Engineering of a membrane-triggered activity switch in coagulation factor VIIa.

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates its EPCR-dependent hemostatic activity in vivo.

Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo. SUMMARY: Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in mice, now allowing its further characterization in vivo. We also illustrate that modulation of the EPCR-FVIIa interaction may lead to improved FVIIa therapeutics.

Effect of non-specific reversal agents on anticoagulant activity of dabigatran and rivaroxaban: a randomised crossover ex vivo study in healthy volunteers.

The new anticoagulants dabigatran and rivaroxaban can be responsible for haemorrhagic complications. As for any anticoagulant, bleeding management is challenging. We aimed to test the effect of all putative haemostatic agents on the anticoagulant activity of these new drugs using thrombin generation tests. In an ex vivo study, 10 healthy white male subjects were randomised to receive rivaroxaban (20 mg) or dabigatran (150 mg) in one oral administration. After a two weeks washout period, they received the other anticoagulant. Venous blood samples were collected just before drug administration (H0) and 2 hours thereafter. Reversal of anticoagulation was tested in vitro using prothrombin complex concentrate (PCC), rFVIIa or FEIBA® at various concentrations. Rivaroxaban affects quantitative and kinetic parameters, including the endogenous thrombin potential (ETP-AUC and more pronouncedly the thrombin peak), the lag-time and time to peak. PCC strongly corrected ETP-AUC, whereas rFVIIa only modified the kinetic parameters. FEIBA corrected all parameters. Dabigatran specially affects the kinetics of thrombin generation with prolonged lag-time and time to peak. Although PCC increased ETP-AUC, only rFVIIa and FEIBA corrected the altered lag-time. For both anticoagulants, lower doses of FEIBA, corresponding to a quarter to half the dose usually used, have potential reversal profile of interest. In conclusion, some non-specific reversal agents appear to be able to reverse the anticoagulant activity of rivaroxaban or dabigatran. However, clinical evaluation is needed regarding haemorrhagic situations, and a meticulous risk-benefit evaluation regarding their use in this context is required.

The quest for Factor VIIa exosite inhibitors.

Coagulation proteases are involved in a highly orchestrated proteolytic cascade which is essential for haemostasis and blood clotting. In particular, the initiator of the coagulation cascade, Factor VIIa, binds to its cofactor, tissue factor, and its substrate, Factor X, via exosite interactions to form a ternary catalytic complex named extrinsic Xase. These exosite interactions have also been shown to allosterically induce the active conformation of the catalytic site of Factor VIIa. We have developed a direct continuous fluorescence polarization-based extrinsic Xase assay, which has been used to screen in excess of 1 million structurally diverse low-molecular-mass compounds as a potential starting point for the development of anticoagulants. The primary screen hits were categorized with deconvolution assays into either active-site or exosite inhibitors. The latter category of hits displayed both competitive and uncompetitive modalities of inhibition with respect to Factor X activation. An uncompetitive mechanism of action is of particular interest as it offers a hypothetical inhibitory advantage in the context of inhibiting a proteolytic cascade such as the blood coagulation pathway.

Crystal structure of Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with trypsin. Implications for KD1 specificity of inhibition.

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nM, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine beta-trypsin were obtained that diffracted to 1.8 A. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Ogamma of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1.

Optimization of a screening lead for factor VIIa/TF.

The structure-based design and progression of a screening lead to a 3nM factor VIIa/TF inhibitor with improved selectivity versus related enzymes is described.

Importance of factor VIIa Gla-domain residue Arg-36 for recognition of the macromolecular substrate factor X Gla-domain.

Macromolecular substrate docking with coagulation enzyme-cofactor complexes involves multiple contacts distant from the enzyme's catalytic cleft. Here we characterize the binding of the Gla-domain of macromolecular substrate coagulation factor X to the complex of tissue factor (TF) and VIIa. Site-directed mutagenesis of charged residue side chains in the VIIa Gla-domain identified Arg-36 as being important for macromolecular substrate docking. Ala substitution for Arg-36 resulted in an increased KM and a decreased rate of X activation. X with a truncated Gla-domain was activated by mutant and wild-type VIIa at indistinguishable rates, demonstrating that Arg-36 interactions require a properly folded Gla-domain of the macromolecular substrate. VIIa Arg-36 was also required for effective docking of the X Gla-domain in the absence of phospholipid, demonstrating that the Gla-domain of VIIa participates in protein-protein interactions with X. In the absence of TF, the mutant VIIa had essentially normal function, indicating that the cofactor positions VIIa's Gla-domain for optimal macromolecular substrate docking. Computational docking suggests multiple charge complementary contacts of the X Gla-domain with TF.VIIa. A prominent interaction is made by the functionally important X residue Gla-14 with the center of the extended docking site created by residues in the carboxyl module of TF and the contiguous VIIa Gla-domain. These data demonstrate the functional importance of interactions of the Gla-domains of enzyme and substrate, and begin to elucidate the molecular details of the ternary TF.VIIa.X complex.