Biological mechanisms underlying inter‐individual variation in factor VIII clearance in haemophilia

Previous studies have highlighted marked inter‐individual variations in factor VIII (FVIII) clearance between patients with haemophilia (PWH). The half‐life of infused FVIII has been reported to vary from as little as 5.3 hours in some adult PWH, up to as long as 28.8 hours in other individuals. These differences in clearance kinetics have been consistently observed using a number of different plasma‐derived and recombinant FVIII products. Furthermore, recent studies have demonstrated that half‐life for extended half‐life (EHL‐) FVIII products also demonstrates significant inter‐patient variation. Since time spent with FVIII trough levels <1% has been shown to be associated with increased bleeding risk in PWH on prophylaxis therapy, this variability in FVIII clearance clearly has major clinical significance. Recent studies have provided significant novel insights into the cellular basis underlying FVIII clearance pathways. In addition, accumulating data have shown that endogenous plasma VWF levels, ABO blood group and age, all play important roles in regulating FVIII half‐life in PWH. Indeed, multiple regression analysis suggests that together these factors account for approximately 34% of the total inter‐individual variation in FVIII clearance observed between subjects with severe haemophilia A. In this review, we consider these and other putative modulators of FVIII half‐life, and discuss the biological mechanisms through which these factors impact upon FVIII clearance in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156160/2/hae14078.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156160/1/hae14078_am.pd

Polyethylene glycol 20 kDa-induced vacuolation does not impair phagocytic function of human monocyte-derived macrophages

Conjugation to polyethylene glycol (PEG) is commonly used to enhance drug delivery and efficacy by extending the half-life of the drug molecule. This has important implications for reducing treatment burden in diseases that require chronic prophylaxis, such as hemophilia. Clearance of PEG molecules with high molecular weights (≥ 40 kDa) has been reported to cause cellular vacuolation in mammals. Rurioctocog alfa pegol (PEGylated recombinant coagulation factor VIII) contains a 20-kDa PEG. This study investigated the effects of exposure to 20-kDa PEG (10 μg/ml to 10 mg/ml) on the morphology and function of human monocyte-derived macrophages (MDMs) in vitro. Exposure to PEG for 24 hours was associated with significant vacuolation only at concentrations of 1 mg/ml or more, which far exceed the levels associated with clinically relevant doses of rurioctocog alfa pegol. Immunofluorescence staining of PEG was detected in the cytoplasm of MDMs, indicating uptake into the cells. No impairment of MDM phagocytic activity (ability to ingest fluorescently labeled Escherichia coli) was observed with 24-hour exposure to PEG, even at concentrations associated with significant vacuolation. Furthermore, PEG exposure did not have significant effects on cytokine secretion in resting or lipopolysaccharide-stimulated MDMs, or on the expression of cell surface markers in stimulated MDMs. Cell viability was not affected by 24-hour exposure to PEG. In conclusion, vacuolation of human MDMs after exposure to 20-kDa PEG only occurred with PEG concentrations far in excess of those equivalent to clinically relevant doses of rurioctocog alfa pegol and did not affect MDM viability or functionality. Together, these results support the concept that PEG-mediated vacuolation is an adaptive cellular response rather than a toxic effect

In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments