S Butenas1, B Parhami-Seren, K G Mann. 1. Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT 05446, USA. sbutenas@uvm.edu
Abstract
BACKGROUND: It has been reported by multiple laboratories that the quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure and the quality of reagents used in the assays. OBJECTIVE: To evaluate the influence of von Willebrand factor (VWF) on FVIII activity in vitro. METHODS: The activated partial thromboplastin time (APTT) and synthetic coagulation proteome assays were used. Citrated FVIII/VWF-depleted substrate plasma (SP) (both antigens < 0.5%) was used in all APTT assays. RESULTS: The concentration of FVIII antigen in pooled plasma from healthy donors [normal plasma (NP)] was 1.5 nm. The SP reconstituted with 1.5 nm recombinant (r)FVIII clotted in 23.8 +/- 0.2 s (standard deviation). The addition of 10 microg mL(-1) VWF to the SP increased the clotting time to 28.7 +/- 0.1 s; that is, the activity of rFVIII (FVIIIc) decreased to 50%. This inhibitory effect of VWF decreased with decreasing rFVIII concentration in SP, and became negligible at rFVIII <or= 10% (150 pm). The FVIIIc of 1.5 nm FVIII in two products formulated with VWF (Immunate and Hemofil M) was not affected by the addition of exogenous VWF to the SP, whereas the FVIIIc of the B-domainless rFVIII product ReFacto was decreased to 50% by the addition of VWF. In the synthetic coagulation proteome triggered with 5 pm tissue factor, VWF had no effect on thrombin generation. CONCLUSIONS: VWF has an inhibitory effect on the measurement of FVIII clotting activity. This effect depends upon the structure and formulation of the FVIII product.
BACKGROUND: It has been reported by multiple laboratories that the quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure and the quality of reagents used in the assays. OBJECTIVE: To evaluate the influence of von Willebrand factor (VWF) on FVIII activity in vitro. METHODS: The activated partial thromboplastin time (APTT) and synthetic coagulation proteome assays were used. Citrated FVIII/VWF-depleted substrate plasma (SP) (both antigens < 0.5%) was used in all APTT assays. RESULTS: The concentration of FVIII antigen in pooled plasma from healthy donors [normal plasma (NP)] was 1.5 nm. The SP reconstituted with 1.5 nm recombinant (r)FVIII clotted in 23.8 +/- 0.2 s (standard deviation). The addition of 10 microg mL(-1) VWF to the SP increased the clotting time to 28.7 +/- 0.1 s; that is, the activity of rFVIII (FVIIIc) decreased to 50%. This inhibitory effect of VWF decreased with decreasing rFVIII concentration in SP, and became negligible at rFVIII <or= 10% (150 pm). The FVIIIc of 1.5 nm FVIII in two products formulated with VWF (Immunate and Hemofil M) was not affected by the addition of exogenous VWF to the SP, whereas the FVIIIc of the B-domainless rFVIII product ReFacto was decreased to 50% by the addition of VWF. In the synthetic coagulation proteome triggered with 5 pm tissue factor, VWF had no effect on thrombin generation. CONCLUSIONS:VWF has an inhibitory effect on the measurement of FVIII clotting activity. This effect depends upon the structure and formulation of the FVIII product.
Authors: Q Shi; E L Kuether; J A Schroeder; C L Perry; S A Fahs; J Cox Gill; R R Montgomery Journal: J Thromb Haemost Date: 2012-11 Impact factor: 5.824
Authors: Danielle N Drury-Stewart; Kerry W Lannert; Dominic W Chung; Gayle T Teramura; James C Zimring; Barbara A Konkle; Hilary S Gammill; Jill M Johnsen Journal: PLoS One Date: 2014-11-19 Impact factor: 3.240