Comparison of Two von Willebrand Factor Collagen-Binding Assays with Different Binding Affinities for Low, Medium, and High Multimers of von Willebrand Factor

Abstract

The use of von Willebrand factor collagen-binding assay (vWF:CBA) as an alternative method for the quantification of the physiological activity of vWF in plasma from patients with von Willebrand disease (vWD) and in blood clotting factor (F) VIII concentrates (FVIII/vWF concentrates) for the therapy of vWD is currently being discussed. We compared two vWF:CBAs that are distinctive with regard to the type and structure of collagen and the coating principle used. After analyzing samples of a plasma pool from normal donors, we received results that were in very good compliance with both methods. However, significantly different results (p < or =0.005) were obtained when FVIII/vWF concentrates were tested. In an attempt to elucidate these discrepancies, vWF multimers were separated by heparin affinity chromatography and analyzed by vWF antigen enzyme-linked immunosorbent assay (ELISA), ristocetin cofactor activity test, both vWF:CBAs, and a multimer analysis. From our data we conclude that the assay with pepsin-digested collagen (human, type III) that was covalently linked to preactivated microtiter plates (vWF:CBAPDC) revealed a higher affinity for low and medium vWF multimers, whereas the assay with collagen fibrils (equine, type I) that were adsorbed to microtiter plates (vWF:CBACF) predominantly bound high vWF multimers. Based on results reported by others, we assume that the discrepancies between both vWF:CBAs were not related to the type and species of collagen used. Taken together our results imply rather that fragmentation of collagen by pepsin digestion or subsequent covalent linkage to the microtiter plate, or both, increased the affinity for low and medium vWF multimers, whereas the fibrillar structure of collagen was required for the binding of high vWF multimers, which exhibit the highest physiological activity in primary hemostasis.

Classification and Characterization of Hereditary Types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (Unclassifiable) von Willebrand Disease

“…Recent data confirmed this observation that the difference between VWF:CB assay using the predominantly type 1 collagen (actually a mixture of type I/III in a ratio of 95%/5%) or VWF:CB using type III collagen can be linked to different sensitivities to the high VWF multimers in plasma and in factor VIII/VWF concentrates (18,19). This is further supported by the demonstration that the VWF:CB assay using type 1 collagen allowed an overall much better discrimination between type 1 and type 2 VWD than the VWF:CB assay using type III collagen (15,18,20). There is also very good evidence that the VWF:CB assay using equine collagen type 1 or a type I/III (95%/5%) mixture is much more sensitive than the VWF:CB assay using type III collagen (18,20) for the measurement of the hemostatic more potent high VWF multimers present in the FVIII/VWF concentrate Haemate-P (7,19).…”

Acquired Von Willebrand syndrome is an early-onset problem in ventricular assist device patients

“…We found pathologic VWF:RCo/ VWF:Ag ratios in 18 out of 55 cumulative tests with missing HMW multimers of VWF, but pathologic VWF:CB/VWF:Ag values in all of them. This high sensitivity of the collagenbinding capacity test at our institution is probably due to the employment of an ELISA with collagen type I. Collagen type I has been shown to have a higher affinity for HMW multimers of VWF compared with collagen type III [23], and is therefore more discriminative for AVWS. Multimeric analysis can prove the AVWS but it is a time-consuming task and requires an experienced investigator for evaluation of the results.…”

Early Diagnosis of Acquired von Willebrand Syndrome (AVWS) is Elementary for Clinical Practice in Patients Treated with ECMO Therapy

“…In addition, we calculated the ratio of VWF:CB to VWF:Ag (VWF:CB/ VWF:Ag, normal: 0.7). These values reflect the biological activity of the available VWF with regard to binding to collagen 12) . VWF multimers were separated on sodium dodecylsulfate (SDS)-agarose low-resolution gels (1.0% agarose) and blotted on a poly (vinylidene fluoride) (PVDF) membrane to assess the HMW multim-…”