Residue Met156 Contributes to the Labile Enzyme Conformation of Coagulation Factor VIIa

Petrovan, Ramona J.; Ruf, Wolfram

doi:10.1074/jbc.m004726200

Cited by 41 publications

(63 citation statements)

References 33 publications

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“…Globally, these exosites are referred to as the 'activation domain' and encompass residues 284-294, 295-304, 332-345 and 363-374 (Eigenbrot et al, 2001). Studies of the mutations of FVII showed that the substitutions M298Q (Petrovan & Ruf, 2001), R304Q (O'Brien et al, 1991), R304W (Matsushita et al, 1994), L305V (Persson et al, 2001a), and M306D (Persson et al, 2001b) in the serine protease domain had adverse effects on the enzyme's activity and TF interaction. Indeed, the R304Q and R304W mutations were associated with reduced affinity for human TF and reduced FVIIa activity, while the L305V, M306D and M298Q mutations increase FVIIa activity even in the absence of TF, without altering the TF affinity.…”

Section: Discussionmentioning

confidence: 99%

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

et al. 2004

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SummaryWe report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild‐type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (Kd from 4·4 nmol/l for FVIIaWT to 17·3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (kcat/Km from 2·3 × 107/mol/l s for FVIIaWT to 8·7 × 105/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen‐like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF‐catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the Km value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.

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Section: Discussionmentioning

confidence: 99%

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

et al. 2004

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“…One of the structural mechanisms is responsible for the stabilization of ion pair formation, as described previously (5, 36, 41, 42). Another structural mechanism is responsible for catalytic efficiency, which could be independent of formation of the ion pair (32,43,44).…”

Section: Discussionmentioning

confidence: 99%

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

Soejima¹,

Yuguchi²,

Mizuguchi³

et al. 2002

Journal of Biological Chemistry

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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...

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“…It is not precisely understood how FVII is cleaved into FVIIa, but a proteolytic role is suggested for either the minute amounts of FVIIa that circulate in the blood (100 pM) (198) or the factor VII-activating protein (FSAP), but recent data argue against the latter (276). At physiological concentrations, FVIIa without TF shows little activity, because of a unique sequence characteristic that retains the FVIIa in a zymogenlike conformation (230). Thus FVIIa activity at physiological levels is entirely TF dependent.…”

Section: Initiation Phasementioning

confidence: 99%

New Fundamentals in Hemostasis

Versteeg

Heemskerk

Levi

et al. 2013

Physiological Reviews

912

831

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Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. After wounding, the hemostatic system engages a plethora of vascular and extravascular receptors that act in concert with blood components to seal off the damage inflicted to the vasculature and the surrounding tissue. The first important component that contributes to hemostasis is the coagulation system, while the second important component starts with platelet activation, which not only contributes to the hemostatic plug, but also accelerates the coagulation system. Eventually, coagulation and platelet activation are switched off by blood-borne inhibitors and proteolytic feedback loops. This review summarizes new concepts of activation of proteases that regulate coagulation and anticoagulation, to give rise to transient thrombin generation and fibrin clot formation. It further speculates on the (patho)physiological roles of intra-and extravascular receptors that operate in response to these proteases. Furthermore, this review provides a new framework for understanding how signaling and adhesive interactions between endothelial cells, leukocytes, and platelets can regulate thrombus formation and modulate the coagulation process. Now that the key molecular players of coagulation and platelet activation have become clear, and their complex interactions with the vessel wall have been mapped out, we can also better speculate on the causes of thrombosis-related angiopathies.

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Residue Met156 Contributes to the Labile Enzyme Conformation of Coagulation Factor VIIa

Cited by 41 publications

References 33 publications

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

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

New Fundamentals in Hemostasis

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