Clearance and Genetic Variability of Von Willebrand Factor Are Major Determinants of the Pharmacokinetic Behavior of Factor VIII Concentrates in the Treatment of Pediatric Hemophilia A
Authors: Ogiwara Kenichi , Swystun Laura L , Georgescu Ilinca , Brown Christine , Tuttle Angie , Tinlin Shawn , Leggo Jayne , Albánez Silvia , Carcao Manuel , Blanchette Victor S. , Male Christoph , Thom Katharina , Lillicrap David 
Published: October 2019

Background: Although Factor VIII (FVIII) concentrates are now routinely used for the prophylactic treatment of hemophilia A (HA), the optimal doses and intervals between administrations are difficult to predict because of variable pharmacokinetics of FVIII (FVIII-PK) between patients. Previous studies in HA have revealed a close relationship between FVIII-PK and the FVIII carrier protein, von Willebrand factor (VWF). A large genome-wide association study from the CHARGE consortium highlighted several novel loci associated with plasma levels of VWF and FVIII in normal subjects, and the five genetic loci associated with FVIII levels coincided with those influencing VWF levels (Smith, 2010).

Objective: To investigate the effects of VWF synthesis, clearance and genetic variability on FVIII-PK in young HA patients. We hypothesized that 1) plasma VWF:Ag levels (VWF secretion and clearance), 2) polymorphic variants within the FVIII binding region of VWF, and 3) the glycosylation pattern of VWF (N-linked and ABO blood group antigen) would influence FVIII-PK.

Methods: HA males recruited at two large academic pediatric hemophilia centers (The Hospital for Sick Children in Toronto and the Medical University of Vienna) were enrolled. Blood was collected at 5 time points (pre, post FVIII-infusion: 1, 9, 24, and 48 h), and FVIII-PK parameters, clearance (CL), volume of distribution (VD) and half-life (HL), were calculated based on a Bayesian model. Plasma levels of VWF (VWF:Ag), VWF propeptide (VWFpp) and FVIII binding ability of VWF (VWF:FVIIIB) were also evaluated. Genetic analysis of the FVIII-binding region and glycosylation sites of VWF was performed.

Results: Samples from 33 boys [median age 10.9 years (range 6.5-17.9)] with severe HA were evaluated. Median values of FVIII-CL, VD and HL were 0.032 dl/h/kg (range 0.018-0.062), 0.47 dl/kg (0.29-0.78), and 10.2 h (6.7-16.8), respectively. VWF:Ag, VWFpp, VWFpp/VWF:Ag ratio and VWF:FVIIIB were 86.6 IU/dl (39.9-141.6), 88.2 U/dl (43.5-156.6), 1.09 (0.33-1.71) and 70.3% (41.2-101.9), respectively. FVIII-CL (r=-0.41, p<0.05) and HL (r=0.56, p<0.001) correlated with VWF:Ag. Interestingly, while VWFpp levels did not correlate with FVIII-HL, the VWFpp/VWF:Ag ratio (a measure of VWF clearance) was negatively correlated (r=-0.47, p<0.01), suggesting that plasma VWF:Ag, especially its rate of clearance, can significantly influence FVIII-HL. Comparing the patients with the longest (13.3-16.8 h, n=8) and shortest (6.7-9.4 h, n=8) FVIII-HLs, significant differences (p<0.05) were observed for VWF:Ag and VWFpp/VWF:Ag ratio, but not for age, VWFpp, and VWF:FVIIIB. Sequencing of the VWF D’D3 region associated with FVIII-binding identified 9 SNPs (5 non-synonymous and 4 synonymous) in exons 17-21 and 24-27. Two SNPs in exon 18 (rs1063856: c.2365A>G, Thr789Ala, and rs1063857: c.2385T>C, Tyr795=) previously identified in the CHARGE study, segregated as a haplotype. In the longest FVIII-HL group there was a 43.8% prevalence of the infrequent alleles and two patients in this group were homozygous for these alleles. In contrast, the shortest FVIII-HL group had no homozygous subjects and only a 12.5% prevalence of the infrequent haplotype. Since c.2365A>C (Thr789Pro) has been reported as a type 2N VWD variant, we studied the VWF:FVIIIB properties of the Thr789Ala variant. The two patients homozygous for the Thr789Ala showed VWF:FVIIIB levels of 85.8% and 85.5% of normal in their plasmas. Recombinant Thr789Ala and Thr789Pro variants derived from 293T cells showed lower (p<0.01) VWF:FVIIIB levels of 67% and <20%, respectively, when compared to recombinant wild-type VWF. This data indicates that the VWF SNP haplotype, rs1063856/rs1063857, appears to be associated with a longer FVIII-HL without increasing FVIII-binding ability. As ABO blood group is associated with VWF glycosylation and its subsequent clearance we analyzed the distribution of blood groups in the patients with the longest and shortest FVIII-HL and found that there were no blood group O patients in the longest FVIII-HL group while 63% of patients in the shortest FVIII-HL group were blood group O (p<0.05). Sequencing of consensus N-glycosylation sites in VWF showed no variants.

Conclusion: In this pediatric HA population, FVIII-PK was significantly influenced by VWF:Ag, rate of VWF clearance and blood group, and a VWF SNP haplotype in the FVIII binding region might also modify FVIII-PK.

Disclosures

No relevant conflicts of interest to declare.