activator (tPA) is a serine protease that has a crucial function in the fibrinolytic process. It catalyses the conversion of the zymogen d(Glu-) plasminogen to the active serine protease plasmin which in turn is responsible for the degradation of fibrin in the thrombus. tPA is synthesized and secreted by vascular endothelial cells. Fibrinolytic activity in blood follows a circadian rhythm with the lowest level of tPA activity in the morning and peak level in the evening. Therefore, it is recommended that blood be collected between 0800 and 0900 to determine the minimal level of tPA activity. Acceptable collection time is 0700-1000. Please record the collection time on the requisition form. Samples for tPA activity must be drawn into a special acid citrate Stabilyte tube. tPA ANTIGEN (Tissue Plasminogen Activator Antigen) Method: Enzyme immunoassay This assay measures total tPA in plasma, including active tPA and tPA/PAI-1 complex. Since PAI-1 activity is usually in excess compared to tPA activity, and the reagent between the two continues after the sample is obtained (in citrate plasma), most of the tPA in this assay is in the form of tPA/PAI-1 complex. Elevated tPA antigen typically is associated with elevated PAI-1 activity and indicates a reduction in fibrinolytic activity. tPA antigen and activity may be elevated after endogenous release or exogenous infusion of tPA. Reduced tPA antigen is usually associated with PAI-1 deficiency. tPA antigen levels increase with age, exercise, and stress. Venous stasis will result in increased tPA levels. 34 VON WILLEBRAND FACTOR ACTIVITY Method: Enzyme immunoassay Antibody directed towards to GPIb complex binding site on von Willebrand factor. Levels parallel total von Willebrand factor antigen levels in normal patients and type 1 von Willebrand disease. Levels are lower that total von Willebrand factor levels in patients with type 2 von Willebrand disease. VON WILLEBRAND FACTOR ANTIGEN Method: Latex immunoassay Decreased von Willebrand factor antigen is associated with von Willebrand disease. See page 9 for a detailed description of von Willebrand disease and its subtypes. VON WILLEBRAND FACTOR MULTIMER ANALYSIS Method: SDS gel electrophoresis Multimeric sizing of the von Willebrand factor protein is used to characterize von Willebrand disease (vWD). Classification of von Willebrand disease is dependent upon a quantitative and/or qualitative defect of the von Willebrand factor protein. In type 1 all the multimers are present but will be decreased in quantity. Type 2 patients lack the high molecular weight multimers and in some situations lack the intermediate weight multimers. Type 3 patients will not have any visible multimers due to the extremely low level or unmeasurable antigen. A low-resolution gel (1% agarose/SDS) permits the high molecular weight, intermediate weight, and low molecular weight multimers to enter the gel and be separated. Once the separation is completed the multimers are transferred to a polyvinyl fluoride membrane using a semi-dry electroblot transfer system. Immunolocalization of the multimeric bands is visualized by application of alkaline phosphatase conjugated antibodies. 35 RESEARCH ASSAYS Listed below are assays offered through our Research and Development division for investigational purposes only. The availability of these assays is dependent upon the number of tests, frequency of testing, and the cost of reagents. In addition to performing these assays, the laboratory offers consultation for research protocol design and data interpretation. If you are interested in any of these assays, please contact Dr. Chandler or Dr. La Spada at (206) 598-6131 for consultation. If you are interested in an assay not listed below, please call Dr. Chandler or Dr. La Spada to determine whether it could be run in our laboratory. This list will change as new assays are being evaluated. In the future some of these assays may be offered as clinical diagnostic assays. Beta thromboglobulin antigen C4b-binding protein antigen CD 40 Ligand (soluble) Chromogenic factor II E-selectin antigen Endothelial Protein C Receptor Factor VII antigen Factor X antigen Fibrinopeptide A High Molecular Weight Kininogen factor activity