title: Plasma glycocalicin as a source of GPIbalpha in the von Willebrand factor ristocetin cofactor ELISA.
abstract: We have previously demonstrated that the von Willebrand factor ristocetin cofactor activity (VWF:RCo), used in the diagnosis of vonWillebrand disease (VWD), can be accurately determined via ELISA by measuring the ristocetin-induced binding of VWF to a captured recombinant fragment of GPIbalpha (rfGPIbalpha, AA 1-289) (Vanhoorelbeke et al., Thromb Haemost 2000; 83: 107-13). This ELISA is more reliable than the currently used platelet agglutination test. Normal plasma contains relatively high concentrations of glycocalicin, a proteolytic fragment of GPIbalpha. We therefore studied whether non-purified plasma glycocalicin can replace rfGPIbalpha in our ELISA. Of 42 anti-GPIbalpha monoclonal antibodies (MAbs) capable of binding plasma glycocalicin, only one MAb captured glycocalicin in a spatial orientation exposing the VWF-binding site in glycocalicin, allowing a specific and dose-dependent ristocetin-mediated VWF-binding. Intra- and interassay variability were comparable with those for the rfGPIbalpha based VWF:RCo ELISA. The VWF:RCo activity of plasma from 33 normal individuals, 19 type 1, 16 type 2A, 9 type 2B, 8 type 2M and 7 type 3VWD patients was determined with this ELISA and allowed a clear identification of VWD patients. Furthermore, determination of the VWF:RCo/VWF:Ag ratio resulted in the discrimination between type 1 and type 2 VWD patients. Results for the glycocalicin based and the rfGPIbalpha based VWF:RCo ELISAs were in good agreement (r=0.943). There was also a good correlation between the glycocalicin based ELISA and the standard platelet agglutination test (r=0.963). In conclusion, to diagnose VWD, a VWF:RCo ELISA based on antibody immobilized plasma glycocalicin can be performed reliably.
authors.affiliation: Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, E. Sabbelaan 53, 8500 Kortrijk, Belgium. Karen.Vanhoorelbeke@kulak.ac.be
authors.forename: Karen
authors.initials: K
authors.lastname: Vanhoorelbeke
authors.forename: Inge
authors.initials: I
authors.lastname: Pareyn
authors.forename: Agota
authors.initials: A
authors.lastname: Schlammadinger
authors.forename: Stephan
authors.initials: S
authors.lastname: Vauterin
authors.forename: Marc F
authors.initials: MF
authors.lastname: Hoylaerts
authors.forename: Jef
authors.initials: J
authors.lastname: Arnout
authors.forename: Hans
authors.initials: H
authors.lastname: Deckmyn
mesh_terms: D003937:Diagnosis, Differential; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006801:Humans; D019038:Platelet Glycoprotein GPIb-IX Complex; D011485:Protein Binding; D015203:Reproducibility of Results; D012310:Ristocetin; D012680:Sensitivity and Specificity; D014842:von Willebrand Diseases / Q000175:diagnosis*; D014841:von Willebrand Factor / Q000032:analysis*
chemical_list: D019038:Platelet Glycoprotein GPIb-IX Complex; C023592:glycocalicin; D014841:von Willebrand Factor; D012310:Ristocetin