This project will explore the mechanism(s) responsible for decreased ristocetin-von Willebrand factor (VWF) interactions due to mutations and polymorphisms in the VWF A1 domain, the region of VWF most critical for interaction with platelet glycoprotein Ib (GPIb). The functional implications of these polymorphisms will be tested in the absence of ristocetin in order to determine their effect on VWF function. In addition, the role of factor VIII (FVIII) release following VWF-platelet binding will be investigated. The objective of this application is to determine the in vivo relevance of A1 domain polymorphisms that lead to decreased VWF activity on functional testing with ristocetin, to ensure that patients are not misdiagnosed with von Willebrand disease (VWD) on the basis of spurious laboratory testing results.
Specific aim 1 will elicit the mechanism(s) behind the decrease in ristocetin-induced VWF-platelet interactions associated with A1 domain mutations through direct binding assays and surface plasmon resonance. Binding strength will be assessed with optical tweezers.
Specific aim 2 will evaluate the effect(s) of key A1 domain mutations on platelet GPIb interactions independent of VWF as well as the mutations'effects on other aspects of VWF function. Both static and flow conditions will be assessed for VWF-collagen and VWF-platelet binding.
Specific aim 3 will determine the impact of factor VIII (FVIII) release in clot formation following VWF-platelet interactions through measurement of FVIII activity and thrombin generation after ligand binding. Clot formation will be examined with in vivo thrombosis models. Bob Montgomery, MD, an experienced researcher in VWF biology, will serve as the primary mentor for this project, along with a committee of research scientists established for the purpose of enhancing the applicant's scientific knowledge and research career growth. This project will utilize the resources of the Medical College of Wisconsin and the Blood Research Institute of the Blood Center of Wisconsin to further the applicant's understanding of coagulation biology and scientific methods for examining coagulation factor function in vitro and in vivo. The applicant will also draw on the resources of a comprehensive clinic for patients with bleeding disorders for better understanding of the clinical behavior of VWF in patients. This environment provides a unique setting for training clinician scientists in the field of coagulation. The ultimate goal of this project is translation and application of these research findings to improve care for patients with VWD and other disorders of coagulation and to further an academic research career for the principal investigator.

Public Health Relevance

Von Willebrand disease (VWD) is a common bleeding disorder, but laboratory testing for this condition is complicated, due in part to use of a non-physiologic agonist, ristocetin, to stimulate von Willebrand factor (VWF)-platelet interactions. This project will explore the effect of polymorphisms in VWF on ristocetin binding and on VWF-platelet interactions independent of ristocetin, with the ultimate goal of improving diagnosis of VWD. Further work will investigate the role of VWF as a carrier protein for factor VIII in clot formation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL102260-03
Application #
8260404
Study Section
Special Emphasis Panel (ZHL1-CSR-U (F1))
Program Officer
Sarkar, Rita
Project Start
2010-07-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
3
Fiscal Year
2012
Total Cost
$135,135
Indirect Cost
$10,010
Name
Medical College of Wisconsin
Department
Pediatrics
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Flood, Veronica H (2014) Perils, problems, and progress in laboratory diagnosis of von Willebrand disease. Semin Thromb Hemost 40:41-8
Flood, Veronica H; Friedman, Kenneth D; Gill, Joan Cox et al. (2013) No increase in bleeding identified in type 1 VWD subjects with D1472H sequence variation. Blood 121:3742-4
Larsen, D M; Haberichter, S L; Gill, J C et al. (2013) Variability in platelet- and collagen-binding defects in type 2M von Willebrand disease. Haemophilia 19:590-4
Flood, Veronica H; Gill, Joan Cox; Friedman, Kenneth D et al. (2013) Collagen binding provides a sensitive screen for variant von Willebrand disease. Clin Chem 59:684-91
Flood, Veronica H; Gill, Joan Cox; Friedman, Kenneth D et al. (2011) Von Willebrand disease in the United States: a perspective from Wisconsin. Semin Thromb Hemost 37:528-34