Influence of high molecular weight factor VIII on the measurement of low molecular weight factor VIII procoagulant in different assay systems

Abstract

Factor VIII procoagulant activity (VIIIC) is exerted by a low molecular weight (LMW) moiety of the factor VIII molecule that can be separated from a high molecular weight (HMW) moiety by high ionic strength buffers. In this investigation the procoagulant activity of the LWM moiety of factor VIII prepared by immunoadsorbent chromatography and its relationship to the HMW moiety of haemophilic plasma was studied by means of different VIIIC assay systems and using different substrates in regard to their content of the HMW VIII moiety. LMW VIIIC was prepared by immunoadsorbent chromatography; HMW VIII without VIIIC was prepared by chromatographing cryoprecipitate from a coagulant antigen negative severe haemophiliac on 4% agarose. The LMW VIIIC obtained by immunoadsorbent c,hromatography gave higher VIIIC values when tested in the one-stage partial thromboplastin time (PTT) system using von WiIlebrand's disease plasma as substrate than using haemophilic plasma as substrate. This finding was shown to be related to the HMW VIII measured as VIII related antigen (VIII: Ag) in the substrate plasmas. When the VIIIR : Ag was removed from the haemophilic substrate plasma by immunoadsorption, the VIIIC values obtained for the LMW VIIIC were higher. Also, adding HMW VIII purified from haemophilic plasma to the von Willebrand's disease substrate plasma resulted in lower VIIIC values for the LMW VIIIC in the PTT system.When the LMW VIIIC was tested in the two stage assay, all VIIIC was adsorbed to A1(OH)3. When unadsorbed LMW VIIIC was assayed by the two-stage method, there

Factor VIII inhibitors: von Willebrand factor makes a difference in vitro and in vivo

“…Previous studies have shown that VWF in the FVIII‐deficient plasmas utilized in one‐stage aPTT assays appears to depress measurements of apparent FVIII activity [23–25]. We wanted to determine whether similar effects were present when inhibitor titers were calculated using a chromogenic assay to measure residual FVIII activity.…”

Assay Discrepancies with Highly Purified Factor VIII Concentrates

“…One specific protein which may have an influence on factor VIII:C assays is vWF, as indicated by Muntean et al [13]. vWF is absent from several immunodepleted plas mas and present at a much higher level in the standards (3-4 times the factor VIII:C level) than in the highly purified products (approx.…”

The “normal” factor VIII concentration in plasma

“…The in vivo half life of FVIII is dependent upon the presence and concentration of VWF and is as short as 1 hour in the absence of this carrier protein [14, 15]. However, in the APTT clotting assay, VWF acts as an inhibitor of FVIIIc and becomes a confounder in this FVIII activity-based assay [4, 16]. …”