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.

Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage.

Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65-270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1ß, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage) in mice.

[Thinking and challenge induced by the hypothesis of breaking stagnant and eliminating blood stasis in Treating acute cerebral hemorrhage by rF VII a].

Definite therapeutic effect has obtained by TCM in treating acute cerebral hemorrhage (ACH) according the TCM theory of "blood circulating outside the vessels is the stasis" using breaking stagnant and eliminating blood stasis (Poxue Zhuyu) method, but no material involving the natural development of stoke in superacue stage (0 - 4 hrs after onset of the disease) being presented so far. It has been proved by randomized, double-blinded multi-centeric clinical trials that recombinant activated factor VII (rF VII a) could decreased the morbidity and disability of patients suffered from ACH, suggesting that use hemostasis treatment in ACH during superacu stage should be stressed, and the drugs for Poxue Zhuyu should be used cautiously in the period of 0 - 4 hrs after onset. The hemorrhagic disorder could be eliminated by using drugs for Poxue Zhuyu and other medicines in rational combination.

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.