Effects of thrombin treatment of preparations of factor VIII and the Ca2+-dissociated small active fragment.

Abstract

A B S T R A C T When human, canine, or bovine factor VIII preparations are chromatographed on 4% agarose at ionic strength 0.2, the factor VIII activity elutes as a single peak in the void volume with slight tailing. Incubation of such preparations with dilute (0.01 U/ml) highly purified thrombin results in some activation of factor VIII. Chromatography of such incubation mixtures, under the same conditions as before, results in elution of two peaks of factor VIII activity, one in the void volume and one much later with marked tailing. The void volume peak has most of the protein and some factor VIII activity. These void volume fractions also contain all the von Willebrand factor activity of thrombin-treated human factor VIII preparations and all of the platelet aggregating factor activity of thrombin-treated bovine preparations. Longer treatment with thrombin, or treatment with stronger thrombin, appears to shift much more of the procoagulant activity to the later eluting peak. Also, when the peak of factor VIII activity, found in the void volume after thrombin treatment, was again incubated with dilute thrombin, an increase in factor VIII activity occurred. Chromatography of this incubation mixture demonstrated only a small amount of activity in the void volume, while the bulk of the activity was present in the second peak. On the other hand, thrombin treatment of factor VIII activity from peak 2 caused a rapid decline of activity instead of a further increase. It is proposed that the residual factor VIII activity found in the void volume represents unreacted factor

Factor VIII-East Hartford (arginine 1689 to cysteine) has procoagulant activity when separated from von Willebrand factor.

“…Concentrations of penicillamine and DTT that maximally enhance VIII:C activity (80 and 1 mM, respectively) had no effect on FVIII-EH light chain cleavage (lanes 3 and 4). As expected, Factor VIII heavy chain cleavage was 120- (29). An anti-Factor VIII monoclonal antibody (ESH-8) was used to identify Factor VIII in these experiments.…”

The Properties of Immune Complexes Formed by Human Antibodies to Factor VIII

“…Among its properties, this protease inhibitor prevents thrombin activation of Factor VIII (17), a process which has been shown to affect the gel filtration pattern of Factor VIII procoagulant activity (18). W. C. antibody (126 U) in 0.5 ml BBS was brought to 0.002 M DFP before the addition of cryoprecipitate prepared from 30 ml of normal plasma (the 5-ml vol was also brought to 0.002 M DFP).…”

“…Dialysis of Factor VIII in 1.0 M NaCl or 0.25 M CaCl2, followed by gel filtration on agarose or by sucrose density gradient centrifugation, has resulted in the separation of two components: a high molecular weight material which retains Factor VIII-related antigenic properties and ristocetin cofactor activity but lacks procoagulant activity; and a low molecular weight material which has Factor VIII procoagulant but lacks ristocetin cofactor activity and does not form immunoprecipitates with rabbit anti-Factor VIII. The identification of the low molecular weight procoagulant activity as Factor VIII has been confirmed by the inhibition of its coagulant activity by human antiFactor VIII (23) and by the ability of thrombin to activate this material (18,25). Because of its very low protein content, this low molecular weight Factor VIII coagulant activity has not yet been characterized chemically.…”

Factor VIII Activity in Chronic Renal Disease

“…The supernatant was stored at -90 C prior to analysis. VIII : C was measured by a one-stage clotting method [3], VIII R:AG by Laurell electroimmunoassay [23] and VI11 : Rcof by ristocetin-induced platelet aggregation [8]. The activity of a standard plasma pool from healthy blood donors corresponded to 100%.…”