Establishment of immunoassay for platelet-derived soluble glycoprotein VI, a novel platelet marker

• We established four anti-GPVI antibodies and two kinds of ELISA, 7S1/10S2-HRP assay and 19D1/21D1-HRP assay.
• There is a large discrepancy between the monomeric and dimeric GPVI conformations.
• 19D1/21D1-HRP had a high affinity for only dimeric GPVI, while 7S1/10S2-HRP showed high affinity for dimeric and monomeric GPVI. 19D1/21D1-HRP had a high affinity for only dimeric GPVI, while 7S1/10S2-HRP showed high affinity for dimeric and monomeric GPVI.
• The 7S1/10S2-HRP assay could sensitively detect platelet-derived sGPVI, whereas the 19D1/21D1-HRP assay could not.

Soluble Glycoprotein VI (GPVI) is an attractive marker for disorders marked by platelet activation, such as thrombotic microangiopathy, myocardial infarction, and stroke. Several groups have already developed an immunoassay for soluble GPVI; however, there are several discrepancies between the groups' assays. In this study, we prepared the two types of recombinant soluble GPVI, the monomeric form GPVI (GPVI-His) and the dimeric form of GPVI (GPVI-Fc), moreover, we generated four anti-GPVI antibodies, F1232-7-1 (7S1), F1232-10-2 (10S2), F1232-19-1 (19D1), and F1232-21-1 (21D1). The former 2 antibodies (7S1 and 10S2) had a high affinity for both GPVI-His and GPVI-Fc, while the latter 2 antibodies (19D1 and 21D1) showed a high affinity for GPVI-Fc but low affinity for GPVI-His. All of the antibodies comparably recognized surface GPVI on resting platelets. Furthermore, we established two immunoassays for soluble GPVI, 7S1/10S2-HRP and 19D1/21D1-HRP (capture antibody/detection antibody). 7S1/10S2-HRP showed equivalent reactivity with GPVI-His and GPVI-Fc, whereas 19D1/21D1-HRP had high affinity for GPVI-Fc but low reactivity with GPVI-His. In terms of reactivity with platelet-derived soluble GPVI, 7S1/10S2-HRP demonstrated sensitive detection whereas 19D1/21D1-HRP was nonreactive. Taken together, 7S1/10S2-HRP is a suitable candidate for a reliable soluble GPVI immunoassay as it has a high affinity for monomeric GPVI.