Establishment of a flow cytometric assay for determination of human platelet glycoprotein VI based on a mouse polyclonal antibody.

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. Recent studies suggest that the surface density of GPVI is related to the activation of platelets by collagen. To measure the level of GPVI on platelets, a mouse polyclonal antibody BJ010 was prepared using an amplified fragment of extracellular domain in GPVI. The specific reactivity of BJ010 was identified by anti-GPVI specific monoclonal antibody 11a12 using immunoprecipitation, sandwich enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The antibody BJ010 recognized both native and denatured human GPVI so that it was used to set up a flow cytometric assay to detect the level of GPVI in normal subjects. The relative level of GPVI on platelets was detected in 101 healthydonors. The median geometric mean fluorescence intensity (GMFI) of platelet GPVI level was 57.7. The variation between minimum and maximum values of platelet GPVI and integrin alpha2beta1 were found to be 3.5- and 4.1-fold, respectively, in the normal subjects. There was a week correlation between the amount of GPVI and integrin alpha2beta1 on platelet surfaces. For the method, the intraassay and interassay coefficient of variation was 6.3% and 8.8%, respectively. The flow cytometric assay described here provides a simple, reliable, reproducible, and readily available means of quantitation of collagen receptor GPVI density on the platelet surface in a larger number of blood samples. (c) 2006 Wiley-Liss, Inc.