Beat A. Perret

Studies on factor VIII-related protein: II. Estimation of molecular size differences between factor VIII oligomers

Human factor VIII-related protein was isolated from cryoprecipitate by agarose (Sepharose CL-2B) gel filtration. Electrophoresis on SDS-2% polyacrylamide-0.5% agarose gels revealed size heterogeneity of factor VIII-related protein which was similar to that shown by SDS-1% agarose gel electrophoresis and electron microscopy. The apparent molecular weights were compared with those of crosslinked IgM oligomers and corresponded to values of up to 20 . 10(6) for factor VIII eluting close to the void volume of our gel filtration column. Measurement of mobility intervals on electrophoretic gels suggested a constant size difference between adjacent bands. Smaller aggregates were found in later eluates from Sepharose columns as well as following partial reduction of factor VIII with cysteine. In order to compare the size difference between small and large aggregates of factor VIII-related protein we calibrated the SDS-2% polyacrylamide-0.5% agarose gels with factor VIII which had been crosslinked with dimethyl suberimidate and subsequently disulfied-reduced with 2-metcaptoethanol. By combination of calibration ranges, constant intervals were measured for large and smaller factor VIII aggregates. The interval between any neighboring protein bands, which were immunologically identified as factor VIII-related protein, was equal to the dimer of the basic factor VIII subunit chain. We conclude that factor VIII aggregates correspond to multimers of a dimeric molecule, i.e. pairs of the basic subunit chain.

Studies on factor VIII-related protein: III. Size distribution and carbohydrate content of human and bovine factor VIII

Human and bovine factor VIII were isolated from cryoprecipitate of fresh frozen plasma by gel filtration on Sepharose CL-2B. The elution diagrams and SDS-agarose electrophoretic analysis of eluted fractions show no significant differences in the size-distribution of factor VIII aggregates between the two species. Agarose gels were stained for carbohydrate by two methods: (1) the dansyl hydrazine reaction following oxidation with periodic acid and (2) staining with fluorescein-labeled concanavalin A. Results of both procedures indicate that in human factor VIII neither the size distribution nor its ristocetin cofactor activity are related to carbohydrate content. Bovine factor VIII contains slightly less sugar than the human preparation as judged from the relative dansyl hydrazine staining intensities. In contrast to human factor VIII, the binding affinity for concanavalin A of bovine factor VIII was gradually decreased with increasing aggregate size. This finding suggests an impaired accessibility of reactive sugar residues in large aggregates of bovine factor VIII.

Studies on factor VIII-related protein: I. Ultrastructural and electrophoretic heterogeneity of human factor VIII-related protein

Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein.

Binding of Colloidal Gold Granules, Coated with Bovine Factor VIII, to Human Platelet Membranes

Summary. Platelet aggregating factor (PAF) activity is preferentially associated with the larger molecular forms of bovine factor VIII and diminishes considerably after dissociation into smaller oligomers by mild disulphide reduction. PAF activity was regained by the binding of small factor VIII oligomers to colloidal gold granules with a mean diameter of 23 nm. In contrast, adsorption of large factor VIII polymers onto gold particles resulted in partial loss of PAF activity. Thus, an optimum multimeric size of bovine factor VIII appears to be required for the maximal expression of PAF activity. Gold granules, coated with reduced factor VIII, can be used as an electron‐dense label. Their interaction with surface receptors for bovine factor VIII on either viable or formalin‐fixed human platelets was demonstrated by transmission and scanning electron microscopy.

Fractionation of individual, biologically active factor VIII multimers

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.

A thiocholine ester of cinnamic acid inhibits crosslinking of fibrin without specific binding to donor lysines.

In the presence of activated factor XIII, 2-diethylbenzyl-aminoethylthiol-14C-transcinnamate bromide completely inhibited the crosslinking of fibrin. However, all three fibrin chains bound the cinnamic acid, and, in the alpha- and gamma-chains, the binding of label was not restricted to the crosslinking donor sites as might be expected. Furthermore, even in the absence of activated factor XIII, fibrinogen and fibrin incorporated cinnamic acid. Thus, as well as reacting with the functional thiol group of factor XIII, the thiocholine ester of cinnamic acid is incorporated non-specifically throughout the fibrinogen and fibrin subunit chains. The thiocholine ester used differs in this respect from dansyl cadaverine which is incorporated enzymatically and exclusively to the (acceptor) sites involved in crosslinking. Thiocholine ester of cinnamic acid cannot be used as a label for localization of specific crosslinking donor sites.