Henrik Østergaard

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

To visualize the formation of disulfide bonds in living cells, a pair of redox‐active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2‐fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non‐invasive fluorimetric measurements. The 1.5 Å crystal structure of the oxidized protein revealed a disulfide bond‐induced distortion of the β‐barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state‐dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox‐active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Inhibitory Serpins from Wheat Grain with Reactive Centers Resembling Glutamine-rich Repeats of Prolamin Storage Proteins CLONING AND CHARACTERIZATION OF FIVE MAJOR MOLECULAR FORMS

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P1-P1′ Gln-Gln, and WSZ2b at P2-P1 Leu-Arg—one of two overlapping reactive centers. WSZ1c with P1-P1′ Leu-Gln was the fastest inhibitor of chymotrypsin (k a = 1.3 × 106 m −1 s−1). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k a∼105 m −1 s−1), which has P1-P1′ Leu-Ser—a reactive center common in animal serpins. WSZ2b inhibited plasmin at P1-P1′ Arg-Gln (k a∼103 m −1 s−1). None of the five serpins inhibited Bacillus subtilisin A,Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

Generation and biochemical characterization of glycoPEGylated factor VIIa derivatives

Prophylaxis with 2-4 times weekly dosing of factor (F)VIII or FIX is established as an efficacious and safe treatment in haemophilia. Although prophylaxis is not readily available for the inhibitor patient, recent studies have demonstrated a reduction in bleeding episodes in inhibitor patients treated with daily infusions of FVIIa. In order to develop a treatment option comparable to prophylaxis with FVIII or FIX we looked to PEGylation which is an established method for prolonging the circulatory half-life of proteins. However, due to the numerous interactions of FVIIa with the cell surface, TF, FIX and FX there are limited options for unspecific chemical modification of FVIIa without loss of activity. Consequently, we explored the GlycoPEGylationtrade mark technology for selective PEGylation of the two N-glycans in the FVIIa light chain and protease domain to generate seven specifically modified derivatives with PEG groups ranging from 2 to 40 kDa. These derivatives were evaluated in vitro for their ability to interact with small synthetic substrates as well as key molecules relevant to function in the coagulation pathway. The results demonstrate that modification of FVIIa using glycoPEGylation has only a very limited effect on the hydrolysis S-2288 and FX activation. However, the modification does to some extend alter the ability of FVIIa to interact with TF and more importantly, reduces the rate of ATIII inhibition by up to 50% which could allow for an extended active half-life in circulation.

The origins of enhanced activity in factor VIIa analogs and the interplay between key allosteric sites revealed by hydrogen exchange mass spectrometry.

Factor VIIa (FVIIa) circulates in the blood in a zymogen-like state. Only upon association with membrane-bound tissue factor (TF) at the site of vascular injury does FVIIa become active and able to initiate blood coagulation. Here we used hydrogen exchange monitored by mass spectrometry to investigate the conformational effects of site-directed mutagenesis at key positions in FVIIa and the origins of enhanced intrinsic activity of FVIIa analogs. The differences in hydrogen exchange of two highly active variants, FVIIaDVQ and FVIIaVEAY, imply that enhanced catalytic efficiency was attained by two different mechanisms. Regions protected from exchange in FVIIaDVQ include the N-terminal tail and the activation pocket, which is a subset of the regions of FVIIa protected from exchange upon TF binding. FVIIaDVQ appeared to adopt an intermediate conformation between the free (zymogen-like) and TF-bound (active) form of FVIIa and to attain enhanced activity by partial mimicry of TF-induced activation. In contrast, exchange-protected regions in FVIIaVEAY were confined to the vicinity of the active site of FVIIa. Thus, the changes in FVIIaVEAY appeared to optimize the active site region rather than imitate the TF-induced effect. Hydrogen exchange analysis of the FVIIaM306D variant, which was unresponsive to stimulation by TF, correlated widespread reductions in exchange to the single mutation in the TF-binding region. These results reveal the delicate interplay between key allosteric sites necessary to achieve the transition of FVIIa into the active form.

A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation

Coagulation factor VIIa (FVIIa) requires tissue factor (TF) to attain full catalytic competency and to initiate blood coagulation. In this study, the mechanism by which TF allosterically activates FVIIa is investigated by a structural dynamics approach that combines molecular dynamics (MD) simulations and hydrogen/deuterium exchange (HX) mass spectrometry on free and TF‐bound FVIIa. The differences in conformational dynamics from MD simulations are shown to be confined to regions of FVIIa observed to undergo structural stabilization as judged by HX experiments, especially implicating activation loop 3 (residues 365–374{216–225}) of the so‐called activation domain and the 170‐loop (residues 313–322{170A–175}) succeeding the TF‐binding helix. The latter finding is corroborated by experiments demonstrating rapid deglycosylation of Asn322 in free FVIIa by PNGase F but almost complete protection in the presence of TF or an active‐site inhibitor. Based on MD simulations, a key switch of the TF‐induced structural changes is identified as the interacting pair Leu305{163} and Phe374{225} in FVIIa, whose mutual conformations are guided by the presence of TF and observed to be closely linked to the structural stability of activation loop 3. Altogether, our findings strongly support an allosteric activation mechanism initiated by the stabilization of the Leu305{163}/Phe374{225} pair, which, in turn, stabilizes activation loop 3 and the S1 and S3 substrate pockets, the activation pocket, and N‐terminal insertion.

The tick-derived inhibitor Ixolaris prevents tissue factor signaling on tumor cells

Summary  Background:  Tissue factor (TF) is frequently overexpressed in cancer cells and correlated with more aggressive tumor phenotypes and poor prognosis. In addition to promoting coagulation‐dependent metastasis and cancer‐associated thrombosis, tumor cell‐expressed TF mediates direct cell signaling involving the protease‐activated receptor (PAR) 2. Ixolaris is a tick‐derived inhibitor of the TF–factor (F)VIIa–Xa coagulation initiation complex which blocks primary tumor growth and angiogenesis in glioblastoma and melanoma models.

Engineering of Substrate Selectivity for Tissue Factor-Factor VIIa Complex Signaling through Protease-activated Receptor 2

The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF·FVIIa complex and by FXa in the ternary TF·FVIIa·FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF·FVIIa-Xa complex normally but were severely impaired in binary TF·FVIIa·PAR2 signaling. The residues identified were located in the model-predicted S2′ pocket of FVIIa, and complementary PAR2 P2′ Leu-38 replacements demonstrated that the P2′ side chain was indeed crucial for PAR2 cleavage by TF·FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF·FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactor-associated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.

Selective factor VIII activation by the tissue factor-factor VIIa-factor Xa complex

Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.