Comparison of two von Willebrand factor collagen-binding assays with different binding affinities for low, medium, and high multimers of von Willebrand factor.
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.
Neugebauer
Brigitte M
BM
Paul-Ehrlich-Institut, Langen, Germany. neubr@pei.de
Goy
Christine
C
Budek
Irmhild
I
Seitz
Rainer
R
D000818:Animals; D002846:Chromatography, Affinity / Q000592:standards; D003094:Collagen / Q000378:metabolism*; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005169:Factor VIII / Q000032:analysis; D006493:Heparin / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D015336:Molecular Probe Techniques / Q000592:standards*; D010446:Peptide Fragments / Q000378:metabolism; D010949:Plasma / Q000737:chemistry; D011485:Protein Binding; D012310:Ristocetin; D014841:von Willebrand Factor / Q000032:analysis* / Q000737:chemistry
D010446:Peptide Fragments; D014841:von Willebrand Factor; D012310:Ristocetin; D005169:Factor VIII; D006493:Heparin; D003094:Collagen