Several distinct populations of factor VIII procoagulant activity (VIIIC) were observed in some clinical concentrates after agarose gel electrophoresis. It is not yet known whether these different forms are the products of proteolytic digestion, or subtle conformational changes of FVIII structure, which may occur during concentrate preparation. Low thrombin concentrations activate FVIII in a time/concentration dependent manner, and the 2 proteins have high affinities for one another. We have studied the properties of the various FVIII forms before and after incubation of concentrate with highly purified α-thrombin. Agarose gel electrophoresis was used to monitor: a) quantitative changes in functional activity (by 2 stage clotting (VIII:C) and amidolytic (VIII: CAm) assays); b) the binding of 125I- anti VIII:C (*IgG) to FVIII: C; c) the crossed immunoelec- trophoretic (CIE) pattern of FVIII: RAg.In the absence of thrombin, FVIII concentrate showed 2 main populations of FVIII: C and VIII: CAm. The radioactivity (VIII: CAg) pattern correlated with the slower moving of these populations. Low thrombin concentrations (0.0001-0.01 u/nl) caused no detectable change in FVIII: C level in the slow peak but additional peaks of activity appeared at the sample well, and between the well and slow peak. This was accompanied by a decrease in the fast moving peak, and the appearance of a third new peak with slightly less mobility. Higher thrombin concentrations (0.5u/ml) caused a marked decrease in the slow moving peak, with a concomitant increase in the well peak, by all methods. Increasing thrombin concentration progressively reduced *IgG binding, and a pattern similar to serum was obtained. Thrombin caused a slightly faster migration of FVIII: RAg, a well peak was sometimes seen. Thrombin therefore appeared to act preferentially on the lower molecular weight FVIII activity, and the high molecular weight peaks that occurred may represent complexes of FVIII with thrombin and/or other contaminant proteins present in concentrate such as fibrinogen or fibronectin.
Studies of Transferrin Polymorphism in Swedish Cattle Using Agarose Gel Electrophoresis