Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA - Its use to follow platelet storage in transfusion

Thrombin. the most potent platelet agonist, plays a central role in haemost asis and in the occurrence of thrombotic events. This agonist activates pla telets by cleaving the PAR G-protein coupled receptors and by binding to gl ycoprotein (GP) Ib and also cleaves GPV at the platelet surface to liberate the soluble 69 kDa fragment GPVf1. Monoclonal antibodies (MoAbs) to GPV we re developed as tools to study the mechanism of platelet GPV cleavage and m easure release of GPV in pathological situations. Specificity of the MoAbs for GPV was confirmed by flow cytometry and immunoprecipitation of proteins from human platelets and Dami megakaryocytic cells. A sensitive immunocapt ure sandwich ELISA for soluble GPV was developed using two MoAbs recognizin g different epitopes of GPV and purified platelet or recombinant GPV as ref erence protein. This ELISA was employed to determine the mean plasma concen tration of GPV in 100 normal individuals (17.3 ng/ml), to demonstrate the d ose-dependent release of GPVf1 from washed platelets stimulated with thromb in and to follow the progressive release of GPVf1 during storage of therape utic platelet concentrates. The present report describes a sensitive GPV EL ISA of direct application to survey the processing and storage of platelet concentrates for transfusion and of potential value to monitor platelet act ivation in thrombotic states.