Recombinant Human Activated Protein C Suppresses Endotoxin Induced Hypotension in Mice Deficient of the Endothelial Protein C Receptor (EPCR).

In Clinical sepsis and human endotoxin studies, administration of the recombinant human form of the anticoagulant activated protein C (rhaPC) demonstrates benefit in parameters of coagulation, inflammation, shock, and sepsis survival. Utilizing transgenic mice deficient in the endothelial protein C receptor, (EPCR), we investigated whether provision of rhaPC improved the hypotensive response and survival subsequent to administration of a lethal dose of endotoxin (LPS). A novel central venous infusion protocol for rhaPC was developed using low expressing EPCR mice (EPCR^δ/δ surface expression <10% of WT EPCR) which were generated in our center. WT C57Bl/6 (>F8) mice were used as controls. An aortic arch continuous blood pressure (B.P.) monitor and right internal jugular vein catheter were placed. The continuous infusion dosing of rhaPC (Eli Lilly) was approximately 1.8 μg/g/hr for 20 hr through the central venous catheter (N=3 per group). The control group received saline. Bolus LPS (10 μg/g i.p.) was administered concomitant to rhaPC. Continuous B.P. measurements were obtained until death or for up to 72 hr. RhaPC kinetic parameters (N=4 per group) collected from WT citrate-benzamidine plasma samples were determined by monoclonal antibody capture assays. In initial kinetic studies plasma levels of rhaPC in WT mice given LPS achieved steady state concentrations (90 ng/mL at 4 hr) similar to that found in human studies (45 ng/mL at 2hr). RhaPC dosing produced no allergic reaction or overt bleeding. The subsequent 72 hour B.P. study achieved the goal of suppressing endotoxin-induced hypotension in both WT and EPCR^δ/δ mice. The two LPS/rhaPC-treated groups were able to overcome the hypotensive stress and survive longer than the two LPS-only treated groups. RhaPC treated EPCR^δ/δ mice were able to recover blood pressure to the same or greater degree than the rhaPC treated WT mice, suggesting either enhanced cell signaling potential by less cell surface EPCR or an alternate aPC receptor. The administered volume of 30 μL/hr (0.6 mL over 20 hr) did not appear to counter the onset of hypotension in EPCR^δ/δ mice, and the rhaPC B.P. effect persisted beyond the 20 hr infusion. The expected sequence of LPS-induced mortalitv, viz., EPCR^δ/δ > WT > WT/rhaPC > EPCR^δ/δ/rhaPC was demonstrated in this study. These transgenic murine studies implicate the protein C pathway as linking coagulation, inflammation and hypotension. By reducing the burden of hypotension in WT and EPCR deficient mice with rhaPC treatment in endotoxin-induced sepsis, this infusion model provides a valuable approach to study effects of aPC in vascular inflammation and hypotension.