In haemophilia A patients factor VIII (FVIII) recovery and half-life can vary substantially. There are parameters known to modulate FVIII pharmacokinetics (PK), but they explain only about 34% of the variability. The aim of this study was to identify new parameters that influence FVIII PK and thus to expand the current knowledge. FVIII PK were determined in 42 haemophilia A patients (37 severe, 5 moderate) without inhibitor. Patients' characteristics and laboratory parameters were evaluated for an association with FVIII PK. We analysed plasma levels of low-density lipoprotein receptor-related protein 1 (LRP1) and protein C (PC) activity, which had been hypothesized to influence FVIII activity. Furthermore, four variations in intron 6 of the LRP1 gene, which had been shown to influence LRP1, were investigated. FVIII half-life differed widely from 6.2 to 20.7 h, with a median of 10.0 h. Patients with blood group O had shorter FVIII half-life compared to patients with non-O blood group (median FVIII half-life 9.0 h vs. 10.4 h, P = 0.018). Age was significantly associated with FVIII half-life (r = 0.32, P = 0.035). Besides age, also VWF antigen (r = 0.52, P < 0.001) and blood group (r = -0.37, P = 0.015) was associated with FVIII half-life. No correlation was found with FVIII- or LRP1-genotype, LRP1 or PC concentrations. Our data showed large differences in FVIII PK between individual patients and revealed age, blood group and VWF levels as important determining factors for FVIII half-life. FVIII genotype or levels of LRP1 or PC had no influence on FVIII PK.

The high rate of patients with BUC despite in-depth haemostatic assessment underlines the incompleteness of available routine laboratory tests. Males with MBDs were more likely to be diagnosed with an established bleeding disorder than females.

The substantial variability in pharmacokinetic parameters in hemophilia patients A poses a challenge for optimal treatment with factor VIII (FVIII) products. We investigated the effect of FVIII-specific immunoglobulin G (IgG) on FVIII half-life in a cohort of 42 adult patients with severe and moderate hemophilia A without inhibitors. Fifteen (35.7%) of 42 patients tested positive for FVIII-binding IgG with titers ‡1:20 in the initial antibody screen, 9 of these 15 patients had FVIII-specific antibodies with titers ‡1:40, mostly low-tomoderate-affinity IgG1 and IgG3, and 1 had high-affinity IgG4 and later developed low-titer FVIII inhibitors. His brother with low-to-moderate-affinity IgG1 and IgG3 also later developed low-titer FVIII inhibitors. The presence of FVIII-specific IgG subclass titer ‡1:40 antibodies was significantly associated with shorter FVIII half-life (median, 7.8 hours [interquartile range, 6.6-9.2 hours]) vs 10.4 hours [interquartile range, 8.9-13.8 hours]); the regression coefficient adjusted for log age and log von Willebrand factor (VWF) antigen was 20.32 (P 5 .004), accounting for 16.9% of the observed variability of FVIII half-life in our cohort. Our data indicate a significant contribution of non-neutralizing FVIII-specific IgG to FVIII half-life reduction in hemophilia A patients. Thus, screening for FVIII-specific IgG could be beneficial in tailoring FVIII prophylactic regimens. (Blood. 2016;128(2):293-296) IntroductionProphylactic treatment of hemophilia A patients with factor VIII (FVIII) products is presently state of the art.1 FVIII pharmacokinetics differ significantly, which poses a challenge for optimal treatment design. It is generally accepted that the von Willebrand factor (VWF) level significantly influences pharmacokinetic (PK) parameters such as FVIII half-life.2-5 Different VWF levels only partially explain the variability in FVIII half-life, which leaves the question of which other parameters are accountable. This study was conducted to evaluate the effect of non-neutralizing, FVIII-specific immunoglobulin G (IgG) antibodies on FVIII half-life. Study designPlasma samples from a cohort of 42 patients with hemophilia A (recently described by Kepa et al 2 ) were screened for FVIII-binding IgG antibodies. Antibodies were analyzed by using fully validated semiquantitative, directbinding enzyme-linked immunosorbent assays (ELISAs) as described in Whelan et al. 6 Details of the validation procedure as well as details of the cutoff determination and the sensitivity of the ELISAs are provided in Whelan et al 6 and Hofbauer et al. 7 Initially, all samples were screened in a 1:20 dilution for FVIII-binding IgG antibodies. Samples that tested positive were characterized for titers of FVIIIbinding IgG subclasses and apparent affinities. The specificity of FVIII-binding antibodies was tested in all samples with antibody titers $1:40 by using an affinity ELISA.7 Antibodies without conclusive apparent affinity were considered unspecific. Antibodies with titers ,1:40 (eg, 1:20)...

Background Factor VIII (FVIII) pharmacokinetics (PK) in adult hemophilia A populations are highly variable and have been previously determined to be influenced by von Willebrand factor:antigen (VWF:Ag), ABO blood group, and age. However, additional genetic determinants of FVIII PK are largely unknown. Objectives The contribution of VWF clearance, VWF‐FVIII‐binding activity, and genetic variants in VWF clearance receptors to FVIII PK in adult patients were assessed. Methods FVIII PK assessment was performed in 44 adult subjects (age 18‐61 years) with moderate or severe hemophilia A. VWF:Ag, VWF propeptide (VWFpp), VWFpp/VWF:Ag, and VWF:FVIII binding activity were measured. The VWF modifying loci CLEC4M, SCARA5, STAB2, and ABO, and the D′D3 FVIII‐binding region of the VWF gene were genotyped. Results VWF:Ag, VWFpp, and VWF:FVIIIB positively correlated with FVIII half‐life and negatively correlated with FVIII clearance. VWFpp/VWF:Ag negatively correlated with FVIII half‐life and positively correlated with FVIII clearance. The correlation between VWFpp/VWF:Ag and FVIII half‐life was stronger for type non‐O patients than for type O patients, suggesting that slower VWF clearance increases FVIII half‐life. Patients heterozygous for the CLEC4M rs868875 variant had increased FVIII clearance when compared with individuals homozygous for the reference allele. The CLEC4M variable number of tandem repeat (VNTR) alleles were also associated with the rate of FVIII clearance. When compared with the quartile of patients with the fastest FVIII clearance, the quartile of patients with the slowest FVIII clearance had a decreased frequency of the CLEC4M 5‐VNTR. Conclusions VWF‐FVIII binding activity and genetic determinants of VWF clearance are important contributors to FVIII pharmacokinetics in adult patients.

In more than 50% of patients with a mild-to-moderate bleeding tendency, no underlying cause can be identified (bleeding of unknown cause, BUC). Data on parameters of fibrinolysis in BUC are scarce in the literature and reveal discrepant results. It was the aim of this study to investigate increased fibrinolysis as a possible mechanism of BUC. We included 270 patients (227 females, median age 44 years, 25–75th percentile 32–58) with BUC and 98 healthy controls (65 females, median age 47 years, 25–75thpercentile 39–55). Tissue plasminogen activator (tPA-) antigen and activity, plasminogen activator inhibitor type-1 (PAI-1), tPA-PAI-1 complexes, thrombin activatable fibrinolysis inhibitor (TAFI), α2-antiplasmin, and D-dimer were determined. While PAI-1 deficiency was equally frequent in patients with BUC and controls (91/270, 34%, and 33/98, 34%, p = 0.996), tPA activity levels were more often above the detection limit in patients than in controls (103/213, 48%, and 23/98, 23%, p < 0.0001). We found lower levels of tPA-PAI-1 complexes (6.86 (3.99–10.00) and 9.11 (7.17–13.12), p < 0.001) and higher activity of TAFI (18.61 (15.80–22.58) and 17.03 (14.02–20.02), p < 0.001) and α2-antiplasmin (102 (94–109) and 98 (90–106], p = 0.003) in patients compared to controls. Detectable tPA activity (OR 3.02, 95%CI 1.75–5.23, p < 0.0001), higher levels of TAFI (OR 2.57, 95%CI 1.48–4.46, p = 0.0008) and α2-antiplasmin (OR 1.03, 95%CI 1.01–1.05, p = 0.011), and lower levels of tPA-PAI-1 complexes (OR 0.90, 95%CI 0.86–0.95, p < 0.0001) were independently associated with BUC in sex-adjusted logistic regression analyses. We conclude that the fibrinolytic system can play an etiological role for bleeding in patients with BUC.