Recombinant VWF Promotes Platelet Adhesion Under Shear Stress Dependent on Its Multimer Size

Abstract

Baxter has developed BAX 111, a recombinant von Willebrand factor (rVWF) currently being evaluated for the treatment of von Willebrand disease based on the results of a phase 3 clinical trial. BAX 111 contains ultra-large molecular weight multimers (ULMWM), which are the most hemostatically active type of multimer. ULMWM demonstrate higher functional activity than the multimers present in commercially available plasma-derived VWF (pdVWF) products in terms of VWF:RCo and VWF:CB activity. To investigate the contribution of VWF multimer size on BAX 111 hemostatic activity, fractions containing distinct portions of VWF multimers were generated and analyzed for their ability to mediate platelet adhesion under shear stress in vitro.rVWF fractions of varying VWF multimer size were purified by size-exclusion chromatography; size distribution was determined using low and high resolution agarose gel electrophoresis. A parallel-plate perfusion chamber system was used to investigate the rVWF-mediated adhesion of platelets from human blood to fibrillar collagen type I under shear stress. For this purpose, a VWF sample of 1.0 IU/mL VWF:Ag was added to healthy donor blood and the time course of surface coverage was determined by microscopy using fluorescent labeled platelets. Multiple pictures were taken every 5 seconds. Perfusion was performed for 3 minutes at +37°C and at a defined wall shear rate of 1500 s-1. Different batches of BAX 111, rVWF fractions of distinct multimer size, and three commercially available pdVWF products with low and high FVIII content were investigated.The endogenous VWF present in human whole blood mediated initial platelet adhesion with approximately 5% surface coverage after 120 seconds of perfusion. Additionally spiking human blood with 1.0 IU/mL of BAX 111 or rVWF fractions of different multimeric size resulted in increased, time-dependent platelet binding. The amount of platelets that became attached to collagen was clearly dependent on the size of rVWF. While highly multimerized BAX 111 effectively promoted platelet binding, lower-sized rVWF fractions containing VWF multimers of lower molecular weight showed decreased platelet adhesion properties. pdVWF products lacking ultra-large molecular weight multimers were less effective in mediating platelet adhesion to collagen, as observed by delayed platelet binding kinetics. After 60 seconds of perfusion, approximately twice the surface coverage was measured for BAX 111 compared to pdVWF products. The amount of FVIII contained in the pdVWF products had no influence on platelet adhesion.These results demonstrate that the improved hemostatic activity of BAX 111 over pdVWF is due to the presence of ULMWM and confirms their importance in mediating intact platelet binding. DisclosuresSchrenk:Baxter Innovations GmbH, Vienna, Austria: Employment. Schreiner:Baxter Innovations GmbH, Vienna, Austria: Employment. Gritsch:Baxter Innovations GmbH, Vienna, Austria: Employment. Turecek:Baxter Innovations GmbH, Vienna, Austria: Employment. Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.