Do. Azorsa et al., Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA - Its use to follow platelet storage in transfusion, THROMB HAEM, 81(1), 1999, pp. 131-138
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.