Increased Platelet Binding of NN1731, a Factor VIIa Variant with Enhanced Tissue Factor-Independent Activity.

Platelet surface activity plays an important role in the efficacy of FVIIa as a bypassing agent in hemophilia. An analog of FVIIa with increased tissue factor (TF) independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain: V158D, E296V, and M298Q. Modeling suggests that Gln in position 298 and Asp in position 158 form a hydrogen bond network with a water molecule that stabilizes the proteolytically active conformation of the molecule. The Val introduced at position 296 avoids electrostatic repulsion between a native Glu residue and the introduced Asp at 158. These changes result in a FVIIa variant that partially mimics the conformation of TF-bound FVIIa. However, in the presence of TF the activity of the wild type FVIIa and NN1731 are indistinguishable. Previous studies suggested that the increased intrinsic activity of NN1731 was not directly reflected in the level of activity on the platelet surface. The goal of the current work was to compare the platelet binding and platelet surface activity of NN1731 and wild type FVIIa.

Mutant FVIIa molecules were prepared as described previously (PNAS 98:13583-8, 2001). We assessed binding to purified human platelets by flow cytometry and activity by measuring FXa generation.

FVIIa and NN1731 had identical binding to phospholipid vesicles designed to have lipid composition similar to platelets. However, the two exhibited different binding to platelets. At all concentrations, more NN1731 bound to thrombin or thrombin-plus-convulxin activated platelets. This was primarily because NN1731 bound to a greater number of sites per platelet. It appears that NN1731 and FVIIa bind similarly to a higher affinity site, but NN1731 binds to an additional low-affinity site. Removal of the Gla domain abolished binding. Thus, the protease domain of NN1731 was insufficient to mediate binding to the low affinity site. Two additional FVIIa mutants were tested: the single M298Q (“Q”) variant, and the double E296V and M298Q (“VQ”) variant. These variants bound to platelets identically to FVIIa.

While the Gla domain is essential for FVIIa binding to platelets, the three amino acid changes in the protease domain in NN1731 enhance platelet binding as well as proteolytic activity. Since NN1731 and FVIIa bind similarly to phospholipid vesicles, characteristics in addition to lipid composition likely contribute to platelet binding. The platelet surface components that contribute to NN1731 binding remain to be defined.

Hoffman:Novo Nordisk: Research Funding. Persson:Novo Nordisk: Employment. Ezban:Novo Nordisk: Employment. Monroe:Novo Nordisk: Research Funding.