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

Abstract

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.

Figure 1.

Schematic diagram of annexin V and its fusion products with various Kunitz-type inhibitors. ANV indicates annexin V; TAP-ANV, Ala-tick anticoagulant peptide linked to annexin V by Gly-Ser dipeptide; ANV-6L15, annexin V linked to 6L15 (a Kunitz inhibitor derived from aprotinin with high affinity for TF/VIIa); ANV-K_APP, annexin V linked to K_APP (Kunitz inhibitory domain of amyloid β-protein precursor); ANV-KK_TFPI, annexin V linked to KK_TFPI (TFPI residues 22 to 161 containing Kunitz-1 and Kunitz-2 domains); C → A, Cys316Ala mutation in ANV; R → H, Arg2His mutation in 6L15.

Figure 2.

SDS-PAGE analysis of purified ANV and its fusion products with various Kunitz-type inhibitors. Samples were analyzed by 12% SDS-PAGE followed by Coomassie blue staining. All samples were boiled for 3 minutes without (A) or with (B) 50 mM dithiothreitol. Approximately 5 μg protein was loaded on each lane. (A-B) Lane 1, molecular weight (MW) markers; lane 2, ANV-KK_TFPI; lane 3, ANV-6L15; lane 4, TAP-ANV; lane 5 ANV-K_APP; lane 6, ANV.

Figure 3.

Stoichiometries of inhibition of bovine Xa and porcine trypsin by purified inhibitors. (A) Inhibition of trypsin by ANV-6L15. (B) Inhibition of trypsin by 6L15. (C) Inhibition of Xa by TAP-ANV. (D) Inhibition of Xa by TAP. (E) Inhibition of trypsin by ANV-K_APP. (F) Inhibition of Xa by ANV-KK_TFPI.

Figure 4.

Effects of various inhibitors in the aPTT assay. Pooled human plasma (180 μL) was mixed with 20 μL of various inhibitors to attain the indicated final concentrations. Clotting times were determined with an ACL 200 coagulation analyzer. The plasma with control buffer had an aPTT of 40.7 seconds. □ indicates TAP-ANV; ○, ANV-K_APP; ⋄, ANV-KK_TFPI; ▵, ANV-6L15; ×, ANV; +, 6L15; •, TAP; ♦, TFPI_1-160.

Figure 5.

Prolongation of time to occlusion by ANV and TAP-ANV in a photochemically induced carotid artery thrombosis model in mice. Experimental conditions are described in “Materials and methods.” ^*P = .32 versus control; ^**P = .013 versus control (2-tailed t test). Error bars indicate mean ± standard deviation.

Figure 6.

Model of the inhibition of a membrane-associated coagulation complex by the ANV-KPI fusion protein. The fusion protein binds to PS on the membrane surface in a Ca⁺⁺-dependent manner via the ANV domain. This facilitates the binding of the KPI domain to the active site of the coagulation complex. Vit. K-dep. indicates vitamin K–dependent.