Molecular mechanism of the interaction between activated factor XIII and its glutamine donor peptide substrate

Summary. Background: Activated factor XIII (FXIII), a dimer of truncated A‐subunits (FXIII‐A₂*), is a transglutaminase that crosslinks primary amines to peptide‐bound glutamine residues. Because in the few natural substrates of FXIII‐A₂* no consensus sequence could be identified around the reactive glutamine, studying the interaction between individual substrates and FXIII‐A₂* is of primary importance. Most of the α₂‐plasmin inhibitor (α₂PI) molecules become truncated by a plasma protease, and the truncated isoform (N1‐α₂PI) is an important substrate of FXIII‐A₂*. The crosslinking of N1‐α₂PI to fibrin plays a major role in protecting fibrin from fibrinolysis. Methods: We studied the interaction of FXIII‐A₂* with its dodecapeptide glutamine donor substrate, N1‐α₂PI(1–12), the sequence of which corresponds to the N‐terminal sequence of N1‐α₂PI. Kinetic parameters for N1‐α₂PI(1–12) and for its truncated or synthetic mutants were determined by a spectrophotometric assay. The interaction of N1‐α₂PI(1–12) with FXIII‐A₂* was investigated by proton nuclear magnetic resonance (NMR) and saturating transfer difference (STD) NMR. Results and Conclusions: Kinetic experiments with peptides in which the Asn1 residue was either truncated or replaced by alanine and proton NMR analysis of the FXIII‐A₂*–N1‐α₂PI(1–12) complex demonstrated that Asn1 is essential for effective enzyme–substrate interaction. Experiments with C‐terminally truncated peptides proved that amino acids 7–12 are essential for the interaction of N1‐α₂PI(1–12) with the enzyme, and suggested the existence of a secondary binding site on FXIII‐A₂*. Hydrophobic residues, particularly Leu10 and the C‐terminal Lys12, seemed to be especially important in this respect, and direct interaction between hydrophobic C‐terminal residues and FXIII‐A₂* was demonstrated by STD NMR.     

A common ancestral mutation (C128X) occurring in 11 non‐Jewish families from the UK with factor XI deficiency

Summary. Factor XI (FXI) deficiency is a mild bleeding disorder that is particularly common in Ashkenazi Jews, but has been reported in all populations. In Jews, two FXI gene (F11) mutations (a stop codon in exon 5, E117X, type II, and a point mutation in exon 9, F283L, type III) are particularly common, but in other populations a variety of different mutations have been described. In the Basque region of France one mutation, C38R in exon 3, was found in eight of 12 families studied, haplotype analysis suggesting a founder effect. In the course of screening 78 unrelated individuals (including 15 Jewish and 12 Asian) we have found 10 Caucasian non‐Jewish patients with the mutation C128X in exon 5. Individuals were investigated because of a personal or family history of bleeding, or finding a prolonged activated partial thromboplastin time. Individuals negative for the type II and type III mutations were screened by a combination of SSCP and heteroduplex analysis. The C128X mutation was found in 10 families (one previously described). Among three individuals with severe FXI deficiency, one was homozygous for the C128X mutation, and two were compound heterozygotes for the C128X and another mutation; other individuals were carriers of the C128X mutation. This is a nonsense mutation producing a truncated protein; individuals have FXI antigen levels concordant with FXI coagulant activity. Haplotype analysis of 11 families, including a further kindred previously reported from the USA, but which originally came from the UK (in which the index patient was homozygous for C128X), suggests a founder effect.     

A cross-reactive material positive variant of coagulation factor XI (FXIP520L) with a catalytic defect

Inherited deficiency of the trypsin-like protease factor (F) XI is associated with a mild to moderate bleeding diathesis. In most cases, FXI protein is reduced in plasma, and examples of dysfunctional circulating FXI variants are rare. We characterized the defect in one such variant with a proline to leucine substitution at residue 520. FXI Pro520 corresponds to chymotrypsin Pro161, and is conserved in most members of the chymotrypsin protease family. Recombinant FXI containing this substitution will be referred to as FXI(P161L). k(cat) for cleavage of chromogenic substrates and for activation of the natural FXIa substrate FIX is approximately 3-fold lower for activated FXI(P161L) (FXIa(P161L)) than for wild-type FXIa (FXIa(WT)), consistent with an abnormal protease active site. Inhibition of FXIa(P161L) by diisopropyl fluorophosphate is 2.4-fold slower than for FXIa(WT), suggesting distortion of the protease oxyanion hole. Binding to p-aminobenzamidine, a probe for the integrity of the S1 substrate-binding site, was similar for FXIa(WT) and FXIa(P161L). Rates of carbamylation of Ile16 were also similar for FXIa(WT) and FXIa(P161L), indicating that the critical salt bridge between Ile16 and Asp194 forms normally during protease activation. Cumulatively, the data demonstrate that Pro161 is required for normal active site oxyanion hole conformation in FXIa. Examination of the FXIa crystal structure and modeling studies indicate that Pro161 forms several hydrophobic contacts with adjacent amino acids that stabilize active site conformation. Leucine can be incorporated at position 161 in FXIa, but would not form the extensive stabilizing network of hydrophobic interactions formed by Pro161.     

Giant spin pumping at the ferromagnet (permalloy) – organic semiconductor (perylene diimide) interface

Pure spin current based devices have attracted great interest in recent days. Spin current injection into non-magnetic materials is essential for the design and development of such pure spin current based devices. In this context, organic semiconductors (OSCs) can be potential non-magnetic materials over widely explored heavy metals. This is due to the relatively low spin–orbit coupling of OSCs, which is essential to host the spin current with a large spin diffusion length and long spin-relaxation time. This research work demonstrates the harvesting of spin currents at the perylene diimide (PDI)/permalloy (Py) based OSC interface. The observed high linewidth broadening of 2.18 mT from the ferromagnetic resonance spectra indicates the presence of giant spin pumping from Py to PDI. The resultant spin-mixing conductance, 1.54 × 10¹⁸ m⁻² quantifies the amount of spin current injected from Py to PDI, which is in a similar range to ferromagnet/heavy metals.

The spin injection from permalloy into an adjacent perylene diimide (PDI) layer is demonstrated via ferromagnetic resonance associated linewidth broadening. The spin mixing conductance is found to be 1.54×10¹⁸ m⁻² in a similar range to FM/heavy metal.     

Deep brain stimulation (DBS) at the interface of neurology and psychiatry

Deep brain stimulation (DBS) is an emerging interventional therapy for well-screened patients with specific treatment-resistant neuropsychiatric diseases. Some neuropsychiatric conditions, such as Parkinson disease, have available and reasonable guideline and efficacy data, while other conditions, such as major depressive disorder and Tourette syndrome, have more limited, but promising results. This review summarizes both the efficacy and the neuroanatomical targets for DBS in four common neuropsychiatric conditions: Parkinson disease, Tourette syndrome, major depressive disorder, and obsessive-compulsive disorder. Based on emerging new research, we summarize novel approaches to optimization of stimulation for each neuropsychiatric disease and we review the potential positive and negative effects that may be observed following DBS. Finally, we summarize the likely future innovations in the field of electrical neural-network modulation.     

Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations

Summary.  Background:  Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665 or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al. , Blood, 112, 2761, 2008). Objectives:  In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effects of these mutations on stability. Methods:  Studies assessing FVIII stability involved monitoring decay rates of FVIII at 55 °C or in guanidinium, decay of FVIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nmol L⁻¹) FVIII concentration. Results and conclusions:  Similar tendencies were observed within each group of variants. Variants with mutations at D519 and either E665 or E1984 (Group A) generally showed significantly better stability as compared with single mutants. Most variants with mutations at E665 and at E1984 (Group B) did not show significant improvement. Triple mutants with mutations at D519, E665 and E1984 (Group C) showed improvement to a similar degree as the Group A double mutants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield FVIII reagents with improved stability parameters.     

Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs

Summary.  Background: The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. Objectives: To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc₂) but not to GPVI monomer (GPVIex) through a phage display method. Results: Ssix Fabs were found: B–F, only reactive with GPVI-Fc₂, and A, mainly reactive with GPVI-Fc₂, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc₂ binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc₂. Adding the anti-GPVI monoclonal antibody 204-11 increased the B _max of m-Fab-F binding to GPVI-Fc₂, suggesting that 204-11 binds to GPVI-Fc₂ molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. Conclusions: GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.     

Preclinical pharmacokinetics and biodistribution of subcutaneously administered glycoPEGylated recombinant factor VIII (N8‐GP) and development of a human pharmacokinetic prediction model

Essentials

• N8‐GP is an extended half‐life recombinant factor VIII (FVIII) for the treatment of hemophilia A.
• Subcutaneous (SC) FVIII dosing might reduce the treatment burden of prophylaxis.
• SC N8‐GP has a favorable PK profile in animal models and disappears from skin injection sites.
• Combined animal (SC) and clinical (IV) data suggest that daily SC dosing may provide prophylaxis.

Summary

Background

N8‐GP is an extended half‐life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous administration of FVIII may reduce the treatment burden of prophylaxis; however, standard FVIII products have low bioavailability after subcutaneous dosing in animals.

Objective

To evaluate the pharmacokinetics, effectiveness and local distribution of subcutaneously administered N8‐GP in preclinical models and predict the human pharmacokinetic (PK) profile.

Methods

The pharmacokinetics of subcutaneously administered N8‐GP were evaluated in FVIII knockout (F8‐KO) mice and cynomolgus monkeys; a human PK prediction model in hemophilia A patients was developed. The hemostatic effect was evaluated in a tail vein bleeding model in F8‐KO mice. The injection‐site distribution and absorption of subcutaneously administered N8‐GP were assessed in F8‐KO mice by the use of temporal fluorescence imaging and immunohistochemistry.

Results

Subcutaneously administered N8‐GP had a bioavailability, a first‐order absorption rate and a half‐life, respectively, of 24%, 0.094 h^?1 and 14 h in F8‐KO mice, and 26%, 0.33 h^?1 and 15 h in cynomolgus monkeys. A dose‐dependent effect of subcutaneously administered N8‐GP on blood loss was observed in mice. A minimal amount of N8‐GP was detected at the injection site 48–72 h after single or multiple dose(s) in F8‐KO mice. Subcutaneously administered N8‐GP was localized to the skin around the injection site, with time‐dependent disappearance from the depot. PK modeling predicted that subcutaneously administered N8‐GP at a daily dose of 12.5 IU kg^?1 will provide FVIII trough levels of 2.5–10% in 95% of patients with severe hemophilia A.

Conclusions

Subcutaneously administered N8‐GP may provide effective hemophilia A prophylaxis. A phase I clinical trial is underway to investigate this possibility.

     

Preoperative plasma D‐dimer predicts 1‐year survival in colorectal cancer patients with absence of venous thromboembolism (VTE): a prospective clinical cohort study

Summary. Background: Fibrin formation is required for tumor angiogenesis, metastasis and invasion. Cancer discovered at the same time as or shortly after venous thromboembolism (VTE) tends to be advanced, and the prognosis poor. Previous studies have demonstrated that plasma D‐dimer – a degradation product of cross‐linked fibrin – correlates with tumor stage and prognosis in patients with colorectal cancer. However, it remains unclear whether D‐dimer is of prognostic significance in colorectal cancer patients with absence of VTE. Objective: To examine whether the preoperative plasma D‐dimer level predicts 1‐year survival in pre‐ and postoperative VTE‐negative colorectal cancer patients admitted for surgery. Methods:  We measured preoperative D‐dimer levels in 157 patients, and computed Kaplan‐Meier survival curves according to the levels of D‐dimer. Cox proportional‐hazard regression analysis was used to compute hazard ratio as a measure of 1‐year mortality rate ratio, controlling for potential confounding factors. The Aalborg Hospital’s standard cut‐off level of 0.3 mg L^?1 was used to distinguish negative and positive D‐dimer results. Results: The overall 1‐year survival rate was 87.3% (95% confidence interval (CI), 81.0–91.6%), with 78.1% survival (95% CI, 65.9–86.4%) in the positive D‐dimer group compared with 93.6% survival (95% CI, 86.2–97.1%) in the negative D‐dimer group. The adjusted hazard ratio of death in the positive D‐dimer group compared with the negative D‐dimer group was 3.6 (95% CI, 1.3–9.9). Conclusion: A positive preoperative D‐dimer is associated with a poor prognosis in colorectal cancer patients with absence of VTE.     

Elucidating the intramolecular contrast in the STM images of 2,4,6-tris(4′,4′′,4′′′-trimethylphenyl)-1,3,5-triazine molecules recorded at room-temperature and at the liquid-solid interface

Star-shaped 2,4,6-tris(4′,4′′,4′′′-trimethylphenyl)-1,3,5-triazine molecules self-assemble at the solid–liquid interface into a compact hexagonal nanoarchitecture on graphite. High resolution scanning tunneling microscopy (STM) images of the molecules reveal intramolecular features. Comparison of the experimental data with calculated molecular charge density contours shows that the molecular features in the STM images correspond to molecular LUMO+2.

Intramolecular contrast in the STM images of 2,4,6-tris(4′,4′′,4′′′-trimethylphenyl)-1,3,5-triazine molecules recorded at room-temperature and at the liquid–solid interface.