Development of a novel flow cytometric immunobead array to quantify VWF: Ag and VWF: GPIbR and its application in acute myocardial infarction.

Abstract

Both von Willebrand disease (VWD) and acute myocardial infarction (AMI) involve quantitative and qualitative changes in von Willebrand factor (VWF). Our objective was to develop a rapid and precise flow cytometric immunobead array (FCIA) to quantify VWF antigen (VWF:Ag) and ristocetin-triggered platelet glycoprotein Ib binding (VWF:GPIbR) and apply it in a clinical setting. Microbeads, coated with monoclonal antibodies for SZ29 or SZ151 IgG, were incubated with diluted plasma. VWF-binding microbeads were detected with FITC-conjugated sheep-anti-human VWF IgG by flow cytometry. Plasma VWF:Ag and VWF:GPIbR levels in normal controls (CTL; n=105), patients with VWD (n=21), and patients with AMI (n=146) were tested by FCIA and ELISA in parallel. ADAMTS13 activity and VWF multimer analyses were also implemented. Our novel FCIA showed a strong correlation with the ELISA results (VWF:Ag, r=.855; VWF:GPIbR, r=.813). The intra-assay coefficient variations (CVs) of VWF:Ag-FCIA and VWF:GPIbR-FCIA were 9.2% and 7.7%, respectively, and the interassay CVs were 12.6% and 13.5%, respectively. Plasma VWF:Ag and VWF:GPIbR levels were significantly higher in patients with AMI than in CTL (P<.0001), whereas the ratios of ADAMTS13/VWF:Ag and ADAMTS13/VWF:GPIbR were significantly lower (P<.0001). Levels of plasma ultra-large VWF (UL-VWF) were dramatically increased in patients with AMI. The novel VWF:Ag and VWF:GPIbR-FCIA assays were found to be simpler, more specific, and more accurate than the classical ELISA method. In addition, elevated VWF:GPIbR and UL-VWF may contribute to the pathogenesis of AMI.