Figure 5
Inflammatory gene regulation by aPC requires procofactor V and pS. (A) RAW cells were incubated for 3 hours in normal human plasma (10% vol/vol) with 100 ng/mL of the indicated aPC variants, function-blocking anti-human pS antibody (anti-pS; 2 μg/mL), or nonimmune immunoglobulin G (IgG; 2 μg/mL), and Peli1 mRNA levels relative to LPS-treated RAW cells cultured without aPC were measured by quantitative PCR. Data are the mean ± standard deviation from 4 independent experiments. *P < .01 by Student t test relative to “no aPC” controls. (B) Survival of LPS-challenged (40 mg/kg; intraperitoneal) wild-type mice infused with mouse L38D-aPC (300 μg/kg; IV) within 30 minutes after LPS administration. (Ci) Plasma-derived fV, fVa, fV Leiden, and recombinant variants with partial B-domain deletions were tested for their ability to restore Peli1 downregulation by wild-type human aPC in RAW cells cultured with 10% human fV-depleted plasma. All variants were tested at 700 ng/mL. Data represent the average of 6 independent experiments ± standard deviation. *P < .01 by Student t test. (ii) Domain structure of intact fV with positions of cleavage sites recognized by aPC or thrombin indicated by arrows. Structures of fV-variants are shown below. (D) Signaling-selective aPC variants cleave procofactor V at R506: plasma-derived fV (i) or thrombin-activated fVa (ii) were added to a final concentration of 700 ng/mL to RAW cell culture medium supplemented with 10% (vol/vol) fV-depleted human plasma, phospholipid vesicles, and 2 U/mL of hirudin. The mixtures were incubated for 15 minutes at 22°C with 10 ng/mL of wt-aPC (wt), 100 ng of wt-aPC (wt 10 ×), or 10 ng/mL of 5A-aPC (5A), 2Cys-aPC (2Cys), or proteolytically inactive S360A-aPC (S360A). Cleavage products were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions, and detected by western blot analysis with anti-human fV antibody AHV-5146 recognizing the 307-506 fragment of fV. Residue numbers of fV fragments are indicated on the left (fragment); molecular weight standards are shown in the center (kDa).

Inflammatory gene regulation by aPC requires procofactor V and pS. (A) RAW cells were incubated for 3 hours in normal human plasma (10% vol/vol) with 100 ng/mL of the indicated aPC variants, function-blocking anti-human pS antibody (anti-pS; 2 μg/mL), or nonimmune immunoglobulin G (IgG; 2 μg/mL), and Peli1 mRNA levels relative to LPS-treated RAW cells cultured without aPC were measured by quantitative PCR. Data are the mean ± standard deviation from 4 independent experiments. *P < .01 by Student t test relative to “no aPC” controls. (B) Survival of LPS-challenged (40 mg/kg; intraperitoneal) wild-type mice infused with mouse L38D-aPC (300 μg/kg; IV) within 30 minutes after LPS administration. (Ci) Plasma-derived fV, fVa, fV Leiden, and recombinant variants with partial B-domain deletions were tested for their ability to restore Peli1 downregulation by wild-type human aPC in RAW cells cultured with 10% human fV-depleted plasma. All variants were tested at 700 ng/mL. Data represent the average of 6 independent experiments ± standard deviation. *P < .01 by Student t test. (ii) Domain structure of intact fV with positions of cleavage sites recognized by aPC or thrombin indicated by arrows. Structures of fV-variants are shown below. (D) Signaling-selective aPC variants cleave procofactor V at R506: plasma-derived fV (i) or thrombin-activated fVa (ii) were added to a final concentration of 700 ng/mL to RAW cell culture medium supplemented with 10% (vol/vol) fV-depleted human plasma, phospholipid vesicles, and 2 U/mL of hirudin. The mixtures were incubated for 15 minutes at 22°C with 10 ng/mL of wt-aPC (wt), 100 ng of wt-aPC (wt 10 ×), or 10 ng/mL of 5A-aPC (5A), 2Cys-aPC (2Cys), or proteolytically inactive S360A-aPC (S360A). Cleavage products were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions, and detected by western blot analysis with anti-human fV antibody AHV-5146 recognizing the 307-506 fragment of fV. Residue numbers of fV fragments are indicated on the left (fragment); molecular weight standards are shown in the center (kDa).

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