Figure 7.

Specific DUSP3 inhibitor MLS-0437605 inhibits platelet activation in response to CRP and rhodocytin. (A) Chemical structure and key properties of MLS-0437605. (B-D) Washed human platelets (B/C) or WT and Dusp3-KO mice washed platelets (D) were pre-incubated for 30 min with DMSO (vehicle) or with MLS-0437605 (3.7 μM), before stimulated with CRP (0.5 μg/mL), rhodocytin (5 or 10 nM), or U46619 (0.15 μg/mL). Representative platelet aggregation curves (B/D) and quantification of platelet aggregation from three healthy human donors (C) are shown. Results were analyzed using one-way ANOVA Bonferroni multiple comparison test and are presented as mean ± SEM; **p<0.01. (E-J) TLs were prepared from vehicle- or MLS-0437605-pretreated human platelets. Cells were non-activated or activated with CRP or rhodocytin for the indicated times. Western blot analysis was performed with 4G10 antibody for global pY of CRP (E) and rhodocytin (F) activated platelets. ERK1/2 was used as a loading control. (G-H) Representative pY blot of Syk immunoprecipitates from TLs of CRP- (G) or rhodocytin-activated (H) platelets. (I-J) pY blot of PLCγ2 immunoprecipitates from TLs of CRP- (I) or rhodocytin-activated (J) platelets. Data are representative of two individual healthy donors.