Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop

Summary. Background: Heterologous expression of factor VIII (FVIII) is about two to three orders of magnitude lower than similarly sized proteins. Bioengineering strategies aimed at different structural and biochemical attributes of FVIII have been successful in enhancing its expression levels. Objective: Disulfide bonds are vital to the proper folding, secretion and stability of most secretory proteins. In an effort to explore additional targeted bioengineering approaches, the role of disulfide bonds in FVIII secretion and function was probed in this study. Methods and Results: Single and paired cysteine mutants were generated by substituting with serine or glycine residues and analyzed by transient transfection into COS‐1 and CHO cells. Seven of the eight disulfide bonds in FVIII were found to be indispensable for proper secretion and function. However, elimination of the disulfide bond formed by C1899 and C1903 within the conserved A3 domain improved the secretion of FVIII. The addition of the C1899G/C1903G mutations to a previously described FVIII variant, 226/N6, with high secretion efficiency increased its secretion by 2.2‐fold. Finally, the addition of the A1‐domain mutation, F309S, in conjunction with the disulfide mutation had an additive effect, resulting in a net improvement in secretion of between 35 and 45‐fold higher than wild‐type FVIII in CHO cells. Conclusion: Such combined targeted bioengineering strategies may facilitate more efficient production of recombinant FVIII and contribute toward low‐cost factor replacement therapy for hemophilia A.     

REDUCED FOREARM BONE MINERAL CONTENT AND BIOCHEMICAL EVIDENCE OF INCREASED BONE TURNOVER IN WOMEN WITH EUTHYROID GOITRE TREATED WITH THYROID HORMONE

We used single-photon absorptiometry to assess forearm bone mineral content (BMC/BW) (arbitrary units normalized for bone width) at a proximal site (PBMC/BW) and at a more distal site (DBMC/BW) in 60 women treated with 25-50 micrograms T3 or 50-100 micrograms T4 for euthyroid goitre, in 13 untreated goitre patients, and in 2 controls matched for age and menopausal state for each goitre patient. BMC/BW was not significantly different between untreated goitre patients and controls. In 36 premenopausal patients, treated for 5.8 +/- 5.4 years (mean +/- SD) a slight decrease in PBMC/BW of about 5% compared to controls to controls was observed (PBMC/BW 1.42 +/- 0.19 vs 1.49 +/- 0.13, P less than 0.05). In 24 postmenopausal patients, treated for 10.0 +/- 5.8 year, a 20% deficit in BMC/BW compared to controls was found (DBMC/BW 0.80 +/- 0.18 vs 1.06 +/- 0.20, P less than 0.001 and PBMC/BW 1.14 +/- 0.20 vs 1.42 +/- 0.19, P less than 0.001). Biochemical indices of bone metabolism in 43 pre and post-menopausal patients and 43 controls showed in the patients a higher serum alkaline phosphatase activity (AP) (P less than 0.01 and P less than 0.05 and serum osteocalcin (NS and P less than 0.05). AP was negatively correlated with TSH levels and, in postmenopausal patients, with DBMC/BW and PBMC/BW. Our results suggest that treatment of euthyroid women with moderate doses of thyroid hormone increases bone turnover with clear adverse effects on bone mineral status in postmenopausal patients.     

Analysis of interdigital spaces during mouse limb development at intervals following amniotic sac puncture

A spectrum of limb abnormalities ranging from adactyly, syndactyly, acrosyndactyly to nail hypoplasia was encountered in mouse embryos subjected to amniotic sac puncture at the corresponding gestational stage when human chorionic villus sampling (cvs) would normally be performed clinically. Previous skeletal studies revealed that, apart from the occasional incidence of fusion of 2 distal phalanges, syndactyly usually only affected the soft tissues within the interdigital spaces. A similar situation was also observed in cases of adactyly; while the skeletal elements of the digits were present, the soft tissues in the interdigital spaces failed to separate. A transient period of bradycardia is induced, possibly secondary to compression of the embryo by the extraembryonic membranes and uterine muscles following amniotic sac puncture. These factors, we believe, produce temporary hypoxia/ischaemia of the distal extremities, and may lead to the modification of the interdigital mesenchymal tissues within the autopods. In order to investigate the mechanism(s) underlying soft tissue syndactyly, limbs recovered at 0.5, 4, 8, 12, 24, or 36 h following amniotic sac puncture (ASP) were examined histologically. Vascular disruption in the form of localised areas of haemorrhage, vascular dilatation and congestion and the presence of fluid-filled cavities occurred in relation to the marginal vein and vascular plexus in the interdigital spaces. It is hypothesised that this interfered with the normal equilibrium of the preset programs of mitosis/cell death and apoptosis within the mesenchymal cells of the interdigital spaces. Apoptosis in these areas was inhibited in the majority of the experimental limbs analysed 4 h after ASP. Instead of undergoing necrosis/apoptosis, increased mitotic activity was usually observed from 8 h following ASP at the sites where apoptosis would normally be expected to be seen. The aberrant fate of the interdigital mesenchyme following ASP and the underlying mechanism(s) involved are discussed, as is the critical importance of an adequate vascular supply to the interdigital spaces during the morphogenesis of the autopod. We believe that this report contributes to understanding the mechanism(s) which lead to syndactyly following ASP, and the limb defects occasionally seen following cvs when this is undertaken during early gestation.