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Egon Persson

Publications and source records attributed to Egon Persson.

17 recordsLinked to original sources
PubMed2007

A variant of recombinant factor VIIa with enhanced procoagulant and antifibrinolytic activities in an in vitro model of hemophilia.

OBJECTIVE: Recombinant factor VIIa (rFVIIa, NovoSeven) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. A rFVIIa analog with mutations V158D/E296V/M298Q (NN1731) exhibits increased procoagulant activity in in vitro and in vivo models. The aim of this work was to define the effects of NN1731 toward factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability. METHODS AND RESULTS: In a cell-based in vitro model of hemophilia, rFVIIa and NN1731 similarly increased factor X activation on tissue factor-bearing cells; however, NN1731 exhibited 30-fold higher factor Xa generation on platelets than similar rFVIIa concentrations. NN1731-mediated thrombin generation depended on platelet activation, but NN1731 did not directly activate platelets. NN1731 produced 4- to 10-fold higher maximal thrombin generation rates than equal rFVIIa concentrations. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at 50-fold lower concentrations than were required of rFVIIa. In fibrinolytic conditions, both rFVIIa and NN1731 increased fibrin formation and stability; however, NN1731 was effective at 50-fold lower concentrations than were required of rFVIIa. CONCLUSIONS: By increasing factor Xa generation, NN1731 promotes the formation of thrombin and a stable clot to a greater degree than rFVIIa.

Antifibrinolytic Agents↗
PubMed2006

A loop of coagulation factor VIIa influencing macromolecular substrate specificity.

Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX).

Amino Acid Substitution↗
PubMed2006

Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor.

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The gamma-carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF.PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF.PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF.PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.

Binding Sites↗
PubMed2006

Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange.

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.

Allosteric Site↗
PubMed2006

Macromolecular substrate affinity for free factor VIIa is independent of a buried protease domain N-terminus.

The initial recognition and binding of macromolecular substrates by factor VIIa (FVIIa) in complex with tissue factor has been shown to be mediated by areas distinct from the active site (so-called exosites). The present aim was to shed light on whether the N-terminal tail of the protease domain of FVIIa influences factor X (FX) binding, and whether the zymogen-like conformation of free FVIIa has a decreased affinity for FX compared to the active conformation. Two derivatives of FVIIa, one (FFR-FVIIa) with a stably buried N-terminus representing the active conformation of FVIIa and one (V154G-FVIIa) with a fully exposed N-terminus representing the zymogen-like conformation, were used as inhibitors of FVIIa-catalyzed FX activation. Their inhibitory capacities were very similar, with K(i) values not significantly different from the K(m) for FX. This indicates that the conformational state of the N-terminus does not affect FX binding or, alternatively, that the activation domain including the N-terminal insertion site is easily shifted to the stable conformation ensuing FX docking to the zymogen-like conformation. The net outcome is that FX binding to the zymogen-like form of FVIIa does not appear to be impaired.

Binding Sites↗
PubMed2005

Sequential coagulation factor VIIa domain binding to tissue factor.

Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the gamma-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced.

Factor VIIa↗
PubMed2005

Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W.

Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gaveriaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTF(R200W)) compared to that of the wild-type protein (sTF(wt)). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTF(R200W) mutant and sTF(wt) were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTF(R200W) indicated a population of partially folded intermediates during denaturation. sTF(R200W) showed a dramatically increased propensity for aggregate formation compared to sTF(wt) at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTF(R200W) in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TF(R200W) carriers, which could protect from thrombotic disease.

Amyloid↗
PubMed2005

Transition state analysis of the complex between coagulation factor VIIa and tissue factor: suggesting a sequential domain-binding pathway.

Injury of a blood vessel exposes membrane-bound tissue factor (TF) to blood, which allows binding of coagulation factor VIIa (FVIIa). This initiation of the coagulation cascade is dictated by a specific multi-domain interaction between FVIIa and TF. To examine the energies involved in the transition state of the FVIIa:TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with a smaller cysteine residue. Determination of Phi values in each of the positions using surface plasmon resonance measurements enabled us to characterize the transition state complex between the resulting sTF variants and FVIIa. We found that the interactions in the transition state seemed to be most pronounced between the protease domain of FVIIa and sTF while detailed specific interactions between the Gla-domain and sTF were missing. Thus, the transition state energy data indicate a sequential binding event between these two macromolecules.

Blood Coagulation↗
PubMed2004

Characterization of recombinant murine factor VIIa and recombinant murine tissue factor: a human-murine species compatibility study.

Tissue factor (TF) is believed to play an important role in coagulation, inflammation, angiogenesis and wound healing as well as in tumor growth and metastasis. To facilitate in vivo studies in experimental murine models, we have produced recombinant murine factor VII (FVII) and the ectodomain of murine TF, TF(1-223). Murine FVII was activated to FVIIa with human factor Xa and upon reaction with FFR-chloromethyl ketone converted into an active site-blocked TF antagonist, FFR-FVIIa. The activity of murine FVIIa was characterized in factor X activation assays as well as in clot assays with murine and human thromboplastin in murine and human plasma. In these assays murine FVIIa exhibited a specific activity equivalent to or higher than human FVIIa. Further analysis showed that murine FVIIa binds with high affinity to both murine and human TF, whereas the association of human FVIIa to murine TF is about three orders of magnitude weaker than the association to human TF. This difference was further emphasized by the effect of murine-and human FFR-FVIIa on bleeding in an in vivo mouse model. Intra-peritoneal administration of 1 mg/kg murine FFR-FVIIa significantly prolonged the tail-bleeding time, whereas no effect on bleeding was observed with a 25-times higher dose of the human FFR-FVIIa. Together, these data confirms the notion of poor species compatibility between human FVII and murine TF and emphasizes the requirement for autologous FVIIa in studies on the role of the TF in experimental in vivo pharmacology.

Animals↗
PubMed2004

Prevention of beta strand movement into a zymogen-like position does not confer higher activity to coagulation factor VIIa.

Coagulation factor VIIa (FVIIa) belongs to the chymotrypsin family of S1 peptidases, whose members require the cleavage of at least one internal peptide bond to attain an active conformation. FVIIa also requires association with tissue factor (TF) to attain full catalytic competency. Without this, FVIIa has very low activity toward peptide and physiologic substrates. Reregistration of beta strands has been suggested to play a part in the activation of FVII, and their positioning is possibly important for the active conformation of FVIIa. To scrutinize this hypothesis, we have designed FVIIa variants which prevent beta strand movement and lock FVIIa in the alleged active conformation. The V299M mutation, alone or combined with the L280I mutation, was introduced to alter the first of two Leu-X-Val motifs in beta strand B2 and thereby prevent reregistration. Along the same line, C164V/V299C-FVIIa has a new disulfide which would keep beta strand B2 in the registration of active FVIIa. The amidolytic and proteolytic activities of V299M-, L280I/V299M-, and C164V/V299C-FVIIa were indistinguishable from or lower than those of wild-type FVIIa, and none of the mutants displayed an altered exposure of the N-terminal amino group of the protease domain. Moreover, the affinities of mutant and native FVIIa for TF increased to a similar extent upon incorporation of an active site inhibitor, and the enzymatic activities were equally stimulated by TF. In conclusion, we found no evidence that the mutants were in a more active state than native FVIIa. Thus, the proposed beta strand reregistration, if part of the regulatory mechanism governing FVIIa activity, apparently does not suffice for the transformation of FVIIa into an enzymatically active conformation. Our data raise the possibility that the structural differences between enzymatically latent (zymogen-like) and active FVIIa resemble those between trypsinogen and trypsin.

Allosteric Site↗
PubMed2004

Augmented intrinsic activity of Factor VIIa by replacement of residues 305, 314, 337 and 374: evidence of two unique mutational mechanisms of activity enhancement.

Coagulation Factor VIIa (FVIIa) lacks the ability to spontaneously complete the conversion to a fully active enzyme after specific cleavage of an internal peptide bond (Arg152-Ile153) in the zymogen. Recently, several variants of FVIIa with enhanced intrinsic activity have been constructed. The in vitro characterization of these variants has shed light on molecular determinants that put restrictions on FVIIa in favour of a zymogen-like conformation and warrants continued efforts. Here we describe a new FVIIa variant with high intrinsic activity containing the mutations Leu305-->Val, Ser314-->Glu, Lys337-->Ala, and Phe374-->Tyr. The variant, called FVIIa(VEAY), processes a tripeptidyl substrate very efficiently because of an unprecedented, 5.5-fold lowering of the K(m) value. Together with a 4-fold higher substrate turnover rate this gives the variant a catalytic efficiency 22 times that of wild-type FVIIa, which is reflected in a considerably enhanced susceptibility to inhibition by antithrombin and other inhibitors. For instance, the affinity of FVIIa(VEAY) for the S1 probe and inhibitor p -aminobenzamidine is represented by an 8-fold lower K(i) value compared with that of FVIIa. Activation of Factor X in solution occurs about 10 times faster with FVIIa(VEAY) than with FVIIa, due virtually exclusively to an increased kcat value. The high activity of FVIIa(VEAY) is not accompanied by an increased burial of the N-terminus of the protease domain. A comparison of the kinetic parameters and molecular properties of FVIIa(VEAY) with those of the previously described mutant V158D/E296V/M298Q-FVIIa (FVIIa(IIa)), and the locations of the substitutions in the two variants, reveals what appear to be two profoundly different structural mechanisms dictating improvements in enzymic performance.

Amino Acid Sequence↗
PubMed2004

Variants of recombinant factor VIIa with increased intrinsic activity.

Following vascular damage, blood clotting is triggered when factor VIIa (FVIIa) forms a complex with tissue factor (TF). In hemophilia A and B, the propagation phase of blood coagulation is disrupted due to the lack of factors VIII (FVIII) and IX (FIX), leading to excessive bleeding. However, high doses of recombinant FVIIa (rFVIIa) can bypass the FVIII/FIX deficiency and ameliorate bleeding problems. Although the precise mechanism of action of rFVIIa at pharmacological doses remains a matter of debate, rFVIIa-catalyzed (TF-independent) activation of factor X (FX) on the surface of the activated platelet appears to be important. Variants of rFVIIa with increased intrinsic (TF-independent) activity have been developed, which may offer improved treatment of bleeding episodes, for example, in hemophiliacs with inhibitory antibodies to FVIII; they can also help us to understand how FVIIa works at the molecular level. This article reviews the properties of these molecules.

Blood Coagulation↗
PubMed2003

Improved hemostasis with superactive analogs of factor VIIa in a mouse model of hemophilia A.

It is currently debated whether the mechanism of action of therapeutic doses of recombinant factor VIIa (rFVIIa, Novo-Seven) relies on the tissue factor (TF)-independent activity of the enzyme. The present study was conducted to investigate the in vivo hemostatic effects of rFVIIa and 3 analogs thereof with superior intrinsic activity (FVIIaIIa, K337A-FVIIaIia, and M298Q-FVIIa) in mice with antibody-induced hemophilia A. A highly significant dose response was observed for the bleeding time and blood loss for each of the rFVIIa variants. The bleeding time and blood loss were normalized after administration of 10 mg/kg rFVIIa, 3 mg/kg K337A-FVIIaIia, and 3 mg/kg M298Q-FVIIa, indicating a potency of these FVIIa analogs 3-4 times above that of rFVIIa in FVIII-depleted mice. The different in vivo potencies of the various forms of FVIIa could not be explained by the pharmacokinetics. Histopathological evaluation of kidneys revealed no signs of treatment-related pathological changes even after treatment with the superactive variants. The fact that FVIIa analogs with enhanced intrinsic activity are more efficacious in the murine hemophilia A model strongly suggests that the TF-independent procoagulant activity of FVIIa contributes to its clinical hemostatic effect.

Amino Acid Substitution↗
PubMed2003

Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains.

We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.

Calcium↗
PubMed2003

Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor.

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.

Amino Acid Substitution↗
PubMed2002

Assignment of molecular properties of a superactive coagulation factor VIIa variant to individual amino acid changes.

The most active factor VIIa (FVIIa) variants identified to date carry concurrent substitutions at positions 158, 296 and 298 with the intention of generating a thrombin-mimicking motif, optionally combined with additional replacements within the protease domain [Persson, E., Kjalke, M. & Olsen, O. H. (2001) Proc. Natl Acad. Sci. USA98, 13583-13588]. Here we have characterized variants of FVIIa mutated at one or two of these positions to assess the relative importance of the individual replacements. The E296V and M298Q mutations gave an increased intrinsic amidolytic activity (about two- and 3.5-fold, respectively) compared with wild-type FVIIa. An additive effect was observed upon their combination, resulting in the amidolytic activity of E296V/M298Q-FVIIa being close to that of the triple mutant. The level of amidolytic activity of a variant was correlated with the rate of inhibition by antithrombin (AT). Compared with wild-type FVIIa, the Ca2+ dependence of the intrinsic amidolytic activity was significantly attenuated upon introduction of the E296V mutation, but the effect was most pronounced in the triple mutant. Enhancement of the proteolytic activity requires substitution of Gln for Met298. The simultaneous presence of the V158D, E296V and M298Q mutations gives the highest intrinsic activity and is essential to achieve a dramatically higher relative increase in the proteolytic activity than that in the amidolytic activity. The N-terminal Ile153 is most efficiently buried in V158D/E296V/M298Q-FVIIa, but is less available for chemical modification also in the presence of the E296V or M298Q mutation alone. In summary, E296V and M298Q enhance the amidolytic activity and facilitate salt bridge formation between the N-terminus and Asp343, E296V reduces the Ca2+ dependence, M298Q is required for increased factor X (FX) activation, and the simultaneous presence of the V158D, E296V and M298Q mutations gives the most profound effect on all these parameters.

Amino Acid Substitution↗