BACKGROUND: Platelet glycoprotein VI (pGPVI) expression is increased in acute coronary syndrome (ACS), reflecting platelet activation. There is no reliable method available to measure pGPVI. Our aim was to develop a bead-based sandwich immunoassay to measure soluble GPVI (sGPVI). METHODS: Based on antibodies for sGPVI developed earlier, we established and validated a bead-based sandwich immunoassay in 2438 consecutive patients with stable angina pectoris (SAP; n = 1371), non-ST-elevation myocardial infarction (NSTEMI; n = 724), and ST-elevation MI (STEMI; n = 343). In a subgroup (n = 1011), we measured surface expression of pGPVI using flow cytometry. RESULTS: The assay revealed a working range of 8-500 ng/L. Intra- and interassay imprecision was <7% and <14%, respectively. Patients with NSTEMI and STEMI showed significantly lower mean sGPVI concentrations than patients with SAP [mean (SD), 8.4 (3.6) mug/L and 8.6 (4.1) mug/L vs 9.8 (4.8) mug/L; P = 0.002], whereas subgroup analysis revealed significantly enhanced pGPVI in NSTEMI (n = 276) and STEMI (n = 80) patients compared with SAP (n = 655) [mean fluorescence intensity (SD), 21.2 (8.1) and 19.8 (6.8) vs 18.5 (7.7); P = 0.002 and P = 0.018]. pGPVI and sGPVI were inversely correlated (r = -0.076; P = 0.023). Area under the ROC curve was 0.716, 95% CI 0.681-0.751, for sGPVI, distinguishing patients with SAP from those with ACS, and was superior (P = 0.044) to the curve of subgroup analysis for pGPVI (0.624, 95% CI 0.586-0.662). sGPVI (P = 0.023) and pGPVI (P = 0.028) had better association with the development of ACS than troponin I (P = 0.055) in the very early stage of disease, based on logistic regression analysis. CONCLUSIONS: This sandwich immunoassay reliably measures sGPVI and may help to identify patients with ACS earlier than other laboratory markers.