VWF Binding to Types I, III or VI Collagen In the ZPMCB-VWD with Identification of Subjects with Selective Reduced Plasma VWF:CB

Abstract 235

Von Willebrand Disease (VWD) is a common bleeding disorder caused by quantitative (types 1 and 3) and qualitative (type 2) abnormalities in von Willebrand factor (VWF). Defective VWF binding to collagen (VWF:CB) has been identified in VWD patients, but the type(s) and amount of collagen vary between recommended assays. We measured VWF:CB using separate assays for types I, III, and VI collagen in plasmas from 233 healthy controls and 315 VWD index cases (261 type 1, 37 type 2 and 17 type 3) recruited into the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD). Additional studies included VWF antigen (Ag), VWF ristocetin-cofactor (RCo), VWF multimer, and VWF gene sequence analysis. VWF:CB was tested using ELISA assays with type I human placental collagen (5 ug/ml), type III human placental collagen (1 ug/ml), and by a commercial human placental type VI VWF:CB assay (Technoclone, Austria). Normal ranges were established for VWF:CB and VWF:CB/Ag ratios with the ZPMCB-VWD healthy control population. Three subjects (2 healthy controls and 1 type 1 VWD subject) were identified with reduced VWF:CB and reduced VWF:CB/Ag ratios to type VI collagen, despite normal results with type I and type III collagen. The two control subjects had VWF:CB/Ag ratios of 0.43 and 0.51 for type VI collagen. The patient with type 1 VWD had absent type VI VWF:CB and a VWF:Ag of 54 IU/dL with a history of clinical epistaxis and an EU Bleeding Score of 8 (normal less than or equal to 3). VWF sequencing demonstrated that these three individuals had an A1 loop polymorphism, R1399H, which has been previously reported as a VWF polymorphism. No other individuals in our study had this sequence change. Of particular note was that the VWF:CB/Ag ratios for types I and III collagen were each normal – suggesting a selective abnormality. As anticipated, collagen binding with all collagens was undetectable in the type 3 VWD subjects. Type 2A and 2B VWD subjects demonstrated reduced VWF:CB and VWF:CB/Ag ratios to all three types of collagen. Type 2M and 2N VWD subjects exhibited normal VWF:CB/Ag ratios to all three types of collagen, but one type 2M patient with an I1425F A1 loop mutation had a reduced VWF:CB/Ag ratio of 0.58. In the VWF A1 domain crystal structure, amino acid 1425 is in close proximity to amino acid 1399, suggesting the conformation of that region may be critical to type VI collagen binding to VWF. Since the frequency of the R1399H polymorphism is estimated to be 2% of the population (Sadler and Ginsburg, 1993), defective binding to type VI collagen may be an important contributor to the variability in the bleeding phenotype of VWD.