Abstract 145: Development of Novel S100A9 Peptide Vaccine for Prevention of Thrombus Formation in Ischemic Stroke in Mice

Abstract

Background and purpose: Nonadherence of antithrombotic medicine is one of major risk factors for recurrent strokes. Immunotherapy, such as antithrombotic vaccination, is expected to solve such a problem due to its long-lasting effects. Here, we focused on platelet-derived S100A9, which has directly regulate thrombosis without influence on hemostasis, and examined the antithrombotic effects of newly developed S100A9 vaccine in mice. Methods: S100A9 vaccine was designed to select the short peptide as an antigen of S100A9 conjugated to keyhole limpet hemocyanin (KLH). Male C57BL/6J mice were immunized with S100A9 vaccine three times at 2 weeks interval. Middle cerebral arteries (MCA) was exposed to topical application of 20% ferric chloride. The time to occlude the MCA was examined measuring cerebral blood flow with a laser doppler flowmeter. Bleeding time was assessed by cutting the tail arteries. The thrombus formation was also assessed using the laminar flow chamber. The influences of vaccine for T-cell activation were assed using an IgG subclass ELISA and an enzyme-linked immunosorbent spot (ELISPOT) assay. Results: S100A9 vaccine successfully produced antibodies specific for S100A9 in serum in a dose-dependent manner. After the third immunization, the time to thrombus formation in MCA was significantly prolonged in the mice vaccinated with S100A9 compared to the non-immunized mice (23.3 ± 4.3 min vs 6.9 ± 0.8 min, p<0.01), but bleeding time was same in both groups. There was no significant difference in platelet count in both groups. The formation of platelet thrombus under ex vivo was significantly reduced in S100A9 vaccine group compared to control group (14.8 ± 0.7 x10 4 /μm 2 vs 17.8 ± 0.9 x10 4 /μm 2 , p<0.05). In the IgG subclass ELISA, the IgG1:IgG2b ratio was greater than 1.0 in the S100A9 vaccine group. In the ELISPOT assay using splenocytes isolated from S100A9 immunized mice, stimulation with S100A9 partial peptide did not induce the production of IFN-gamma and IL-4 in comparison with KLH. Conclusion: The S100A9 vaccine could successfully prevent thrombus formation in MCA without influence on hemostasis nor eliciting an autoimmune response. S100A9 vaccine might shed light on the development of safe antithrombotic vaccine in ischemic stroke.