Study of a therapeutic concentrate of factor VIII/vWf prepared in a closed system

An original procedure of preparation in a closed system of high purity Factor VIII concentrate is presented. Starting from cryoprecipitates, this method involves a first step of partial removal of fibrinogen by glycine precipitation (1.6 M) and a second step of Factor VIII concentration by cryoprecipitation. The yield is 16.5% of plasmatic F VIII:C (0.8 mu/ml.). Several batches of concentrates thus prepared are compared "in vitro" to 9 other commercially available concentrates from 8 different manufactories. The results show that most of the characteristics of our concentrate are within the range of specifications of other commercially available high-purity F VIII concentrate: F VIII: C activity (CRTS Lille concentrate: 25-40 U/ml.; other concentrates: 25-50 U/ml) solubility, specific activity (CRTS lille concentrate; 1.0-1.82 U F VIII:C/mg protein and 1.79-4.8 U F VIII: C/mg clottable proteins; other concentrates: 0.53-2.79 U F VIII:C/mg protein an 1.39-4.84 U F VIII:C/mg clottable proteins), isoagglutinin titers (CRTS Lille concentrate: 2-8 anti-A, 0.16 anti-B; other concentrates: 0-64 anti-A, 8-16 anti-B) F VIIIC/F VIII R: Ag ratios (CRTS Lille concentrate: 0.18-0.49; other concentrates: 0.20-0.42). Furthermore F VIII R:Ag electrophoretic mobility studied by crossed immunoelectrophoresis add F VIII R: RCo assays provide evidence that very high molecular weight multimeric forms of F VIII/vWf which support vWf activity are present in our concentrate. "In vivo" study and clinical efficacy in vWd patients confirm these results and show that our concentrate is appropriate for the treatment of patients with F VIII:C or V VIII R:RCo deficiency.     

The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets

The binding of 35S-labeled recombinant human Factor VIII to activated human platelets was studied in the presence and absence of exogenous plasma von Willebrand factor. In the absence of added von Willebrand Factor, platelets bound 210 molecules of Factor VIII/platelet when the unbound Factor VIII concentration was 2.0 nM (Kd = 2.9 nM). As the von Willebrand factor concentration was increased, the number of Factor VIII molecules bound/platelet decreased to 10 molecules of Factor VIII bound/platelet at 24 micrograms/ml of added vWF. Addition of an anti-vWF monoclonal antibody that inhibits the vWF-Factor VIII interaction attenuated the ability of vWF to inhibit binding of Factor VIII to platelets. In contrast, addition of a control anti-vWF antibody that does not block the vWF-Factor VIII interaction did not affect the ability of vWF to inhibit Factor VIII binding to platelets. From the vWF concentration dependence of inhibition of Factor VIII-platelet binding, a dissociation constant for the Factor VIII-vWF interaction was calculated (Kd = 0.44 nM). To further elucidate the role that vWF may play in preventing the interaction of Factor VIII with platelets, the platelet binding properties of a Factor VIII deletion mutant (90-73) which lacks the primary vWF-binding site was studied. The binding of this mutant was unaffected by added exogenous vWF. These observations demonstrate that Factor VIII can interact with platelets in a manner independent of vWF but that excess vWF in plasma can effectively compete with platelets for the binding of Factor VIII. In addition, since cleavage of Factor VIII by thrombin separates a vWF-binding domain from Factor VIIIa, we propose that activation of Factor VIII by thrombin may elicit release of activated Factor VIII from vWF and thereby make it fully available for platelet binding.     

In vitro characterization of commercial Factor VIII concentrates: long-term follow-up

Fifty batches of Factor VIII concentrates from 12 producers were characterized in a long-term follow-up. The following parameters were measured: Factor VIII: C, Factor VIIIR: AG, Factor VIII: Rcof, specific activity (U Factor VIII: C/mg protein), fibrinogen, IgG, IgM, IgA, isoagglutinins, Hbs-AG, heparin-like activity, thrombin-like activity, antithrombin III, Factor VIII-stability at room temperature, and the rate of complete dissolution of the lyophilizate. In most preparations there was an unacceptable batch-to-batch variation of both Factor VIII complex and contaminating proteins, which exceeded the inter-assay coefficient of variation of the applied test systems. Nevertheless, different brands could be recognized by their typical protein pattern. The results obtained suggest that the standardization of Factor VIII concentrates of unknown composition is still accompanied by considerable risks.     

The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets

Recombinant-derived human Factor VIII was labeled intrinsically with [35S]methionine, and its binding to washed human platelets was studied. Binding measurements were performed by incubating Factor VIII and platelets for 15 min at room temperature in Tyrode's solution supplemented with Ca2+ (5.0 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (5.0 mM), 0.50% bovine serum albumin, and the Factor Xa and thrombin inhibitors 5-dimethylaminonaphthalene-1-sulfonylglutamylglycinylarginyl chloromethyl ketone and 5-dimethylaminonaphthalene-1-sulfonyl-arginine-N-(3-ethyl-1, 5-pentanediyl)amide. Separation of free from bound Factor VIII was accomplished by centrifugation through oil, and nonspecific binding was determined with excess unlabeled Factor VIII. Binding was saturable, reversible, and stimulated 20-fold after platelet activation with thrombin. Furthermore, binding was specific in that bound labeled Factor VIII could be displaced by excess unlabeled Factor VIII, but not by Factor V. Scatchard analysis indicated a single class of binding sites with Kd = 2.9 nM and 450 sites/activated platelet. The time course of displacement indicated a t1/2 of bound Factor VIII of approximately 5 min. When platelets were incubated in Ca2+, both the heavy and light chains of Factor VIII were bound, whereas exposure to EDTA resulted in the binding of the light chain only. These results demonstrate the specific reversible binding of Factor VIII to human platelets, likely mediated through the light chain.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

Standardization of factor VIII: establishment and use of secondary standards

Two secondary standards for use in routine assays of Factor VIII in therapeutic concentrates and in patients, plasmas, respectively, have been established in a multicenter collaborative study. In order to assess the effect of the adoption of these preparations as common Secondary Standards a comparative assay has been performed: one sample of a Factor VIII concentrate of intermediate purity and one plasma sample have been tested in two laboratories for Factor VIII:C activity using as reference, among others, the common working standard. Analysis of the results shows that with the plasma sample the differences of the estimates obtained with any of the references in our two laboratories were not statistically significant (P greater than 0.3), while with the concentrate sample the differences were always statistically significant (P less than 0.005). The study shows that the adoption of common working standards (besides the uniformity in assay method, reagents and basic equipment) is not sufficient to eliminate interlaboratory variation in the measurement of Factor VIII:C.     

Residual Factor VIII-like cofactor activity of thioredoxin and related oxidoreductases

Background

Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood.

Methods

We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay.

Results

We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol–disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme.

Conclusion

Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases.

General significance

The possible involvement of thiol–disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.     

Double cryoprecipitated factor VIII concentrate from heparinised plasma and its heat treatment

In an attempt to implement the small pool concept in Factor VIII purification, cryoprecipitate derived from heparinised plasma was reprecipitated in the cold providing a factor VIII concentrate for freeze drying and heat treatment. There was considerable purification; only 1% of the original plasma proteins was left in the final product. Factor VIII:C concentration was about 19 IU/ml. Factor VIII related antigen (RAg) appeared heterogeneous, with a broad base and asymmetry on crossed immunoelectrophoresis. Fibrinogen content was 15 g/l. In contrast to high-purity commercial concentrates, fibronectin was considerably concentrated. Immunoglobulin contents were similar to a high-purity commercial product. The amount of other plasma proteins was very small, varying from less than 0.2% for C3 complement to 2.3% ceruloplasmin. In some respects the preparation may be considered as an intermediate-purity Factor VIII concentrate. Following addition of 2% sucrose before freeze drying, Factor VIII, total protein and fibrinogen remain virtually stable (less than 15% loss) during heating of the material to 60, 64 or 68 degrees C for 24 to 72 h without changes of protein spectrum following heating. The heated product when stored at 4 degrees C remains stable for at least 3 months. In two severe haemophiliacs receiving this heat treated product, in vivo Factor VIII recovery was 100% with a mean half life of 10.2 h.     

Use of monoclonal antibody and colloidal gold in E.M. localization of von Willebrand factor in megakaryocytes and platelets

The subcellular localization of Factor VIII/von Willebrand protein (VIII R:Ag) is studied with monoclonal antibody and gold immunocytochemical technique. Monoclonal antibody against purified VIII R:Ag is brightly fluorescent on megakaryocytes and platelets. In E.M., gold immunolabeling is performed on thin cell sections of human megakaryocytes and platelets. Different embedding materials are used to preserve the antigenicity : Epon embedded megakaryocytes show a high concentration of VIII R:Ag in alpha-granules using 4F9 monoclonal antibody. In comparison, lowicryl K4M embedded material does not improve the same specificity, only a few platelets granules were stained. This subcellular localization, in full agreement with biochemical results appears visualized for the first time in E.M.     

Immunological abnormalities in haemophilia: are they caused by American factor VIII concentrate?

Scottish patients with haemophilia, most of whom had received no American factor VIII concentrate for over two years, were found to have immunological abnormalities similar to those in their American counterparts--that is, a reduced proportion of T helper cells, an increased proportion of T suppressor cells, and a reduced response to concanavilin A. Factor VIII from both the United States and Scotland severely inhibited the in vitro lymphocyte response to mitogens in patients and controls. The American and Scottish concentrates could not be distinguished in terms of either patient usage or their effect in vitro. These results argue against a disease vector specific to American blood products.     

Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)     

The dielectrophoresis of blood platelets as affected by hemophilic traits

We find that there are moderate differences in the electrical polarizabilities of normal and various hemophilic canine blood platelets. The technique of dielectrophoresis, using the effect of nonuniform fields on neutral bodies, was used to perform rapid assays of the platelets. At present the dielectrophoretic test can only distinguish reliably between relatively large groups of animals on a statistical basis. The present technique shows a unique ability however, to distinguish even between normal canine platelets and the transmitter female canine platelets. In studies of the effects of various chemical agents upon the dielectrophoresis of platelets, inhibitors such as NaF, sodium iodacetate, NaCN, and NaN₃ had marked effects at low concentration. Ions such as Na⁺ K⁺, Mg⁺⁺, and La⁺⁺⁺, as well as NO₃ ⁻, SO₄ ⁼, and mellitic ion had lesser effects. In some cases the presence of trace quantities of the chemical agent “stabilizes” the cellular dielectrophoretic response, enabling the platelet to continue to be attracted by the nonuniform field for longer than usual. The CN⁻ and F⁻ ions appear to do this. This may have useful application. From the shape of the frequency spectrum of the dielectrophoretic response we suggest that the peaks at about 0.1 to 10 MHz imply a Maxwell-Wagner type of response, typical of an interface between bulk regions of differing conductivity, as at the cell boundary. From a lack of low frequency response, we suggest that the platelet interface with the surrounding aqueous medium must be singularly free of ionic double layers — or at least that the ionic double layers present must be of unusually low charge density. The technique of dielectrophoresis has been used in comparative study of canine blood platelets, from (1) normal dogs, (2) female dogs that transmit Factor VIII deficiency to their offspring, (3) male dogs with Factor VIII deficiency, and (4) female dogs with Factor VIII deficiency. The study showed that differences exist between the 4 groups of dogs in the average dielectrophoretic responses at 1 MHz. The effect of several chemical agents, i.e., NaCN and NaF on normal canine platelets was to effectively stabilize the platelets against deteriorationin vitro.     

Cyclic peptide analogs of 558–565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII–Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558–565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa–Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.     

Effects of high-molecular-weight cryoprotectants on platelets and the coagulation system

The objective of this study is to examine the effects of the most widely used high-molecular-weight cryoprotectants on the coagulation system. Dextran, hydryoxyethyl starch (HES), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), and albumin were added at different concentrations in the range between 0.01-1% (w/v) to solvent/detergent-treated plasma. Using a STA/STA Compact coagulation analyzer the following clotting tests were performed: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Factor V, and Factor VIII percentage of activity. PVP and PEG caused a significant increase in APTT, a decrease in Factor VIII percentage of activity, and a slight decrease in TT, while PT and Factor V percentage of activity remained unchanged. Dextran, HES, and albumin did not effect the clotting tests. The effect of high-molecular-weight cryoprotectants on platelets was assessed by platelet-induced clot retraction (PICR) and aggregation with thrombin and agglutination with ristocetin. Platelet aggregation and agglutination were unaffected by all cryoprotectants tested; however, PICR was significantly reduced in the presence of PVP or PEG. Possible mechanisms by which PVP and PEG interfere with the coagulation system are discussed. We also raise issues concerning the development of one-step blood cryopreservation techniques which do not require cryoprotectant removal prior to transfusion.     

Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells

The effects of increased synthesis of secreted proteins expressed from stably integrated heterologous genes in Chinese hamster ovary (CHO) cells following treatment with sodium butyrate was studied. Butyrate treatment increased expression of mRNA transcribed from the adenovirus major late promoter in combination with the SV40 enhancer for Factor VIII, von Willebrand factor, and erythropoietin. Increased levels of mRNA were compared to increases in intracellular primary translation product and secreted protein. While von Willebrand factor and erythropoietin were efficiently secreted, Factor VIII was not. Increased expression of all these proteins induced expression of the glucose-regulated proteins, GRP78 and GRP94. However, increased Factor VIII synthesis was correlated with an 80-fold increase in GRP78 mRNA and a 10-fold increase in GRP94 mRNA. These data suggest that elevated levels of newly synthesized secretion-competent protein as well as misfolded protein induce the glucose-regulated proteins.     

Experience with a blood component program in surgical hemotherapy

Within the component concept applied during the past 6 years at the University Hospital in Berne (Switzerland), 85% of all red cell units are transfused as concentrates with a hematocrit of 70%, and the remaining 15% as fresh whole blood. The rationale in surgical patients is to exploit the different "critical levels" of the blood volume, hematocrit, total serum protein, plasmatic coagulation factors, and platelets. A colloid plasma substitute compensating for the plasma deficit of the red cell concentrates is an integral part of the system. A carefully checked, retrospective study in 372 patients revealed no disadvantages for the postoperative course. During the first 4 1/2 years after its introduction it did not increase the demand for human plasma protein solutions. With the red cells as a pacemaker for the number of blood donations, this system can simultaneously cover a reasonable national demand for albumin and factor VIII.     

Interaction of factor VIII-von Willebrand Factor with phospholipid vesicles.

The interaction of Factor VIII-von Willebrand Factor with phospholipid vesicles has been studied by using sucrose-density-gradient ultracentrifugation. When purified Factor VIII-von Willebrand Factor was run alone. Factor VIII activity and Factor VIIIR-Ag sedimented together to the lower half of the tube. Addition of phosphatidylserine/phosphatidylethanolamine vesicles at concentrations above 250 microgram/ml resulted in complete separation of Factor VIII activity and Factor VIIIR-Ag, the former appearing with the phospholipid on the top of the tube and the latter sedimenting as before. This separation was obtained even in the presence of proteinase inhibitors. Activation of Factor VIII-von Willebrand Factor by thrombin resulted in formation of a slow sedimenting component containing essentially all the Factor VIII activity, whereas the Factor VIIIR-Ag sedimented towards the bottom of the tube as before. The thrombin-induced Factor VIII activity was strongly bound to phospholipid vesicles as determined by density-gradient centrifugations at various Factor VIII concentrations and low concentrations of phospholipid. Based on certain assumptions a dissociation constant of 2.5 nM was calculated, a mechanism for the formation in vivo of the Factor X-activator complex is suggested.     

Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor

The acidic region of the Factor VIII light chain was studied with regard to structural requirements for the formation of a functional von Willebrand factor (vWF)-binding site. Factor VIII mutants lacking the B domain, with additional deletions and an amino acid replacement within the sequence 1649-1689 were constructed using site-directed mutagenesis and expressed in Cos-1 cells. These mutants, which were recovered as single-chain molecules with similar specific activities, were compared in their binding to immobilized vWF. Deletion of amino acids 741-1648 or 741-1668 did not affect the binding of Factor VIII to vWF. However, a mutant with a deletion of residues 741-1689 was no longer capable of interacting with vWF. This indicates a role for residues within the sequence 1669-1689 in the formation of a vWF-binding site. When recombinant Factor VIII was expressed in the presence of chlorate, an inhibitor of protein sulfation, the resulting Factor VIII displayed strongly reduced binding to vWF. vWF binding was completely abolished when within the sequence 1669-1689 the tyrosine residue Tyr1680, which is part of a consensus tyrosine sulfation sequence, was replaced by phenylalanine. The Factor VIII sequence 1673-1689 was identified as a high affinity substrate for tyrosylprotein sulfotransferase (Km = 57 microM) in cell-free sulfation studies. It is concluded that sulfation of Tyr1680 is required for the interaction of Factor VIII with vWF. Two synthetic peptides that represent the sequence 1673-1689, but differ with respect to sulfation of Tyr1680 are shown to have vWF binding affinity that is considerably lower than the Factor VIII protein. Several models to accommodate our findings are discussed.