Effect of nanobodies on the APC-mediated cleavage of SEAP-PAR1. (A) APC and nanobodies were added to HEK293 cells expressing SEAP-PAR1 and endothelial protein C receptor. Nanobodies were found to modulate PAR1 cleavage to different degrees. LP8, LP17, LP19, and LP21 showed the most potent inhibition. In contrast, LP3, LP9, LP11, and LP18 showed minimal inhibition of APC-mediated PAR1 cleavage even at the highest concentration of 500 nM (n = 3 independent experiments). PAR1 cleavage were expressed as the percentage of the total SEAP activity present on the cells vs background. (B) R41Q-SEAP-PAR1 and R46Q-SEAP-PAR1 cell line designs used to evaluate the cleavage pattern of APC (50 nM) at R46 and R41, respectively, in the presence of cleavage-inhibiting and cleavage-sparing nanobodies. (C) PAR1 cleavage-inhibiting nanobodies, LP8 and LP20, inhibited the cleavage at both R46 and R41 sites by APC in a dose-dependent manner. At high concentrations of LP8 and LP20, the cleavage ratio shifted to below 1, a less cytoprotective PAR1 cleavage profile (n = 3 independent experiments). (D) PAR1 cleavage-sparing nanobodies LP11 and LP18 induced a curvilinear cleavage response at both R46 and R41. The R46 to R41 cleavage ratios were above 1 for the entire concentration range of the assay, suggesting LP11 and LP18 may enhance cytoprotection by APC (n = 3 independent experiments).