1. Development of a Collagen Binding Assay for von Willebrand factor (VWF:CBA) von Willebrand disease (VWD) is the most common inherited bleeding disorder. Individuals with VWD have defects in, or reduced levels of von Willebrand Factor (VWF), an adhesive plasma protein with two primary hemostatic roles (permits adhesion of platelets to sites of vascular damage and acts to stabilize Factor VIII). VWD is a heterogeneous disorder, and patients are subtyped according to pathophysiology, and using different clinical and laboratory criteria. Discrimination of Type 1 and 2 VWD is important because patients may then be differentially managed with regard to their therapy. VWF:CBA is reported to be effective in discriminating Type 2 VWD because of its ability to preferentially bind high molecular weight VWF, which is absent or deficient in Type 2A and 2B VWD. VWF:CBA is then a pertinent test to assess the quality of various preparations of VWF because of this characteristics. We have so far tested one protocol of VWF:CBA using human placenta-derived, pepsin-digested type III collagen covalently immobilized on microtiter plates as the capture for VWF. The bound VWF is then detected with a peroxidase conjugated rabbit anti-human VWF antibody. Initial results indicate that there is dose dependence between signal and VWF but the conditions of the reaction need to be optimized. This project is done with the technical assistance of Donald Lebel. 2. Development of an International Standard for Thrombin with a Unified Unit. Dr. Andrew Chang initiated this project in collaboration with WHO and NIBSC. The purposes of which are (a) replenishing the much depleted inventory of Lot J, the current national thrombin standard; and (b) unifying the unit for thrombin to eliminate the inconvenience of having to convert between the U.S. unit and the international unit. Two suppliers have expressed interest to donate sufficient thrombin concentrate to be evaluated for a new international standard. Eleanor Koo and I will perform physical and biochemical characterization on these two candidate thrombin concentrates with reference to Lot J and the International standard for thrombin (human) (89/588). The tests include thrombin clotting activity, active site titration, SDS-PAGE analysis, thrombin antigen determination for specific activity, and storage stability at different temperatures. 3. Prospective assays for Fibrinogen. We plan to explore additional methods to correlate the percentage of clottable protein in fibrinogen preparations with the quality of the resultant clot. We will also need an assay to establish the correct assembly of fibrinogen hexamer, especially for recombinant products where the three chains are expressed separately. In addition, the amount of plasminogen in Fibrinogen products may be relevant to safety issues since a report suggests preferential binding of the disease-associated prion protein (PrPSC) to plasminogen (Fischer, M.B. et al. (2000) Nature 408, 479-483).
