Inhibition of Factor VIIa-Tissue Factor by Protein C Inhibitor.

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that inhibits several proteases in blood coagulation including thrombin, thrombin-thromobomodulin and activated protein C. Although PCI has been shown to inhibit these key blood coagulation proteases, a clearly defined hemostatic role for PCI remains to be elucidated. Preliminary results from our laboratory show that exogenously added PCI prolongs venous thrombus formation upon blood vessel injury in a murine model of vascular injury. These results suggest that PCI may be responsible for inhibiting other proteases in blood coagulation, possibly including factor VIIa. Blood coagulation is initiated when factor VIIa binds to TF on cell surfaces. We investigated the ability of PCI to inhibit factor VIIa, both in the presence and absence of TF in vitro, and in a cellular environment. It has been shown previously and we have confirmed that the serpin antithrombin (AT) inhibits both factor VIIa and factor VIIa/TF; however, the ability of PCI to inhibit this protease has not been fully investigated. PCI and AT inhibit factor VIIa in the presence of TF and heparin (AT: k²=1.1 × 10⁴ M⁻¹ s⁻¹; PCI: k²=7.2 × 10³ M⁻¹s⁻¹). In the absence of heparin the inhibition of factor VIIa/TF by PCI is reduced 10-fold. Interestingly, PCI is unable to inhibit VIIa in the absence of TF with or without heparin. Furthermore, both PCI and AT form SDS-stable complexes with factor VIIa (in the presence of TF). We also investigated whether PCI is able to inhibit VIIa-mediated cell migration of the breast cancer cell line MDA-MB-231 that constitutively expresses TF. AT and PCI are able to inhibit cell surface derived factor VIIa/TF. Interestingly, the rate of factor VIIa inhibition on MDA-MB-231 cells is 1.2 and 0.4 × 10³ M⁻¹s⁻¹ for PCI and AT, respectively. The factor VIIa-mediated cell migration of MD-MB-231 cells is attenuated in the presence of PCI, but not in the presence of the PCI inactive mutant (T341R). Given that both AT and PCI are able to inhibit factor VIIa/TF both in vitro and in a cellular environment, suggests that more potent derivatives (prepared by site-directed mutagenesis) of these serpins may be useful alternatives as novel therapeutics anticoagulants that target the initial step in the coagulation pathway.