APC variants 35–39 exhibit normal ability to inhibit apoptosis and endothelial barrier protection. A, confluent EAhy926 cells were incubated with Protac-generated plasma purified APC, recombinant wild type APC, and APC Gla domain variants for 17 h. A rat monoclonal anti-EPCR antibody (RCR-252) was added (400 μm) alongside wild type APC to determine EPCR dependence. Apoptosis was induced in EAhy926 cells by incubation with 20 μm staurosporine for 4 h. RNA was extracted and reverse-transcribed as described under “Experimental Procedures.” Reverse transcription-PCR was performed using specific Bax, Bcl-2, and β-actin primers. Experiments were performed in triplicate, and data are presented as the mean ± S.E. Unpaired two-tailed t tests were used to determine significance (**, p < 0.005 compared with staurosporine-only treated EAhy926 cells). hAPC, human APC. B, the protective effect of APC on the endothelial cell barrier was determined for wild type APC and each Gla domain variant. EAhy926 cells were preincubated with 20 nm wild type or variant APC (black bars) for 3 h. Untreated cells (white bar) were used as a negative control. EAhy926 cells were then treated with 5 nm thrombin in serum-free media for 10 min (cells treated with thrombin only, gray bar), and endothelial barrier permeability was assessed after 30 min using Evans Blue-bovine serum albumin (see “Experimental Procedures”). Unpaired two-tailed t tests were used to determine significance (*, p < 0.05 compared with thrombin-only treated EAhy926 cells). C, endothelial barrier protection was assessed over time as described above: untreated EAhy926 cells, •; 5 nm thrombin, ♦; WT-APC, ▴; APC-D35T, ▪; APC-D36A, ▵; APC-L38D, ⋄; APC-A39V, ▾.