CHS Research Grants for 2008
- Platelet type von Willebrand disease: An underdiagnosed cause of excessive mucocutaneous bleeding?
- Factor VII(a) clearance behaviour
- Structural and genetic analysis of the complexes between factor VIII and low density lipoprotein receptor-related protein: Toward a long-acting factor VIII
- The role of X-inactivation in the expression of hemophilia A in women
- An evaluation of the prevalence and functional significance of non-neutralizing antibodies to factor VIII
- Carrier frequency among mothers of children with hemophilia
- Transfusions and regional anesthesia of the knee
Platelet type von Willebrand disease: An underdiagnosed cause of excessive mucocutaneous bleeding?
Adjunct Assistant Professor, Department of Pathology and Molecular Medicine
Queen’s University - Kingston, Ontario Second year funding
Co-investigator: Dr. David Lillicrap
Platelet type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease have similar clinical as well as laboratory features while resulting from abnormalities in two different genes. Type 2B VWD results from a defective von Willebrand factor (VWF) and PT-VWD is caused by platelet GP1BA gene defect. The discrimination between the two disorders is often a challenge. The general tests necessary for the identification require particular technical experience and necessitate fresh blood samples. Definitive diagnoses can be made by revealing the gene defect in either the VWF or the GP1BA. The correct diagnosis and discrimination between the two disorders has important implication on treatment decision. The VWF/FVIII concentrate, the major treatment in type 2B-VWD, may worsen platelet count and bleeding conditions in PT-VWD. Platelet concentrate is the ideal treatment for PT-VWD. In this study, we propose that patients with PT- VWD are being under / miss diagnosed among type 2B VWD and we adopt the use of a genetic approach for a definitive diagnosis of PT-VWD. The DNA analysis will include the related area of the VWF gene responsible for type 2B VWD and also the platelet GP1BA gene responsible for PT-VWD in 100 patients initially diagnosed with type 2B VWD from Canada and worldwide. Under the auspices of the International Society for Thrombosis and Hemostasis: Scientific and Standardization Committee on von Willebrand Factor, we established an online database/disease registry now accessible at www.pt-vwd.org. The project /registry will address the question of how many PT-VWD cases are being misdiagnosed among the type 2B VWD cases, increase awareness among physicians, specialists and patients about this diagnostic problem and will indicate the significance of the genetic analysis in these patients with subsequent implication on treatment decision.
Factor VII(a) clearance behaviour
Associate Professor, Department of Pathology and Molecular Medicine
McMaster University - Hamilton, Ontario
Second year funding
Co-investigator: Dr. B.J. Clarke
The hemophilia community knows all too well that the replacement therapy that can restore hemophiliacs to an active life is not always problem-free. For individuals with hemophilia A, replacement therapy with purified recombinant factor VIII (fVIII) can sometimes be complicated by inhibitor formation. Inhibitors are antibodies directed against the injected fVIII. Fortunately, administration of recombinant factor VIIa (fVIIa) can bypass this potentially serious problem. However, fVII is one of the coagulation factors that leaves the circulation the most rapidly. This clearance behaviour is not completely understood. For this reason, Drs. Bill Sheffield and Bryan Clarke, of McMaster University and Canadian Blood Services have received a CHS research grant to study the clearance of fVII(a) in mice genetically altered to have hemophilia A. The investigators will use DNA and cell culture technology to make both human fVII identical to current clinical products, and new forms of fVII in their laboratories. Once purified, how long the fVII-related proteins last in the mouse circulation, and how well they control bleeding will be determined. The most unusual products to be tested are “fusions” - in which fVII and human serum albumin, ordinarily separate proteins, are combined into a single chain. The researchers propose that fVII will continue to work in this new format, and will remain in the circulation for much longer than the current product. The longer it remains, the more effective it should be in combating bleeding. Sheffield and Clarke hope that their research will lead to better understanding of the mechanism of action of rfVIIa, and to the design of improved rfVII(a) products to provide caregivers with novel tools to help hemophiliac patients with inhibitors.
Structural and genetic analysis of the complexes between factor VIII and low density lipoprotein receptor-related protein: Toward a long-acting factor VIII
Department of Chemistry and Biochemistry
Concordia University – Montreal, Quebec
First year funding
Co-investigator: Dr. Tongpil Min (CO-PI)
Scientific developments have provided a new generation of safer factor VIII (FVIII) biopharmaceuticals that are free of blood-born pathogens. However, the high cost of FVIII still remains a big hurdle in hemophilia treatment. Only certain countries can afford prophylaxis or on-demand treatment while many others cannot even meet on-demand requirements. Clearly, FVIII has to be further developed to yield a product with a prolonged half-life.
Our project that is being supported by the CHS Dream of a Cure Research Program will provide the critical structural information to inform the development of longer acting FVIII. It is well known that low-density lipoprotein receptor-related protein (LRP), a receptor molecule, is responsible for eliminating FVIII from blood. FVIII clearance by LRP requires that the two proteins bind to each other. Thus, if the FVIII-LRP binding interaction is decreased, FVIII will remain in the blood stream longer. However, FVIII/LRP binding mechanisms have to be well understood to avoid possible side effects such as the induction of inhibitory antibodies. We will generate a 3D model of the FVIII/LRP binding interfaces using x-ray crystallography. Complexes between specific FVIII domains and LRP clusters will elucidate at the molecular level how LRP binds and eliminates FVIII. Such atomic-level structural details of the binding interfaces will indicate how the sequence of FVIII should be modified to decrease its interaction with LRP. Reduced affinity between FVIII and LRP will be evaluated using state-of-the art biophysical techniques. The results will guide the development of the next generation of FVIII biopharmaceuticals with a prolonged biological half-life. Longer lasting FVIII will allow treatment for all and bring us one step closer to a cure.
The role of X-inactivation in the expression of hemophilia A in women
Dr. Wenda L. Greer, FCCMG
Professor, Department of Pathology
Dalhousie University - Halifax, Nova Scotia
First year funding
Co-investigators: Dr. Sue Robinson, Dr. Sarah Dyack
Hemophilia A (HA) is an X-linked bleeding disorder resulting from mutations in the F8 gene. One functional F8 gene is sufficient for normal clotting. Thus, typically XY males who interit one mutated F8 gene are affected while heterozygous XX females are not. Rarely, heterozygous females with HA have been described. These can arise due to unfavourably skewed X chromosome inactivation (XCI). This is a dosage compensation mechanism that causes one X in every female cell to be inactivated early in development. In most females, approximately half of their cells inactivate their maternal and half their paternal X. In rare cases, XCI is skewed. If it is skewed toward the activation of a mutated gene, a heterozygous female can be affected. A family has presented with several males and several females affected with HA. In the females, most cells were expressing the mutated paternal X chromosome. It is unlikely that chance could account for the dramatic skewing of XCI leading to 3 affected females.
Our objective is to understand why these females are expressing HA. Our hypothesis is that is is due to skewed XCIR resulting from an inherited predisposition for the mutated X-chromosome to remain active.
Our aims are to:
- determine XCI patterns and correlate these with disease expression
- determine whether XCIR skewing is more consistent with genetically influenced or random XCI.
- delineate, through microsatellite analysis, a region of the X-chromosome that is associated with varied susceptibility of XCI
- consider potential candidate genes from this region.
- identify the clinical symptoms and standards of care of carrier females, and their experiences with medical treatment by interviewing carrier females and healthcare professionals.
An evaluation of the prevalence and functional significance of non-neutralizing antibodies to factor VIII
Department of Pathology and Molecular Medicine
Queen’s University – Kingston, Ontario
First year funding
Co-investigators: Dr. Jerome Teitel, Dr. Georges-Étienne Rivard, Dr. Irwin Walker
Funded through an unrestricted research grant to the Canadian Hemophilia Society from Bayer HealthCare
The development of antibodies to factor VIII (FVIII) represents the main treatment-related complication of current therapies for hemophilia A. For the past 30 years, this complication has been evaluated by a laboratory test that detects only those antibodies that interfere with FVIII’s clotting function. There is growing evidence to suggest that in addition to antibodies that inhibit the functional activity of FVIII, some “non-neutralizing” antibodies are also generated by FVIII exposure. The prevalence of these antibodies, their overlap with neutralizing inhibitors and their clinical significance are all factors that are very poorly characterized and form the basis for the studies proposed in this research project. In this project, we will establish a laboratory test to detect and quantify the levels of antibodies that bind to FVIII in plasma. The test plasma samples have already been characterized with a functional antibody test (the Bethesda assay) and thus overlapping patterns of reactivity will be apparent. Finally, when detected, we will attempt to correlate the presence of non-neutralizing antibodies with in vivo FVIII recovery and half-life.
Carrier frequency among mothers of children with hemophilia
University of Western Ontario
Under the supervision of Dr. Manuel Carcao
Hospital for Sick Children in Toronto - Toronto, Ontario
Funded through a research grant to the Canadian Hemophilia Society from Bayer HealthCare.
David Feldman worked at the Hospital for Sick Children from May-August 2008 on a study examining the concordance between hemophilia mutation and DDAVP responsiveness in patients with mild and moderate hemophilia A. He was also involved in a study which examined the carrier status of mothers of patients with hemophilia A. All patients included in these studies were followed in the hemophilia clinic at the Hospital for Sick Children, Toronto.
DDAVP responsiveness and genetics
David was responsible for the collection and organization of the raw data for his primary project which examined the correlation between DDAVP responsiveness and hemophilia genotype. He compiled a list of patients (n=105) that met the inclusion criteria of the study. David then reviewed the charts of all of these eligible patients. Of the 105 patients, 74 met both the inclusion and the exclusion criteria. All the data was incorporated into a database which David designed. David was responsible for all aspects of collecting, entering and checking the data, which he completed before the end of the summer term.
David submitted an abstract to the Summer Student Research (SSuRe) Program Symposium at the Hospital for Sick Children. At the symposium he presented a poster using preliminary data from this study.
Data collection is now closed for the study. David started the process of writing a paper before completing his work term. He is currently involved in finishing up the paper so that it can be submitted to a peer reviewed journal – either Haemophilia or the British Journal of Haematology.
The results of this study have revealed a tremendous concordance of DDAVP responsiveness in individuals with the same genetic mutation responsible for their hemophilia. It also showed that DDAVP response is associated with older age at testing and with higher baseline factor VIII levels. Awareness of this should allow physicians to be able to predict who will and who will not respond to DDAVP and as such may result in less need for DDAVP challenge tests to be done. It also alerts physicians to the need to repeat DDAVP testing in patients tested at young ages and found not to respond to DDAVP.
The incidence of carrier status in mothers of children with hemophilia
The second project on which David worked examined the incidence of carriership in mothers of children with hemophilia. For this project David reviewed a previously developed genetics data base at the Hospital for Sick Children and then updated this database.
Preliminary results have demonstrated that approximately 95% of mothers of children with hemophilia are carriers. Final analysis of the data is pending the completion of the DDAVP project. After analysis has been done the plan is to write a paper to be published in a peer reviewed journal reporting the results.
Transfusions and regional anesthesia of the knee
Université de Montréal
Under the supervision of Dr. Jean-François Hardy
Centre Hospitalier de l’Université de Montréal (CHUM)
Protocol and Objectives:
To determine prospectively through observation the minimum level of hemoglobin necessary to allow for optimal recovery after major orthopedic surgery.
It will be possible for us to determine the level of hemoglobin below which functional recovery becomes difficult. This value of hemoglobin will be used in phases 3 and 4 of the TRIOS project (Transfusion strategies in orthopedic surgery of the lower limb within a rehabilitative context).