Fig 1: MASP-1 and platelet activation. Whole blood supplemented with corn trypsin inhibitor and acetylsalicylic acid was re-calcified and incubated with rMASP-1cf (10 µg/ml and 50 µg/ml) and fixed at different time points. The entire platelet population was identified with an anti-CD41 antibody conjugated with APC. The activated population was identified with an anti-CD62P antibody conjugated with BV421. (A) Addition of rMASP-1cf increased the percentage of platelets expressing CD62P (shown as mean with SD) in a time- and dose-dependent manner. Statistical analysis: The Shapiro-Wilk test confirmed normal distribution, and p-values for differences between groups were determined with an ANOVA test (ns not significant, * p = 0.05, ** p = 0.01, *** p = 0.001). Number of experiments: Control: n=8; 10 µg/ml MASP-1: n=3; 50 µg/ml MASP-1: n=6). (B) The effect of 50 µg/ml rMASP-1cf was tested in the presence of PAR4 inhibitor BMS986120 and/or hirudin. The samples were fixed after 15min. Addition of the PAR4 inhibitor significantly reduced the effect of rMASP-1cf, while addition of hirudin canceled it out completely. Statistical analysis: The Shapiro-Wilk test confirmed normal distribution, and p-values for differences between groups were determined with an ANOVA test (ns not significant, * p = 0.05, ** p = 0.01, *** p = 0.001). Number of experiments: Control: n=8; MASP-1 only: n=6; MASP-1+ BMS: n=3; MASP-1+ Hirudin: n=4; MASP-1+ BMS + Hirudin: n=4.