[21] Secretion of von Willebrand factor from platelets

This chapter presents procedures for the isolation and analysis of platelet von Willebrand factor (vWF) and its release from the secretory pool. Investigations of the platelet release reaction and other platelet functions and parameters require a system from which plasma components have been removed. There are different methods to wash platelets free of the plasma environment. Two washing procedures are presented in the chapter. The first is designed for the study of the structure of vWF in platelet lysates and includes the centrifugation and resuspension of the cells in buffers containing inhibitors to prevent platelet aggregation and the proteolysis of the platelet vWF. The other procedure is used for release experiments and involves platelet separation by washing and gel filtration in Sepharose 2B. This system provides functionally intact platelets and is quick, simple, and reproducible. Less than 0.5% total starting vWF is present in the platelet-free supernatant, thereby indicating little carryover of soluble plasma vWF. Radioquantitative immunoelectrophoresis is used for quantifying vWF.