Conditions: Hemophilia Bone Disease
Keywords: hemophilia bone disease clinical study biomarkers
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 16
Healthy volunteers: no

Brief summary:
One of the major shortcomings in studying bone disease in hemophilia is the lack of fracture outcome data demonstrating the clinical significance of decreased BMD and altered bone biomarkers in the hemophilia population. This study demonstrates that PwH have an increased risk of fracture compared to the general population and that the issue of bone health will increase in importance as the PwH population ages.

Detailed description:
This is a pilot study to determine the impact of factor replacement on bone biomarkers in up to 20 hemophilia A subjects. Subjects will be recruited over 1 year for the 5-day protocol.

Following a 72-hour washout period, factor levels and bone biomarkers will be followed before and after 50 units/kg replacement on Day 1 and 20 units/kg replacement on Day 3. Each subject can serve as their Figure 4. Fracture rates in PwH compared to historic controls.

Arms:
arm 1: Everyone receives Advate (antihemophilic factor) on Day 1 and 3.

Interventions: intervention 1: Advate
Intervention descriptions: intervention 1: Patients who are currently taking Advate as their factor replacement will be eligible for the 5-day study.

Protocol document:
Prot_SAP_000.pdf: 
Page 1 of 10 / Version: 28 March 2017 
 
PROSPECTIVE BIOMARKERS OF BONE METABOLISM IN HEMOPHILIA A 
 
Hypothesis 
Hemophilia is associated with decreased skeletal health. We hypothesize biomarkers of bone disease in 
hemophilia A will normalize with factor VIII (FVIII) replacement. 
 
Introduction 
The landscape of hemophilia treatment has changed dramatically over the last several decades.  
We are now in an era where safe factor replacement is widely available for preventing and treating 
bleeding episodes.  Rates of HIV and HCV are essentially absent in those less than 28 years of age.  
Furthermore people with hemophilia (PwH) have a normal life expectancy 1.  The focus of hemophilia 
management must now turn to addressing long-term complications of this condition 1,2.  Unfortunately 
PwH are seldom screened for decreased bone mineral density (BMD) despite having several major risk 
factors for bone loss. 
 
Decreased BMD and increased risk for fractures has been identified in PwH.  Studies in adults 
showed 70% of patients have low BMD, over 40% had osteopenia and 27% had osteoporosis 3,4. Several 
studies have demonstrated that children with hemophilia have significantly lower bone density than age 
matched control populations 5-7.  All of these studies included patients with HIV and HCV infections and 
patients had varying degrees of hemophilic arthropathy; all of which are risk factors for decreased BMD 
5,8, 9.  Thus it has been generally believed that decreased skeletal health in PwH is secondary to the risk 
factors of hemarthroses, chronic disease and immobility.   
 
Using a FVIII-deficient mouse model we have recently demonstrated that decreased skeletal 
health is independent of these risk factors and that bone disease is intrinsic to FVIII deficiency10.  As 
shown in Table 1, 20-week old (the age of peak bone mass) mice have decreased BMD and 
biomechanical strength compared to wild-type control. In a subsequent study we demonstrated through 
quantitative histology (histomorphometry) that the difference is caused by increased bone resorption in 
the factor deficient mice11 (data not shown). 
 
Table 1.  Femoral bone measurements of 20-week old mice showing statistically significant 
differences in BMD, cortical thickness, stiffness, and ultimate breaking force between WT 
and FVIII-deficient animals.  Standard deviations are in parenthesis.  
 
 
Femoral 
BMD 
(mg/cm2) 
Cortical 
Thickness 
(mm) 
Ultimate 
Force 
(N) 
Stiffness 
(N/mm) 
 
FVIII-
deficient 
(n = 23) 
 
56.49 
(2.66) 
 
0.1597 
(0.006) 
 
20.08 
(1.41) 
 
98.50 
(9.83) 
 
WT 
(n = 20) 
 
58.48 
(3.36) 
 
0.1638 
(0.006) 
 
22.31 
(2.24) 
 
108.2 
(11.22) 
 
p value 
 
0.036 
 
0.039 
 
0.0003 
 
0.005 


Page 2 of 10 / Version: 28 March 2017 
 
We demonstrated no differences in markers of bone formation (osteocalcin and alkaline phosphatase) nor 
in a key regulatory pathway of bone metabolism (receptor activator of nuclear factor kappa-B ligand 
[RANKL] and osteoprotegerin [OPG] (figure 1) between the FVIII-deficient and wild-type mice.  We did 
however identify differences in cytokine production, with decreased interferon- and interleukin-1 in 
the FVIII-deficient compared to wild-type 
mice (data not shown). 
 Based in part on these findings Baxter 
Bioscience funded a retrospective study 
investigating the bone biomarkers of PwH 
from the biorepository at the University 
of Colorado. We evaluated the bone 
biomarkers of 79 males with severe 
Hemophila A, 20 males with severe 
Hemophilia B and 51 healthy male 
controls. The results of the study were 
presented at the American Society of 
Hematology meeting in December 2013 
in poster form (please see the Appendix 
for the full poster).  There were several 
key findings in our study. First, 
biomarkers of bone disease were 
markedly different in individuals with 
developing bone (<16 years of age) 
compared to developed bone (age 16+) for both healthy controls and individuals with hemophilia A.  
There was increased CTX-1, a biomarker for increased bone resorption, in individuals with hemophilia A 
and mature bone compared to control.  Finally, samples with factor replacement the day prior had 
 
Figure 1: Bone Biomarkers. RANKL binding to the 
RANK receptor induces the proliferation and 
differentiation of osteoclasts resulting in increased bone 
resorption. OPG competitively inhibits RANKL binding 
to RANK, blocking these osteoclastic effects. CTX-1 is 
formed through the degradation of type 1 collagen in 
bone and thus is a direct marker of bone resorption. 
 
Figure 2. Factor replacement within 1 calendar day prior to blood draw increases OPG in 
FVIII-deficient subjects with mature bone.  p values are shown relative to controls. 
 
Control
FVIII Total
FVIII with Factor
FVIII without
Factor
0-15
16+
0.39
0.62
0.00054
0.73
0.065
0.52
Osteoclast 


Page 3 of 10 / Version: 28 March 2017 
 
increased OPG (Figure 2) and normalized CTX-1 (Figure 3) compared to controls. These results suggest 
that factor replacement ameliorates bone disease in hemophilia A.  Supporting the clinical significance of 
this study, an additional study demonstrated a correlation between bone biomarkers and BMD in 
individuals with hemophilia A12. 
Preliminary (unpublished) data 
Since the American Society of Hematology annual meeting additional data has been obtained 
from the biorepository samples.  We found no difference in osteocalcin (a biomarker for bone formation) 
between the various groups. Several cytokines are known to play a significant role bone metabolism 
(Table 2).  Using a multiplex system we simultaneously measured the plasma levels of key cytokines 
including: IFN, TNF, MSCF, IL-10, IL6, IL-12, IL-17, IL-1, IL-1. There was significant decrease 
in IL-10, IL-12 and TNF in the plasma of patients with hemophilia A compared to controls (P < .01), 
which normalized with recent factor infusion.  Since IL-10 and IL-12 have opposing effects on osteoclast 
function compared to TNF it is difficult to interpret the results.   Further evaluation of how cytokines 
vary with factor use and other variables in hemophilia is needed.  
Cytokine 
Osteoclast Activation 
IL-1 
MCSF 
TNF- 
IL-6 
IL-10 
IL-12 
IL-17 
IFN-γ 
IFN-β 
++++ 
+++ 
+++ 
+++ 
Inhibitory 
Inhibitory 
+++ 
Inhibitory 
Inhibitory 
Table 2:  Known effects of cytokines on osteoclast activation and bone resorption (adapted from 13).  Plus 
signs indicate relative activation potential of the cytokine 
 
Figure 3.  Factor replacement within 1 calendar day prior to blood draw normalizes CTX-1 in 
FVIII-deficient subjects.  p values are shown relative the controls. 
 
Control
FVIII Total
FVIII with Factor
FVIII without
Factor
0-15
16+
0.27
0.05
0.12
0.69
0.043
0.54


Page 4 of 10 / Version: 28 March 2017 
 
 
One of the major shortcomings in studying bone disease in hemophilia is the lack of fracture outcome 
data demonstrating the clinical significance of decreased BMD and altered bone biomarkers in the 
hemophilia population.  We undertook a retrospective analysis of the fracture rate of all PwH at our 
hemophilia treatment center over a 10-year period ending in December 2012.  A total of 47 fractures in 
387 subjects over 738,000 subject days were identified for a PwH fracture rate of 23.2 fractures per 1000 
subject years, over twice the rate of 9.58 fractures per 1000 subject years in the general population14. As 
demonstrated in the figure, the fracture rate in PwH also increased with age. Subjects with hemophilia A 
and hemophilia B had similar fracture rates (23.2 v 23.7 fractures per 1000 subject years).  This study 
demonstrates that PwH have an increased risk of fracture compared to the general population and that the 
issue of bone health will increase in importance as the PwH population ages. 
 
 
Specific Aim 
1.  Characterize the impact of FVIII replacement on the levels of biomarkers of bone metabolism in 
subjects with severe Hemophilia A. 
2. Correlate the levels of biomarkers of bone metabolism with subject history, physical examination and 
bone mineral density.  
 
Study Design 
This is a pilot study to determine the impact of factor replacement on bone biomarkers in up to 20 
hemophilia A subjects.  Subjects will be recruited over 1 year for the 5-day protocol. 
 
Following a 72-hour washout period, factor levels and bone biomarkers will be followed before and after 
50 units/kg replacement on Day 1 and 20 units/kg replacement on Day 3.  Each subject can serve as their 
 
Figure 4. Fracture rates in PwH compared to historic controls. 


Page 5 of 10 / Version: 28 March 2017 
 
own control using baseline washout biomarker levels to compare with subsequent values after factor 
replacement.  The subjects will also undergo two DEXA scans (spine and hip) and answer several survey 
questions about their history and factor use.   
 
Recruitment Methods 
Subjects who are currently using any factor FVIII replacement therapy will be recruited from the 
Hemophilia Center at Oregon Health & Science University. Subjects will be approached during a 
scheduled clinic visit, via email or over the phone and a research visit will be scheduled.   
 
The age of bone maturation depends on what criteria used but generally occur occurs around 16 years of 
age15.  Biomarkers of bone metabolism display a significant difference during development compared to 
bone that is more mature (Figure 2-3).  To verify that 16 years is an appropriate cutoff age to compare 
biomarkers in the hemophilia A population subanalysis CTX-1 levels from biorepository data was 
performed.  CTX-1 showed significant different between plasma samples in subjects > 16 compared to < 
16 (Figure 3).  Furthermore to prevent potential confounding effects of recent factor replacement samples 
from subjects that received factor replacement the day prior were removed from this analysis. There was a 
highly significant statistical difference in CTX-1 plasma concentration in the severe hemophilia A 
population comparing 14-15 to 16-17 year old subjects (p = 0.009), while there was no statistical 
difference in CTX-1 plasma concentration comparing16 to 17-19 year old subjects with severe 
hemophilia A. Thus 16 will be the cutoff age for enrollment in this study. 
 
Consent Process 
All participants will be both verbally instructed and provided with a written description of how the data 
will be collected, stored, used for future study, and how their privacy and confidentiality with be 
protected by de-indentifying the data.  If consent is obtained, we will ask the subjects to have at least a 
72-hour washout. 
 
Methods 
Inclusion criteria: 
1. Males with a diagnosis of hemophilia A with a historic baseline FVIII level ≤ 2%. 
2. Age > 16 years old 
3. Currently using any FVIII replacement therapy 
 
Exclusion criteria: 
1. Subject or guardian is unwilling or unable to give written informed consent and/or assent 
2. Joint or muscle bleeding within 2 weeks of Study Day 1 
3. Presence of a current factor inhibitor (>0.6 BU/mL via Nijmegan-modified Bethesda assay) 
4. Known collagen vascular bone disease. 
5. Current acute infection, per investigator discretion   
 
Subjects will be withdrawn from the study if a bleeding event occurs during the 5-day study visit period. 
 
 
 


Page 6 of 10 / Version: 28 March 2017 
 
Procedures 
The study will take place over two weeks and each subject will return daily to OHSU to complete study 
Visits 2-6. The study staff will call the subjects the day before study Visit 2 to confirm eligibility.  If 
subject has a current acute infection or has had a bleed in the last two weeks. 
 
Blood draws  
As outlined in Table 2, time points for blood draws include: time 0, 0.5, 4, 24, 48, 48.5, 72, and 96 hours. 
At each time point two tubes of citrated blood will be drawn.  One tube will be sent to the clinical 
laboratory to determine FVIII levels. The second tube will be centrifuged to obtain platelet poor plasma, 
divided into 400 l aliquots and placed into a -80 degree freezer within 30 minutes of the blood draw. A 
third tube will be drawn at time 0 to measure Vitamin D and C-reactive protein. FVIII inhibitor assay, von 
Willebrand Factor (vWF) antigen, and vWF activity will also be performed at the time 0 timepoint. 
 
Bone biomarker assays to be run at each timepoint include: OPG and RANKL (see Figure 1); CTX-1 and 
TRAP 5b (markers of bone resorption); osteocalcin and bone-specific alkaline phosphatase (markers of 
bone formation); and cytokine multiplex (see Table 2 and Preliminary Data). 
 
Factor infusion 
ADVATE [Antihemophilic Factor (Recombinant), Plasma/Albumin-Free Method] is an antihemophilic 
factor approved by the Food & Drug Administration for control and prevention of bleeding episodes in 
adults and children with Hemophilia A.  Subjects who are currently using any factor VIII replacement 
therapy will be eligible for enrollment in this study.  Subjects will receive ADVATE on Day 1 Hour 0 at a 
dose of 50 units/kg replacement.  Subjects will subsequently receive ADVATE on Day 3 Hour 0 at a dose 
of 20 units/kg factor replacement. The participants in this study are have all seen exogenous factor VIII 
previously and data demonstrates that in this population the risk of these serious side effects, especially in 
a short term study is <1%.” 
 
Two separate factor dosing levels will be used to help determine dose response of biomarkers to factor 
replacement and assess the level of factor replacement needed for normalization of biomarkers 
  
Subject Questionnaires  
On Day 1 subjects will answer questionnaires to obtain medications, past medical history and a complete 
bleeding history. Addition questionnaires will be given on quality of life and factor compliance. For visits 
3-6, the subjects will be given a short daily questionnaire asking about recent bleeding, changes in health 
and physical activity.  [Appendix A-G] 
 
List of questionnaires: 
Quality of life (Haemo-QoL) 
Hemophilia Activities List (HAL) 
Bleeding history  
Factor compliance (VERITAS-PRN) 
Daily health and pain assessment (EQ-5D-3L) 
Daily protocol specific questionnaires (Day 1 and Day 2-5) 
 
 


Page 7 of 10 / Version: 28 March 2017 
 
Physical examination 
One study Visit 2, subjects will have a physical exam by a physician. A joint assessment by a physical 
therapist using the Hemophilia Joint Health evaluation (Version 2.1) will be completed within one week 
of Visit 2.  The joint assessment score will be correlated with health history, BMD and bone biomarkers.  
[Appendix H] 
 
Gait Map Assessment 
Subjects will undergo a gait map evaluation for gait abnormality within 1 week of Study Day 1. Subjects 
will be asked to walk the full length of the mat 4 to 12 times, at least 2 twice with shoes on and at least 2 
twice with shoes off. This assessment will help identify abnormalities that may impact the BMD and 
physical activity. 
 
Dual-energy X-ray Absorptiometry (DEXA) 
Two DEXA scans will be performed on each subject within 1 week of the Study Day 1. DEXA scans are 
being performed in order to correlate BMD with the bone biomarkers. The imaging will be done on the 
spine and hip. 
 
Table 2: Study Procedure Chart 
  
Consenting 
Visit 
 
Visit 2 
Day 1 
Visit 3  
Day 2 
Visit 4 
Day 3 
Visit 5  
Day 4 
Visit 6 
Day 5 
 
 
0 hours 
30 
min 
 4 
hours 
24 hours 
48 hours 
48.5 
hours 
72 hours 
96 hours 
Informed 
consent review 
x 
 
 
 
 
 
 
 
 
 
Physical Exam 
 
 
x 
 
 
 
 
 
 
 
Weight 
x 
 
x 
 
 
 
 
 
 
 
Factor 
Replacement 
 
 
50 
units/kg 
  
  
  
20 
units/kg 
  
  
  
Biomarkers 
 
 
x 
 x 
x 
x 
x 
x 
x 
x 
Factor Level 
 
 
x 
x  
x 
x 
x 
x 
x 
x 
vWF antigen  
and activity 
 
 
x  
  
  
  
  
  
  
  
Gait Map 
 
 
Must be completed within one week of Day 1 
DEXA scans 
 
 
 Must be completed within one week of Day 1  
Questionnaires 
 
 
x 
  
  
x 
x 
 
x  
x 
 
Compensation 
Subjects will be compensated $500 to complete the entire 5study visit protocol, DEXA scans and gait 
mapping. 
 
Institutional approval 
This protocol will obtain full institutional approval, including the Institutional Review Board, prior to 
initiation of the study. 
 
 
 


Page 8 of 10 / Version: 28 March 2017 
 
Data Analysis and Evaluation 
A statistician will evaluate the data using multivariable analysis to compare baseline bone biomarkers 
with those following FVIII infusion as well as to patient history, joint health and bone mineral density. 
For instance, repeated measures ANOVA and survival analysis will test for differences in biomarker 
levels in response to factor replacement therapy. 
 
RedCap 
A portion of the data for this project will be stored in OCTRI's installation of REDCap, a highly secure 
and robust web-based research data collection and management system. 
 
Features of REDCap that protect participants' privacy and data security include: 
 
Physical Security: OCTRI's REDCap software is housed on servers located in ITG's Advanced 
Computing Center providing locked physical security. 
 
Electronic Security: The REDCap servers are housed behind both the OHSU firewall and a 
second ACC firewall. All web-based data transmissions are encrypted with industry-standard SSL 
methods. 
 
Controlled User Access: REDCap is employs a robust multi-level security system that enables 
researchers to easily implement "minimum necessary" data access for their research staff, 
including specification of data fields that are identifiers. This feature includes “single click” 
ability to provide completely deidentified (removing all identified data fields and shifting dates) 
for analysis or other purposes.  User activities are logged to enable auditing of all data access.   
Access is integrated with OHSU's network such that users who are also OHSU employees are 
authenticated against their OHSU network credentials.   
 
Data Integrity: REDCap is jointly managed in accordance with OHSU Information Security 
Directives by ACC staff and members of OCTRI's Biomedical Informatics Program, ensuring 
fidelity of database configuration and back-ups.  User activities are logged to enable auditing of 
all data changes. 
 
References 
1. Plug I, Van Der Bom JG, Peters M, et al. Mortality and causes of death in patients with hemophilia, 
1992-2001: A prospective cohort study. J Thromb Haemost. 2006;4(3):510-516. 
2. Siboni SM, Mannucci PM, Gringeri A, et al. Health status and quality of life of elderly persons with 
severe hemophilia born before the advent of modern replacement therapy. J Thromb Haemost. 
2009;7(5):780-786. 
3. Gerstner G, Damiano ML, Tom A, et al. Prevalence and risk factors associated with decreased bone 
mineral density in patients with haemophilia. Haemophilia. 2009;15(2):559-565. 


Page 9 of 10 / Version: 28 March 2017 
 
4. Wallny TA, Scholz DT, Oldenburg J, et al. Osteoporosis in haemophilia - an underestimated 
comorbidity? Haemophilia. 2007;13(1):79-84. 
5. Barnes C, Wong P, Egan B, et al. Reduced bone density among children with severe hemophilia. 
Pediatrics. 2004;114(2):e177-81. 
6. Gallacher SJ, Deighan C, Wallace AM, et al. Association of severe haemophilia A with osteoporosis: A 
densitometric and biochemical study. Q J Med. 1994;87(3):181-186. 
7. Tlacuilo-Parra A, Morales-Zambrano R, Tostado-Rabago N, Esparza-Flores MA, Lopez-Guido B, 
Orozco-Alcala J. Inactivity is a risk factor for low bone mineral density among haemophilic children. Br J 
Haematol. 2008;140(5):562-567. 
8. Amorosa V, Tebas P. Bone disease and HIV infection. Clin Infect Dis. 2006;42(1):108-114. 
9. Anagnostis P, Vakalopoulou S, Vyzantiadis TA, et al. The clinical utility of bone turnover markers in 
the evaluation of bone disease in patients with haemophilia A and B. Haemophilia. 2013. 
10. Liel MS, Greenberg DL, Recht M, Vanek C, Klein RF, Taylor JA. Decreased bone density and bone 
strength in a mouse model of severe factor VIII deficiency. Br J Haematol. 2012;158(1):140-143. 
11. Recht M, Liel MS, Turner RT, Klein RF, Taylor JA. The bone disease associated with factor VIII 
deficiency in mice is secondary to increased bone resorption. Haemophilia. 2013;19(6):908-912. 
12. Kempton CL, Antun A, Antoniucci DM, et al. Bone density in haemophilia: A single institutional 
cross-sectional study. Haemophilia. 2013. 
13. Lee SK, Lorenzo J. Cytokines regulating osteoclast formation and function. Curr Opin Rheumatol. 
2006;18(4):411-418. 


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14. Brinker MR, O'Connor DP. The incidence of fractures and dislocations referred for orthopaedic 
services in a capitated population. J Bone Joint Surg Am. 2004;86-A(2):290-297. 
15. Roche AF, Roberts J, Hamill PV. Skeletal maturity of youths 12--17 years racial, geographic area, and 
socioeconomic differentials. united states, 1966-1970. Vital Health Stat 11. 1978;(167)(167):1-98. 
 
 
Appendix A: Quality of life questionnaire (Haemo-QoL) 
Appendix B: Hemophilia Activities List (HAL) 
Appendix C: Bleeding history 
Appendix D: Factor compliance questionnaire (VERITAS-PRN) 
Appendix E: Daily health and pain questionnaire (EQ-5D-3L) 
Appendix F: Day 1 Questionnaire 
Appendix G: Days 2-5 Questionnaires 
Appendix H: Hemophilia Joint Health Assessment