In mouse podocytes, aPC induces heterodimerization of PAR-1 and PAR-3, which are both required for aPC-mediated apoptosis inhibition. (A) Representative images show expression of PARs and EPCR in mouse podocytes, mouse GEnCs, and mouse placenta (positive control) as determined by semiquantitative RT-PCR. PAR-1 and PAR-3 are the receptors predominantly expressed in mouse podocytes. (B-D) Bar graphs summarizing the frequency of apoptosis in PAN-stressed mouse podocytes treated with PAR-APs (B), murine wild-type or mutant aPC (3K3A aPC, which lacks anticoagulant function, C), or PAR-blocking antibodies (D). Activation of PAR-1 and PAR-3 conveys the antiapoptotic effect of aPC independent of its anticoagulant function. (E) Representative immunoblots of immunoprecipitates showing heterodimerization of PAR-1 and PAR-3 after treatment with aPC (20nM) at different time points (top panel) and bar graph summarizing results (bottom panel). aPC induces heterodimerization of PAR-1 and PAR-3 in mouse podocytes. (F) Representative immunoblots of immunoprecipitates showing heterodimerization of PAR-1 and PAR-3 in mesangial cells transiently transfected with V5-tagged wild-type PAR- 3 (PAR-3-wt–V5) or V5-tagged mutant PAR-3 lacking the known cleavage site (PAR-3-m–V5). Proteolytic activation of PAR-3 by aPC is required for PAR-1/PAR-3 heterodimerization. PAR AP indicates protease-activated receptor agonist peptide; and PAR Ab, protease-activated receptor blocking antibody. Data are mean ± SEM of at least 3 independent experiments performed in duplicates. *P < .01 vs control (ANOVA).