Plasma glycocalicin as a source of GPIbα in the von Willebrand factor ristocetin cofactor ELISA

Summary

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 ofVWF to a captured recombinant fragment of GPIb α (rfGPIb α ,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 GPIb α . We therefore studied whether non-purified plasma glycocalicin can replace rfGPIbα in our ELISA. Of 42 anti-GPIbα monoclonal antibodies (MAbs) capable of binding plasma glycocalicin, only one MAb captured glycocalicin in a spatial orientation exposing theVWF-binding site in glycocalicin,allowing a specific and dosedependent ristocetin-mediated VWF-binding. Intra- and interassay variability were comparable with those for the rfGPIbα basedVWF:RCo ELISA.TheVWF: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 ofVWD 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 rfGPIb α basedVWF: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.

Keywords

Plasma glycocalicin - von Willebrand disease (VWD) - von Willebrand ristocetin cofactor activity (VWF:RCo) assay

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