Molecular coevolution of coagulation factor VIII and von Willebrand factor.

Ancestral sequence reconstruction provides a unique platform for investigating the molecular evolution of single gene products and recently has shown success in engineering advanced biological therapeutics. To date, the coevolution of proteins within complexes and protein-protein interactions is mostly investigated in silico via proteomics and/or within single-celled systems. Herein, ancestral sequence reconstruction is used to investigate the molecular evolution of 2 proteins linked not only by stabilizing association in circulation but also by their independent roles within the primary and secondary hemostatic systems of mammals. Using sequence analysis and biochemical characterization of recombinant ancestral von Willebrand factor (VWF) and coagulation factor VIII (FVIII), we investigated the evolution of the essential macromolecular FVIII/VWF complex. Our data support the hypothesis that these coagulation proteins coevolved throughout mammalian diversification, maintaining strong binding affinities while modulating independent and distinct hemostatic activities in diverse lineages.

Recombinant VWF fragments improve bioavailability of subcutaneous factor VIII in hemophilia A mice.

Conventional treatment of hemophilia A (HA) requires repetitive IV injection of coagulation factor VIII (FVIII). Subcutaneous administration of FVIII is inefficient because of binding to the extravascular matrix, in particular to phospholipids (PLs), and subsequent proteolysis. To overcome this, recombinant dimeric fragments of von Willebrand factor (VWF) containing the FVIII-stabilizing D3 domain were engineered. Two fragments, called VWF-12 and VWF-13, demonstrated high binding affinity to recombinant human FVIII (rhFVIII) and suppressed PL binding in a dose-dependent manner. High concentrations of VWF fragments did not interfere with the functional properties of full-length VWF in vitro. The HA mouse model was used to study the effects of VWF-12 or VWF-13 on the in vivo pharmacokinetics of rhFVIII, demonstrating (1) no significant impact on rhFVIII recovery or half-life after a single IV administration; (2) enhanced bioavailability (up to 18.5%) of rhFVIII after subcutaneous administration; and (3) slow absorption (peak concentration, 6 hours) and prolonged half-life (up to 2.5-fold) of rhFVIII after subcutaneous administration. Formation of anti-FVIII antibodies was not increased after administration of rhFVIII/VWF-12 subcutaneously compared with rhFVIII IV. A single subcutaneous dose of rhFVIII/VWF-12 provided protection in the HA tail-bleeding model for up to 24 hours. In summary, recombinant VWF fragments support FVIII delivery through the subcutaneous space into vascular circulation without interfering with VWF or FVIII function. Slow resorption and excretion of FVIII after subcutaneous administration highlight the potential application of VWF fragments for subcutaneous FVIII prophylaxis in HA.

Mass Spectrometry-Based Method for Detection and Identification of Free Thiol Groups in Proteins.

Many proteins contain free sulfhydryl groups which can be involved in a variety of biochemical reactions. Reactive thiol groups can either reside within the active center of oxidoreductases or represent a part of a thiol-based redox switch in proteins. Therefore, the exact position of a free sulfhydryl within a protein is mostly very important.This chapter describes a mass spectrometry-based method to determine the location of protein sulfhydryl groups exemplary shown for a synthetic decapeptide and the plasma glycoprotein von Willebrand factor (VWF). We outline (1) labeling of free sulfhydryl groups, (2) enrichment of labeled peptides, and (3) detection and identification of labeled peptides by mass spectrometry.

Comparative N-Glycosylation Analysis of the Fc Portions of a Chimeric Human Coagulation Factor VIII and Immunoglobulin G1.

Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion to Fc-immunoglobulin (Ig)G that mediate protection from clearance or degradation via binding to the neonatal Fc receptor. While human-like N-glycosylation of recombinant FVIII is known to be crucial for the clotting factor's quality and function, the particular glycosylation of the fused Fc portion has not been investigated in detail so far, despite its known impact on Fcγ receptor binding. Here, we analyzed the N-glycosylation of the Fc part of a chimeric FVIII-Fc protein compared to a commercial IgG1 purified from human plasma. Fc parts from both samples were released by enzymatic cleavage and were subsequently separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Corresponding protein bands were referred to PNGase F in-gel digestion in order to release the respective N-glycans. Analysis via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed structural differences of both N-glycan patterns. Labeling with 2-aminobenzamide (2AB) and analysis via hydrophilic interaction liquid chromatography (HILIC) allowed a quantitative comparison of the respective N-glycosylation. Observed variations in Fc glycosylation of the chimeric FVIII fusion protein and human plasma-derived IgG1, e.g., regarding terminal sialylation, are discussed, focusing on the impact of the clotting factor's properties, most notably its binding to Fcγ receptors.