Endothelial Cell Protein C Receptor-Mediated Redistribution and Tissue-Level Accumulation of Pharmacologically Administered Recombinant Human Factor VIIa

Abstract 532

Recombinant factor VIIa (FVIIa) has been demonstrated to be an effective hemostatic agent for treatment of severe hemophilia patients. Recent clinical studies suggest that the prophylactic use of FVIIa markedly reduces the number of bleeding episodes in hemophilic patients with inhibitors. Clinical evidence from hemophilic patients during prophylaxis and post-prophylaxis suggest that the hemostatic effect of FVIIa surpassed the short circulatory half-life of FVIIa. At present, mechanisms by which this phenomenon occurs are not precisely known. Our laboratory has formerly established that FVIIa is capable of binding to the endothelial protein C receptor (EPCR) on endothelial cells, yet the significance of this interaction in hemostasis, and especially in prophylaxis, is not fully understood. Recent studies from our laboratory using a murine model have demonstrated that pharmacologically administered FVIIa rapidly associates with the endothelium, enters into extravasculature where it can interact with TF, and remains functionally active in tissues for an extended period of time. The aim of the present study was to determine if interaction of FVIIa with EPCR on the endothelium and subsequent EPCR-dependent transcytosis serves as a mechanism by which pharmacologically administered FVIIa is transported to tissues and retained for greater periods of time compared to its circulatory half-life. We have employed transgenic murine lines, including EPCR-over expressing, EPCR-deficient, and wild-type (WT) mice, to investigate the function of EPCR in the biodistribution of FVIIa and its extended stay in tissues. Mice were injected with human FVIIa (120 μg/kg b.w.) via tail vein and at 30 min, 3 h, 8 h, and 24 h following FVIIa administration, blood was collected via cardiac puncture and various tissues were harvested for antigen and immunohistochemical analyses. Our studies reveal that, following intravenous injection, FVIIa rapidly disappears from plasma (plasma levels of FVIIa in WT mice at 30 min and 3 h post-injection were ∼ 15% and <1%, respectively, of that which was injected) and associates with the endothelium in an EPCR-dependent manner. At 30 min post-injection, plasma levels of FVIIa were significantly higher in EPCR-deficient mice as compared to WT mice whereas plasma FVIIa levels were significantly lower in EPCR-over expressing mice compared to WT mice, suggesting that EPCR facilitates the sequestration of FVIIa from the bloodstream. This EPCR-dependent decrease in plasma FVIIa coincides with an EPCR-dependent increase in antigen levels of FVIIa in tissue homogenates. FVIIa antigen in the lungs of EPCR-over expressing mice at 30 min following FVIIa administration was approximately 6-fold higher than that which was found in WT mice. At the corresponding time, FVIIa antigen in EPCR-deficient mice was approximately 35% lower than that which was measured in WT mice. FVIIa antigen in the lung tissue of EPCR-over expressing mice remained relatively stable or slightly increased at 3, 8 and 24 h post FVIIa administration. FVIIa levels in tissue homogenates of WT and EPCR-deficient mice remained significantly lower compared to EPCR-over expressing mice at all time points. Immunohistochemical analysis of lung, skin, and bone-joint sections revealed that association of FVIIa with the endothelium is more pronounced in EPCR-over expressing mice as compared to lesser, albeit appreciable, levels in WT mice; EPCR-deficient mice showed negligible association of FVIIa with the endothelium. Association of FVIIa with the endothelium in EPCR-over expressing mice was maximal at 30 min and was thereafter progressively reduced at each time point; FVIIa association with the endothelium was undetectable at 24 h post FVIIa administration. The observation that EPCR-dependent association of FVIIa with the endothelium is most pronounced soon after injection and subsequently declines time-wise, combined with the retention of FVIIa in tissue homogenates for extended periods, implies that FVIIa bound to the endothelium enters tissues in an EPCR-dependent manner. These findings put forward an EPCR-dependent mechanism by which pharmacologically administered FVIIa can relocate to and be retained within tissues where TF resides, which conceivably may prime coagulation thereby contributing to hemostasis.