; p=0.028 for fostamatinib + CVF). Syk staining was significantly less intense in the IVIG group than in the ITP group (p=0.0001). P-Syk levels in all IVIG groups were significantly higher than in other groups (p<0.001). The intensity of PECAM staining was significantly higher in the ITP group than in the sham group (p=0.0001). In ITP and all treatment groups, PECAM staining score was higher than in healthy controls (p<0.001). PECAM scores of all fostamatinib groups (fostamatinib, fostamatinib + LC, and fostamatinib + CVF) were similar to that of the ITP group. PECAM scores in the IVIG group were significantly higher than all other groups (p<0.001). CX3CR1 staining in spleens of the ITP group was more intense than the sham group (p=0.0001). CX3CR1 staining in fostamatinib and fostamatinib + LC groups was less intense than in ITP group (p<0.001). Fostamatinib + CVF and ITP groups had similar CX3CR1 expression. CX3CR1 staining in the fostamatinib group was less intense than in the IVIG group (p<0.001). IVIG+LC and IVIG+CVF groups had more intense CX3CR1 staining than IVIG group (p<0.001). The percentage of VLA-4(+) T cells in bone marrows of all groups were similar. The mean fluorescence intensity of VLA-4 on T cells in the bone marrows of the groups were not significantly different.

Conclusions: Our study showed that the Syk pathway played a role in the pathogenesis of ITP. The Syk inhibitor, fostamatinib, improved the thrombocytopenia in ITP by acting on the Syk pathway. In addition, fostamatinib decreased the migration of T lymphocytes to the spleen, but not to the bone marrow. IVIG treatment, similar to fostamatinib treatment, demonstrated a suppressive effect on the Syk pathway. To the best of our knowledge, this is the first study demonstrating some effect of fostamatinib on an ITP mouse model.