[Seroprevalence of hepatitis C virus in hemophiliacs in Switzerland]. / Hepatitis-C-Virus-Serostatus der Hämophilen in der Schweiz.

The sera of patients with hemophilia and von Willebrand factor deficiency, collected during clinical trials with virus-inactivated coagulation factor preparations, were retrospectively screened for the presence of antibodies against hepatitis C virus (HCV). Using the anti-HCV c100-3 assay, 10 out of 35 study patients had no HCV antibodies when entering the studies. The samples originally negative for anti-HCV were retested with a second-generation assay: all negative samples except two were positive on retesting. We conclude that the HCV seroprevalence in Swiss hemophiliacs must be of the order of > 90%. These results confirm that the first-generation assay for HCV antibodies is not sensitive enough; to obtain more accurate information on the seroprevalence of hemophiliacs, second-generation tests should be used.

[Clinical testing of Premofil M SRK, a blood coagulation factor VIII concentrate purified from human plasma using monoclonal an antibodies]. / Klinische Prüfung von Premofil M SRK, einem mit monoklonalen Antikörpern hochgereinigten Gerinnungsfaktor VIII-Konzentrat aus Humanplasma.

Premofil M SRK, licensed in Switzerland by the IKS since mid-1990, was clinically tested in close collaboration with 7 hemophilia treatment centers. Up to May 1991, we collected results of in vivo recovery from 17 patients, half-life determinations from 12, and safety data from 9 who were exclusively treated with the preparation for several months. The mean in vivo recovery of factor VIII was calculated to be 77 +/- 14%; the rise in factor VIII activity in plasma following injection of one unit/kg body weight was 1.6 +/- 0.3 units/dl. The mean halflife was 11.9 +/- 4.6 hours. No side reactions were registered throughout the study period. None of the patients showed any signs of HIV or hepatitis infection. It can bei concluded that Premofil M SRK fulfills the requirements related to tolerance, efficacy and safety for a factor VIII concentrate.

[No HCV seroconversion in hemophilia following substitution with virus-inactivated coagulation factor VIII and IX concentrates]. / Keine HCV-Serokonversion bei Hämophilen nach Substitution mit Viren-inaktivierten Gerinnungsfaktor VIII- und IX-Konzentraten.

Since 1986 the factor VIII and IX concentrates of the Central Laboratory, Swiss Red Cross Blood Transfusion Service have been virus inactivated with tri-(n-butyl) phosphate and Tween 80. Clinical studies had shown that both preparations were well tolerated and hemostatically effective; no HIV infection was transmitted. However, safety from transmission of non-A/non-B hepatitis could not be shown since the study included no previously untreated patients. In the meantime, a laboratory test has become available which allows retrospective testing for anti-hepatitis C antibodies in frozen sera of the study patients. 5 of the 26 patients, observed during a 2-year follow-up study, had no HCV antibodies before entering the long-term trial. During this trial, each of these 5 patients substituted an average quantity of 40,200 coagulation factor units (7500-69,000) from 45 production lots. None of these 5 patients developed anti-HCV antibodies, nor did any of them show clinical signs of infection with hepatitis. This suggests that virus inactivation using solvent/detergent treatment reduces the risk of transmission of HCV.

Safety of intravenous immunoglobulin preparations: a prospective multicenter study to exclude the risk of non-A, non-B hepatitis.

The risk of non-A, non-B hepatitis transmission by an intravenous immunoglobulin (IVIG) preparation was assessed in a prospective multicenter trial in 68 patients with primary immunodeficiency disorders (40 children or adolescents and 28 adults). During the 4-week prestudy evaluation period the clinical examinations and liver function tests including alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, and bilirubin were normal in all patients. The treatment consisted of three infusions of 200 mg IVIG (pH 4; pepsin procedure) per kilogram body weight at 2-week intervals. During the observation period of 24 weeks following the first infusion of the study IVIG, the patients were monitored at regular time intervals. No clinical and laboratory signs of hepatitis or liver dysfunction were noticed. All patients completed the study. In 5 patients, one isolated alanine aminotransferase value and in another patient one gamma-glutamyl transpeptidase value were moderately elevated, but always below 2.5 times the upper limit of the reference range. Similar isolated and transient elevations were observed for aspartate aminotransferase and alkaline phosphatase. It was concluded that the IVIG preparation did not transmit non-A, non-B hepatitis or other viral liver diseases.

Laboratory workshop on the characterization of anti-platelet antibodies in immune thrombocytopenic purpura.

The aim of this laboratory workshop was to evaluate the state of knowledge concerning the demonstration of membrane glycoprotein specific anti-platelet antibodies. The main interest lay in investigating whether specific antibody detection offers possibilities to distinguish the chronic from the acute form of ITP. In five laboratories four different methods were applied to demonstrate such antibodies. These methods are briefly described and compared. In all, except two, of the 45 ITP samples anti-platelet antibodies could be detected by at least one participating laboratory, in 85% of the samples antibodies were found by two or more laboratories. For seven out of eight control samples no positive results were reported. The comparison of glycoprotein specific anti-platelet antibodies shows partly considerable differences which may be due to the different methods as well as the different antibodies used (monoclonal antibody against membrane glycoprotein and antihuman globulin sera). This laboratory workshop leads to the conclusion that by exchange of reagents and patient samples the different methods may be compared and evaluated. The results obtained allowed no further characterization of ITP. All participants agreed on the usefulness of further similar laboratory workshops.

Von Willebrand factor-dependent agglutination of washed fixed human platelets by insoluble collagen isolated from bovine aorta.

Adult bovine aortic tissue was homogenized in a neutral phosphate buffer containing proteinase inhibitors. The insoluble residue was rehomogenized in Tris-buffered 6 mol/L guanidinium chloride (pH 7.4). An insoluble fibrillar protein, floating above the main pellet after recentrifugation, was harvested. This material agglutinated washed fixed human platelets in the presence of either normal human plasma or purified von Willebrand factor (vWF). No such reaction was seen when either buffer or plasma from patients with severe von Willebrand's disease was added instead. The extent of platelet agglutination was measured photometrically, similarly to the ristocetin cofactor assay. The agglutination reaction was strongest at neutral pH and was impaired after either addition of EDTA or previous digestion of the fibrillar material by collagenase or pepsin. By light microscopy platelets were seen to adhere onto isolated fibers. Amino acid composition, subunit polypeptides, substrate properties, and interaction with fibronectin of this fibrillar protein were comparable to those of collagen. Therefore, we tentatively denote the induction of platelet agglutination by vWF protein in the described test system as "vWF-collagen cofactor" activity. Comparison of this activity in 65 plasma samples, containing various concentrations of vWF, with ristocetin cofactor activity showed good correlation between results obtained in both tests (r = 0.91).

Reactivity of small molecular forms of human factor VIII/von Willebrand factor with botrocetin and anti-factor VIII-coated latex particles.

Two recently developed tests for measurement of factor VIII/von Willebrand factor (FVIII/vWF), i.e. platelet agglutination by botrocetin and a kinetic latex antigen assay, were compared with ristocetin cofactor and electroimmunoassay, in respect with FVIII/vWF size-distribution. FVIII/vWF was measured in six cases of atypical von Willebrand's disease (type II), in gel-filtered fractions of normal cryoprecipitate and in the course of depolymerization of purified normal FVIII/vWF by disulfide reduction. Small molecular forms of FVIII/vWF from normal and variant type II plasma, and those derived by disulfide reduction of high-molecular weight FVIII/vWF, showed remarkably decreased reactivity in ristocetin-, botrocetin- and latex-assay. We conclude that for botrocetin-induced platelet agglutination, as well as for agglutination of antibody-coated latex particles, multiple interactions with repeating subunits of FVIII/vWF are required. As a practical consequence, the combined measurement of FVIII/vWF by the latex test and electroimmunoassay provides a simple tool for discriminating between the classical von Willebrand's disease and its variants.

Isolation of small molecular forms of Factor VIII/von Willebrand factor from plasma.

Cryoprecipitated factor VIII/von Willebrand factor (FVIII/vWF), freed of fibrinogen by clotting with calcium and Defibrase, was chromatographed on Sepharose CL-2B. Fractions containing lower-molecular-weight forms of FVIII/vWF comprised coprecipitated plasma proteins of similar molecular weights. The major contaminants, fibronectin and IgM, were removed by affinity chromatography on gelatin- and anti-IgM-agarose, respectively. Finally, pure low-molecular-weight FVIII/vWF protein was harvested in the void volume fraction of a Sepharose CL-6B column. The smallest multimers had the size of the tetramer of the basic subunit chain of FVIII/vWF.

Silver staining of high molecular weight proteins on large-pore polyacrylamide gels.

A large-pore gel for electrophoresis in the presence of sodium dodecyl sulfate, composed of 2.55% polyacrylamide crosslinked with 2.75% methylenebisacrylamide, is described. This gel has a resolving power for very high molecular weight proteins and can be stained with silver. The gel is suitable for fractionation of factor VIII/von Willebrand factor directly from plasma samples. Visualization by silver staining revealed a series of covalently bound multimers with molecular weights of up to 8 X 10(6). The procedure described should be useful also for studies on other very high molecular weight proteins and nucleic acids.

Binding of bovine factor VIII-coated colloidal gold particles to receptors on platelet membranes.

Bovine factor VIII/platelet aggregating factor was adsorbed into gold granules and the protein-gold complex added to either formalin-fixed or fresh washed human platelets. Following aggregation, binding of gold granules to the platelets was measured by monitoring the optical density of colloidal gold remaining in the supernatant. Scatchard analysis of binding data indicated that multiple classes of binding sites were present. The number of high affinity binding sites per formalin-fixed platelet depended on the concentration of ristocetin: 420 gold granules were calculated to bind at 1.4 mg/ml of ristocetin, 610 at 0.6 mg/ml of ristocetin and 875 when no ristocetin was added. Fresh washed platelets bound 1350 granules per cell in the absence of ristocetin. We conclude that during platelet aggregation, induced by bovine factor VIII, the binding sites on the platelet surface are only partially occupied.

Von Willebrand activity of low molecular weight human Factor VIII increases by binding to gold granules.

Human factor VIII/von Willebrand protein is a population of multimers which vary in size but contain apparently identical subunits. Large-molecular-weight forms possess higher ristocetin cofactor/von Willebrand activity than the native smaller oligomers. Disulfide reduction of large factor VIII multimers results in progressively decreasing molecular size and a loss of ristocetin cofactor activity. Small molecular forms of factor VIII were adsorbed onto gold granules (average diameter 20-30 nm) and thereby increased their ristocetin cofactor activity. The amount of adsorbed material and the extent of activation were dependent on the pH of the coiled suspension. The maximum recovery of von Willebrand activity was observed at pH 4.75. Aggregation of fixed human platelets by factor VIII-coated gold particles was dependent on ristocetin concentration and was not competitively inhibited by unbound low-molecular-weight factor VIII. These results suggest that the subunits of the native small factor VIII species possess potential binding affinity for platelet receptors, which is manifested following formation of large factor VIII polymers. We conclude that an optimal size of remarkably high molecular weight is required for efficient aggregation of platelets by factor VIII as occurs during the primary phase of hemostasis.

Binding of colloidal gold granules, coated with bovine factor VIII, to human platelet membranes.

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.

[Increased von Willebrand activity of low molecular-weight factor VIII following binding to gold granules]. / Erhöhung der von Willebrand-Aktivität von kleinmolekularem Faktor-VIII-Protein durch Bindung an Goldpartikel.

High molecular weight factor VIII was partially reduced with beta-mercaptoethanol. Disulfide reduction resulted in progressive dissociation of the multimeric protein with a concomitant decrease in von Willebrand activity. The binding of reduced small factor VIII oligomers to gold particles with an average diameter of 20--25 nm led to marked "activation" of their von Willebrand activity. A similar increase in activity was also observed following adsorption of native small molecular weight forms of factor VIII to gold granules. Binding of initially highly active high molecular weight factor VIII to gold granules resulted in inhibition of von Willebrand activity, probably by formation of superaggregates. The platelet aggregating function of factor VIII in the circulation appears to depend largely on the size of this multimeric protein, which must be in the range of several millions for its maximum expression.

Studies on factor VIII-related protein. IV. Interaction of galactose-specific lectins with human factor VIII/Von Willebrand factor.

Factor VIII of human cryoprecipitate was purified and fractionated on Sepharose CL-2B into three fractions of progressively decreasing multimer size and ristocetin cofactor activity. Following complete disulfide reduction, the resulting subunits were electrophoresed on 3% polyacrylamide gels and subsequently stained with two galactose-specific, fluorescein-labelled lectins from Ricinus communis (RCAI and RCAII). Measurements of fluorescence indicated that the reduced chains, derived from the largest factor VIII multimers, have stronger binding affinity for both lectins than those obtained after reduction of smaller factor VIII species. Ristocetin cofactor activity of purified factor VIII was competitively inhibited by both Ricinus lectins and by concanavalin A. RCAI-lectin was found to be a considerably more efficient inhibitor than RCAII or concanavalin A. Following removal of sialic acid from factor VIII, the inhibiting effect of RCAII-lectin was markedly potentiated, probably by exposing additional galactose residues, some of which must be located close to the ristocetin cofactor 'active site' of factor VIII. Ristocetin cofactor activity was also strongly inhibited with RCAII-lectin for binding sites which are located on the surface factor VIII multimers. Our results suggest that RCAI-lectin, which contains galactose-specific binding sites per molecule, and anti-factor VIII antibodies inhibit ristocetin cofactor activity by crosslinking and aggregation of factor VIII multimers.

[Is there a correlation between sugar content, thrombocyte aggregating activity and molecular size of factor VIII?]. / Besteht ein Zusammenhang zwischen Zuckergehalt, plättchenaggregierender Aktivität und Molekülgrösse von Faktor VIII?

Human and bovine factor VIII, isolated from cryoprecipitates of fresh plasma by gel filtration on Sepharose CL-2B, gave similar elution patterns and showed comparable distribution of oligomers on SDS agarose electrophoretic gels. The carbohydrate content of individual factor VIII bands, measured by reaction with dansyl hydrazine or binding of glucose/mannose specific concanavalin A, was not directly related to the size or von Willebrand activity of factor VIII oligomers. Staining of disulfide-reduced factor VIII subunits, in polyacrylamide gels, with galactose-specific fluorescein-labelled Ricinus communis lectins, showed an increased binding affinity with increasing size and von Willebrand activity of the parent factor VIII. The von Willebrand activity was strongly inhibited by reaction with Ricinus RCAI lectin, whereas concanavalin A inhibited platelet aggregation only at concentrations above 1 mg/ml. These results suggest that galactose residues are involved in the aggregation of platelets by factor VIII.

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.