Effect of dextran 70 on factor VIII and platelet function in von Willebrand's disease.

Dextran given intravenously to two patients with mild von Willebrand's disease and two healthy controls impaired the factor VIII properties measured, i.e. it decreased the factor VIII-related antigen and the ristocetin co-factor activity. In the patients the variables fell from borderline to abnormally low levels accompanied by a prolongation of the bleeding time. Infusion of factor VIII concentrate reversed the dextran-induced impairment of factor VIII and the prolonged bleeding time. The findings lend support to the view that dextran induces a defective platelet function in vivo by interfering with the factor VIII molecular complex. They also show that patients with von Willebrand's disease should not be given dextran as it might potentiate their bleeding diathesis.     

Effect of carbohydrate modifications of factor VIII/von Willebrand factor on binding to platelets

This study compares the ability of unmodified and carbohydrate-modified forms of factor VIII/von Willebrand factor (FVIII/vWF) protein to bind to platelets in the presence of ristocetin or thrombin. Treatment of intact FVIII/vWF with alpha-D-neuraminidase results in more than 95% desialylation. Asialo FVIII/vWF retains total activity in ristocetin- and thrombin-mediated binding to platelets as demonstrated by direct and competitive binding assays. Examination of its multimeric pattern by sodium dodecyl sulfate-agarose electrophoresis reveals a normal multimeric structure. Treatment of intact FVIII/vWF with beta-D-galactosidase results in the removal of 20% of galactose (agalacto FVIII/vWF) whereas 55% of galactose is released from asialo FVIII/vWF (asialo agalacto FVIII/vWF). Agalacto and asialo-agalacto FVIII/vWF are both unable to bind to platelets in the presence of ristocetin. In contrast, they still bind to thrombin-stimulated human (except thrombasthenic) platelets. Removal of either ultimate (agalacto FVIII/vWF) or ultimate and penultimate (asialo-agalacto FVIII/vWF) galactose results in the same loss of the larger molecular weight multimers and in an increase of smaller multimers. These results suggest (1) that sialic acid does not play a significant role in ristocetin- or thrombin-mediated FVIII/vWF-platelets interactions and multimeric structure of FVIII/vWF (2) that ultimate beta-linked galactose residues are essential for the maintenance of a normal multimer organization (3) that ristocetin- and thrombin-mediated binding of FVIII/vWF to platelets differ in FVIII/vWF galactose requirement.     

The factor VIII/von Willebrand factor ratio discriminates between reduced synthesis and increased clearance of von Willebrand factor

It is often stated that a decrease in Von Willebrand factor (VWF), the carrier protein of factor VIII, results in a concordant change in factor VIII. Clinical data suggest that this is not always the case and we hypothesized that the ratio between factor VIII and VWF depends on the genetic defect that causes the VWF deficiency. We have analyzed the ratio between plasma factor VIII and VWF in several subtypes of Von Willebrand Disease and we show that the ratio is increased when VWF synthesis is reduced, but that the ratio remains one when VWF clearance is increased. These observations could be of clinical importance as an increased factor VIII/VWF ratio in combination with a borderline VWF level may indicate the presence of a true genetic defect, possibly a VWF null allele.     

Studies on the factor VIII/von Willebrand factor antigen on human platelets

A purified protein having both Factor VIII activity (coagulant assay) and von Willebrand factor activity (platelet retention and ristocetin aggregation assays) was used to immunize a goat. The resulting antiserum neutralized Factor VIII coagulant activity, decreased platelet retention of normal blood and blocked ristocetin aggregation of normal platelet rich plasma.

This same antiserum acted as an “anti-platelet” antibody in serotonin release, platelet aggregation and immunofluorescence with normal, hemophilic and von Willebrand platelets. However, after suitable absorption with small numbers of platelets this antiserum recognized Factor VIII/von Willebrand factor antigen only on normal and hemophilic platelets but not on platelets from two patients with severe von Willebrand's disease.

It is possible that antisera produced against Factor VIII purified from plasma not rendered completely free of platelets contains antibodies to platelet membrane material.     

The role of fibronectin in factor VIII/von Willebrand factor cryoprecipitation

To evaluate the role of fibronectin (Fn) in factor VIII (FVIII) and von Willebrand factor (vWf) cryoprecipitation, factor VIII procoagulant activity, factor VIII coagulant antigen, factor VIII-related antigen and von Willebrand ristocetin cofactor activity were measured in cryoprecipitate and cryosupernatant from normal and Fn-depleted plasmas. Following cryoprecipitation of normal plasmas, most of the FVIII and almost all the FvWf recovered were found with a part of Fn and of fibrinogen in cryoprecipitate. Fn-depleted plasmas prepared either by affinity chromatography on gelatin or by immunoadsorption on monoclonal anti-Fn antibodies behaved differently : although their cryoprecipitate contained normal fibrinogen levels, neither FVIII nor FvWf was precipitated. Experiments performed with Fn-depleted plasma to which purified fibronectin had been added, and samples of plasma with decreased Fn levels (0.01 to 0.2 g/l) suggest that there is a relation between initial Fn level and the extent of FVIII/vWf cryoprecipitation. We conclude that Fn, like fibrinogen, is necessary to induce cryoprecipitation of FVIII/vWf and that an initial plasma level of 0.2 g/l is sufficient to obtain good recovery of FVIII/vWf in cryoprecipitate.     

Heterogeneity of molecular size of factor VIII/von Willebrand factor in von Willbrand's disease

Patterns of VIIIR:AG in the plasma and its fractions, cryoprecipitate and cryosupernatant, from various types of von Willebrand's disease (vWd) were observed by SDS 1.5% polyacrylamide gel electrophoresis - cross immunoelectrophoresis (SDS PAGE - CIE). VIIIR:AG in normal cryoprecipitate showed several precipitin peaks which correspond to molecular weights ranging from 8 X 10(5) to 1 X 10(7) daltons and are similar to those in normal plasma. Normal cryosupernatant VIIIR:AG gave smaller molecular weights from 8 X 10(5) to 2 X 10(6) daltons. VIIIR:AG in the plasma and cryoprecipitate from 2 patients with classical vWd gave low precipitin peaks with molecular weights in normal range. VIIIR:AG from 2 patients with subgroup A variant which showed fast anodal migration on the conventional CIE, presented 3 peaks with molecular weight of 8 X 10(5) to 3 X 10(6) which are similar to those in normal cryosupernatant. VIIIR:AG from 2 patients with subgroup B variant which showed normal migration on the CIE, gave normal patterns through all fractions.     

The interaction of the factor VIII/von Willebrand factor complex with hematin

Intravenous infusion of hematin, used in the treatment of acute porphyria, induces a decline in the plasma factor VIII/von Willebrand factor complex (VIII/vWF) and thrombocytopenia. We investigated this problem by studying the interaction between hematin, purified VIII/vWF, and platelets in vitro. Hematin was labeled with either 59Fe or 3H and characterized by gel chromatography. Hematin self-aggregated, forming a complex with an average molecular weight of approximately 10,000 daltons. When incubated with VIII/vWF for 30 min at 37 degrees C and applied to Sepharose CL-4B, the hematin eluted with the VIII/vWF in the void volume. Hematin inhibited the dissociation of factor VIII antigen (VIII:Ag) from the von Willebrand antigen (vWF:Ag) in 0.25 M CaCl2, and reversed the aggregation of VIII:Ag induced by 0.1 M 6-aminocaproic acid. Both hematin and the hematin-VIII/vWF complex bound to washed normal platelets and to platelets from a patient with Bernard-Soulier syndrome. Thrombasthenic platelets were not aggregatable by hematin, and bound significantly less hematin-VIII/vWF than normal platelets suggesting that hematin-induced platelet activation was required for binding. Likewise, binding was inhibited by PGE1 which also prevented aggregation. We conclude that hematin forms complexes with VIII/vWF, alters the functional activity and dissociation of this compound, and participates in the binding of VIII/vWF to platelets.     

A comparative multi-laboratory assessment of three factor VIII/von Willebrand factor concentrates

Five expert laboratories have participated in a cross-laboratory study to co-evaluate and compare three commercial Factor VIII/von Willebrand factor (VWF) concentrates. A total of nine factor concentrate lots were evaluated, comprising AHF (High Purity) (AHF HP; x3), Biostate (x3) and Humate/Haemate (x3). All laboratories blind tested for FVIII: C, VWF: Ag and VWF: CB, four tested for VWF: RCo, and one performed VWF: Multimers. The study yielded inter-laboratory CVs for VWF: Ag and FVIII:C around 10-15%, and for VWF:CB and VWF:RCo around 20%, significantly lower than those of previous multi-laboratory surveys. All three lots of AHF HP contained in the vicinity of 25 U/ml FVIII:C, around 60-75 U/ml of VWF:Ag, but only 30-45 U/ml of VWF:CB and 40-50 U/ml of VWF:RCo (thus, CB/Ag ratio around 0.5-0.6 and RCo/Ag ratio around 0.6-0.7). Study determined that FVIII: C and VWF: RCo levels were similar to manufacturer assigned levels. Some loss of the high molecular weight (HMW) multimers was observed, together with an intense low molecular weight (LMW) VWF band consistent with some reduction or proteolysis of HMW VWF. All three lots of Humate/Haemate contained in the vicinity of 23-32 U/ml of FVIII:C, 70-105 U/ml of VWF: Ag, 50-90 U/ml of VWF: CB and VWF: RCo (i.e. CB/Ag ratio around 0.6-0.9 and RCo/Ag ratio around 0.6-1.1). Study-determined FVIII: C and VWF: RCo levels were similar to manufacturer-assigned levels. The LMW multimer band seen with AHF HP was also observed with Humate/Haemate. All three lots of Biostate contained in the vicinity of 40-55 U/ml of FVIII:C, 105-170 U/ml of VWF:Ag, 90-150 U/ml of VWF:CB, and 90-135 U/ml of VWF:RCo (i.e. CB/Ag and RCo/Ag ratios around 0.7-1.0). Study-determined FVIII:C levels were similar to manufacturer-assigned levels. The LMW multimer band seen with AHF HP was not observed with Biostate. The defined pattern of increasing CB/ Ag from AHF HP to Humate/Haemate and Biostate was consistently observed in study data from each of the five laboratories. In conclusion, study findings indicate some differences in the retention of functional/ HMW VWF between factor concentrates, and this is expected to have significant implications in terms of clinical efficacy for therapy in VWD.     

Effect of exercise, DDAVP, and epinephrine on the factor VIII:C/von Willebrand factor complex in normal dogs and von Willebrand factor deficient Doberman pinscher dogs

Endothelial cells in biopsied blood vessels from von Willebrand factor (vWf)-deficient Doberman pinscher dogs contain immunologically detectable vWf. These dogs and normal dogs were treated with DDAVP (0.6 microgram/kg) and epinephrine (0.5 microgram/kg/min for 30 minutes) and were exercised, using 5 different exercise protocols, (3-4 m/s for 5-40 minutes at 0-5% grade) to determine if treatments reported to increase plasma factor VIII:C/vWf complex in humans would elevate canine plasma vWf. Following the two most strenuous exercise conditions--30 and 40 minutes--plasma von Willebrand factor antigen (vWf:Ag) increased in normal dogs by 30% and 70%, respectively. Factor VIII:C was increased 47% by the most strenuous exercise conditions. The vWf-deficient dogs would not exercise beyond 30 minutes and neither vWf:Ag nor factor VIII:C activity increased. Following DDAVP, plasma vWf:Ag increased in the normal dogs by 47% and factor VIII:C activity was increased by 48%. Factor VIII:C activity increased by 30% in the vWf-deficient dogs, but there was only a slight change in vWf:Ag. Bleeding time decreased in 5 of 6 vWf-deficient dogs. In the normal dogs vWf:Ag increased by 14% after epinephrine infusion, but factor VIII:C activity did not change; neither parameter was altered in the vWf-deficient dogs. While the factor VIII:C/vWf:Ag complex was increased in the normal dog by exercise and DDAVP, the increase is not as pronounced as has been reported for humans. It is not known whether the poor response of the vWf-deficient dog is due to low levels of vWf in their endothelium or to a release defect.     

The dynamic inter-relationship between factor VIII and von Willebrand factor.

A monospecific heterologous antibody which specifically inhibited von Willebrand factor (W.F.) but had no factor VIII procoagulant (VIIIc) neutralising properties was insolubilised by attachement to sepharose beads. When fresh normal plasma was mixed with such beads, VIIIc, W.F. and factor VIII related antigen (VIIIAg) were removed in equal proportions, with the VIIIc lost from the plasma being detectable attached to the sepharose. This VIIIc was removed from the sepharose and largely recovered in the eluate after treatment with 0.2M calcium chloride. Subsequent addition of plasma resulted in a high transfer of VIIIc from the plasma onto the sepharose beads without accompanying W.F. and VIIIAg. Similar experiments, with initial treatment of antibody attached sepharose beads with serum, resulted in no independent segregation of VIIIc on subsequent addition of plasma. It is suggested that VIIIc and W.F. exist in plasma as separate, but attached molecules in a dynamic equilibrium. In serum, W.F. loses its ability to attach VIIIc.     

Development of a heat-treated factor VIII/von Willebrand factor concentrate prepared from heparinized plasma

A high yield, intermediate purity factor VIII concentrate derived from heparinized plasma has been developed which can be heat-treated at 60 degrees C, 68 degrees C or 80 degrees C/72 h to permit inactivation of viral contaminants which may be present. After cold reprecipitation of the heparinized cryoprecipitate (CRC), the resolubilized CRC precipitate was adjusted to 25-30 mg/ml protein and pH 6.35 +/- 0.1 and incubated for 1 h at 8 degrees C. After centrifugation to remove the precipitated fibrinogen and fibronectin, a factor VIII-rich supernatant can be recovered which contains greater than 500 units of VIII:C per liter of starting plasma (Method I product) at a purity of 1.5 U/mg protein. Adjusted to 50 mM glycine and pH 6.8, the product can be lyophilized and heat-treated at 60 degrees C/72 h without a significant loss of VIII:C activity. However, at 68 degrees C or 80 degrees C/72 h, temperatures now reported to be more effective in viral inactivation, the recoveries were reduced to 68 and 33% respectively. Significantly improved recoveries after heat-treatment (HT) at 68 degrees C or 80 degrees C/72 h were achieved if the 8 degrees C supernatant product was prepared by a modified procedure (Method II). This further reduces the fibrinogen content of the product while maintaining VIII:C yields greater than 500 U/l at a purity of 1.9 U/mg. When adjusted to 50 mM glycine and 1-2% (w/v) sucrose (pH 6.8), lyophilized and heat treated at 60 degrees C, 68 degrees C or 80 degrees C/72 h, the VIII:C recoveries of Method II product were 88-100%, 79-84% and 80-83% of pre-HT levels respectively. The yield of VIII:C was greater than 400 U/l at a purity of 1.6-1.4 U/mg at 1-2% (w/v) sucrose even after the severe heat-treatment at 80 degrees C. In addition, the von Willebrand factor multimers are similar in size and triplet pattern to those observed in routine cryoprecipitate preparations.