Fig 1: Plasma PEPD is degraded exclusively by FVIIaA., B. hPEPD (9.2 pmol) was incubated at RT with plasma (100 µl) with or without FIIa or FXa (10 pmol) for 24 h and after centrifugation to remove the precipitates, remaining PEPD was measured by enzymatic activity analysis. The precipitates were washed by PBS and checked for presence of hPEPD by IB, using FXIIIa and pure hPEPD as a binding control and standard, respectively. C. Plasma samples from untreated WT mice were incubated with an antibody that binds to both FVII and FVIIa; the immunocomplexes were pulled down with protein A sepharose. The supernatant fraction along with a regular plasma sample was analyzed for FVII/FVIIa level by IB. FVIIa was undetectable in the samples. D. hPEPD (9.2 pmol) was incubated with regular plasma or FVII/FVIIa-depleted plasma (100 µl) in the absence or presence of FIIa (10 pmol) at RT for 24 h; remaining hPEPD was measured by hPEPD enzymatic activity. Notably, plasma samples used in A-D were prepared from blood drawn from mice without an anticoagulant. E., F. Plasma PEPD and coagulation factors in control mice and mice at 6 h after hPEPD treatment; PEPD concentration was measured by ELISA, and coagulation factors were analyzed by IB (7.5 µl plasma per sample). Arrows indicate cleaved fragments. Error bars in A, D and E indicate SD (n = 3). Data were analyzed by one-way ANOVA in A or two-way ANOVA in D and E, followed by Tukey multiple comparisons test. Data in E were log transformed before ANOVA. * p < 0.05; **** p < 0.0001.