Probable regulation of factor VIIa–tissue factor and prothrombinase by factor Xa–TFPI and TFPI in vivo

Given that factor VIIa–tissue factor (TF) probably initiates coagulation in vivo , this study investigated the relationship between plasma concentrations of factor VIIa and prothrombin fragment 1+2 in plasma (the latter as an index of prothrombinase activity in vivo ). The relationships between these two parameters and the concentrations of tissue factor pathway inhibitor (TFPI) and factor Xa–TFPI in plasma were also investigated. TFPI inactivates factor Xa in a reaction accelerated by heparin, whereas factor Xa–TFPI inactivates factor VIIa–TF and prothrombinase. Established enzyme-linked immunosorbent assays (ELISAs) were used to quantify TFPI and prothrombin fragment 1+2, whereas we developed an ELISA to quantify factor Xa–TFPI using affinity purified rabbit (anti-human TFPI)-IgG and chicken anti-(human factor Xa–TFPI)-IgY as the capture and detector antibodies, respectively. Plasma factor VIIa was quantified using truncated tissue factor. The concentrations of factor VIIa and prothrombin fragment 1+2 increased in parallel in the plasmas of up to 145 healthy adults assayed ( P  = 0.007), as did the concentrations of factor VIIa and TFPI ( P  = 0.0039), and prothrombin fragment 1+2 and TFPI ( P  = 0.013). In contrast, there was an inverse relationship between the concentrations of free factor Xa–TFPI and factor VIIa ( P  <0.0001) and free factor Xa–TFPI and prothrombin fragment 1+2 ( P =0.0095). These results are consistent with factor Xa–TFPI regulating factor VIIa–tissue factor and prothrombinase in vivo .     

Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants

The anionic phospholipid, phosphatidyl-L-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid beta-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor-initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents.