von Willebrand factor antigen latex immunoassays are affected to a different extent by rheumatoid factor

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

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.status: publishe

The A/T1381 polymorphism in the A1-domain of von Willebrand factor influences the affinity of von Willebrand factor for platelet glycoprotein Ibalpha

Many polymorphisms in vonWillebrand factor (VWF) have been reported and their association with VWF plasma levels or cardiovascular diseases has been investigated. The aim of this study was to examine whether the amino acid polymorphism A/T1381 in the VWF A1-domain would affect VWF binding to platelet GPIbalpha. Sixty-one normal individuals were genotyped at the A/T1381 locus. Twenty-one A/A1381 homozygotes, 30 A/T1381 heterozygotes and 10 T/T1381 homozygotes were identified. Remarkably, when compared to VWF of A/T1381 and A/A1381 individuals, VWF of individuals carrying the T/T1381 variant showed an increased affinity for its platelet receptor GPIbalpha under static conditions, as reflected by an increased sensitivity to low concentrations of ristocetin or botrocetin. In addition, also the rVWF-T1381 demonstrated a higher affinity for GPIbalpha than rVWF-A1381. Interestingly, this enhanced affinity of the T/T variant over the A/T and A/A variant was, however, too subtle to affect platelet adhesion under physiological flow conditions, which fully corroborates the normal haemostatic phenotype of all individuals. We demonstrate that the VWF A/T1381 polymorphism plays an important role in inter-individual variability of the affinity of VWF for GPIbalpha, with T/T variants having a higher affinity than A/A and A/T variants, at least under static conditions in vitro. Further genetic linkage and association studies are necessary to establish whether the A/T1381 polymorphism could correlate with an increased risk of thrombotic events.status: publishe