The 99 and 170 Loop-modified Factor VIIa Mutants Show Enhanced Catalytic Activity without Tissue Factor

Abstract

To elucidate the functions of the surface loops of VIIa, we prepared two mutants, VII-30 and VII-39. The VII-30 mutant had all of the residues in the 99 loop replaced with those of trypsin. In the VII-39 mutant, both the 99 and 170 loops were replaced with those of trypsin. The k cat /K m value for hydrolysis of the chromogenic peptidyl substrate S-2288 by VIIa-30 (103 mM ؊1 s ؊1 ) was 3-fold higher than that of wild-type VIIa (30.3 mM ؊1 s ؊1 ) in the presence of soluble tissue factor (sTF). This enhancement was due to a decrease in the K m value but not to an increase in the k cat value. On the other hand, the k cat /K m value for S-2288 hydrolysis by VIIa-39 (17.9 mM ؊1 s ؊1 ) was 18-fold higher than that of wild-type (1.0 mM ؊1 s ؊1 ) in the absence of sTF, and the value was almost the same as that of wild-type measured in the presence of sTF. This enhancement was due to not only a decrease in the K m value but also to an increase in the k cat value. These results were in good agreement with their susceptibilities to a subsite 1-directed serine protease inhibitor. In our previous paper (Soejima, K., Mizuguchi, J., Yuguchi, M., Nakagaki, T., Higashi, S., and Iwanaga, S. Coagulation factor VIIa (VIIa) 1 is a plasma serine protease that is essential for the initiation of extrinsic blood coagulation (1). When tissue factor (TF) is expressed after injury of the vessel wall, it forms a complex with factor VIIa, and blood coagulation can be initiated. Forming a complex with TF markedly enhances the ability of VIIa to activate factors IX and X. In vitro, the formation of the active complex can be evidenced by measuring the esterolytic and amidolytic activities of VIIa (2, 3); these activities are also enhanced in the presence of soluble TF (sTF) and calcium ions (3-5).Human zymogen VII is a single chain enzyme precursor with an NH 2 -terminal Gla domain, followed by two EGF-like domains, EGF 1 and EGF 2, and a COOH-terminal serine protease domain. , which is composed of a light chain (residues 1-152) with Gla, EGF 1, and EGF 2 domains, and a heavy chain with a serine protease domain (residues 153-406) (6). The crystal structures of the molecular complex of the active site occupying VIIa, with and without sTF, as well as the crystal structure of zymogen VII, are known (7-12). The shape of VIIa could be described as a tulip, with the catalytic domain as the flower, the light chain as the stem, and the Gla domain as the bulb. TF winds around the light chain (the stem) and the Gla domain (the bulb) of FVII.Conversely, the catalytic domain of the coagulation proteases such as VIIa, IXa, Xa, and ␣-thrombin have active sites and internal cores that are similar to those of trypsin. Although these protease domains have a highly homologous three-dimensional structure, they display significant differences in specificity and catalytic activity. Furthermore, each protease requires a specific cofactor to express enhanced catalytic activity and physiological function, which differs from digestive serine proteases such as trypsin a...

Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus

“…The Km and kcat for FX activation by ETC, FVIIa/phospholipids and FVIIa/sTF in the absence of exactin (Tables S1, S2 and S3) are similar to those reported previously3844. The lower Km for FX activation by ETC (14 nM) could be attributed to TF (Innovin) used in our assays.…”

Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey: comment

“…Observation 4: Shortening of the 170-loop by grafting the trypsin loop into h-FVIIa seems to affect the structural integrity of the TF-binding helix and enhances activity [14][15][16]. We and others have shown that shortening of the 170-loop ( Fig.…”

“…Observation 5: The combination of sequence elements (mutations and grafts) that modulate protease activity and TF-induced allostery, in particular those mentioned in Observations 1-4 above, generally produces expected additive effects, albeit with some possibility of synergy [7,9,13,15,16]. This combination heuristic is supported by biochemical evidence whose finer nuances are addressed in the cited articles.…”

Cofactor-induced and mutational activity enhancement of coagulation factor VIIa

“…This demonstrates that the influence of the 170 loop on the intrinsic FVIIa activity is more complex and not only dictated by loop length. Additional grafting of the socalled 99 loop from trypsin had a slight opposite effect on the ability to activate factor X although it further improved the amidolytic activity [82].…”