The role of Willebrand factor in platelet - blood vessel interaction, including a discussion of resistance to atherosclerosis in pigs with von Willebrand's disease

Von Willebrand pigs have all the manifestations of the severe human disease. The role of Willebrand antigen (VIII R:AG) and ristocetin cofactor (VIII: RWF) was assessed in these pigs by (1) transfusion and (2) "in vitro" bleeding time assay. The skin bleeding time became normal when the level of transfused Willebrand factor (VIII R:AG/RWF) was raised in the plasma above 30 U/dl. After single or repeated transfusions, skin capillary endothelium and platelets were still distinguished from normal by VIII R:AG deficiency. When incisions in excised porcine skin ("in vitro" bleeding time) were perfused with blood and plasma fractions, haemostasis occurred when plasmatic Willebrand factor exceeded 30 U/dl whether the skin or platelets came from normal or from von Willebrand pigs. The platelet plug occluding the skin incision contained VIII R:AG by immunofluorescence. Willebrand factor appears to coat surfaces and to serve as a platelet attachment protein. These bleeder pigs are resistant to atherosclerosis. If platelets are involved in early atherosclerotic lesions, the role of Willebrand factor in platelet - blood vessel interaction may be important.     

Plasma exchange and human factor VIII concentrate in managing haemophilia A with factor VIII inhibitors.

Plasma exchanges were combined with human factor VIII concentrate therapy in the treatment of major bleeding episodes in five patients with haemophilia A and factor VIII inhibitors. All patients had a good clinical response to combined treatment. Inhibitor levels showed satisfactory falls before rapid secondary increases of inhibitor levels took place. A sixth patient with von Willebrand''s disease and a factor VIII clotting activity inhibitor was successfully prepared for operation using plasma exchange. Postoperative haemostasis and healing were normal. In two patients the plasma exchanges were relatively more effective than the administered human factor VIII in reducing the levels of factor VIII inhibitor. Combined plasma exchange and human factor VIII treatment may offer a rapidly effective means of reducing factor VIII inhibitor levels in this group of patients, together with significant saving of costs.     

An inhibitor in the Willebrand-Jürgens syndrome and its effect on the factor VIII molecule properties]

In a patient with a clinically serious Willebrand-Jürgen's syndrome an inhibitor appeared at the age of 1 1/2 years, which, contrary to inhibitors in haemophilia A, was directed against all properties of factor VIII molecule. In a quantitative test the height of the inhibitor level to factor VIIIag was determined. Administrations of plasma concentrate resulted in a titre increase which could not be suppressed even by an immunosuppressive therapy with cyclophosphamide. After a long break in the substitution a severe bleeding could be successfully treated and the substitution effect for factor VIIIc, factor VIIIag and Ristocetin co-factor could be identified for several days.     

Donor selection for the production of factor XIII preparations

For the purpose of establishing a programme of plasmapheresis for manufacturing preparations containing factor VIII, investigations were made to check the expediency of selecting donors. Factor VIII activity (Factor VIII: C) was determined in a total of 165 donors of plasmapheresis. Dependencies of factor-VIII: C on blood group, age and influence of smoking were examined. Depending on the blood group (0 less than A, B, AB) the results found by us showed significant differences in factor-VIII: C. The results are discussed and compared with data in literature. A selection of donors is considered to be reasonable. Increases in gaining factor-VIII are possible by making a pre-selection according to blood groups or better by exact testing respectively.     

The clinical significance of different routes of desmopressin (DDAVP) administration in various bleeding disorders

In our study, we wanted to evaluate the clinical effect of s.c. DDAVP on haemostasis in different kinds of bleeding disorders. A total of 109 patients was treated with DDAVP s.c. at a dose of 0.4 microgram/Kg body weight. An effect of DDAVP on F VIII modalities was found after s.c. injection in all patients, but in comparison to i.v. DDAVP, the effect seems to be somewhat less. From our data, the patients blood groups do not influence the amount of F VIII modalities. Furthermore, the s.c. injection of DDAVP was found to be effective in patients with disorders of primary haemostasis. A number of operations was performed under the use of s.c. DDAVP in 24 patients with different kinds of bleeding disorders, in none of them, bleeding complications occurred.     

The treatment of haemophilia A inhibitor with high dose intravenous immunoglobulin

In patients with Haemophilia A, the development of inhibitor is a life-threatening complication of treatment. These patients are at high risk for dangerous bleeding as a result of this acquired resistance to human Factor VIII concentrate. Although treatment of bleeding complications has been improved with the introduction of an activated prothrombin complex preparation, therapy remains unsatisfactory. Two patients with Haemophilia A inhibitor were treated with high dose intravenous immunoglobulin in the expectation of an immunosuppressive effect. A rise in the antibody titre at the same time as the administration of factor VIII concentrate showed that this treatment was ineffective in patients with Haemophilia A inhibitor.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Desmopressin and bleeding time in patients with cirrhosis.

Desmopressin acetate 0.3 microgram/kg was given intravenously to nine patients with chronic liver disease and to a further six such patients in a double blind controlled study versus placebo. Desmopressin acetate significantly shortened the bleeding time compared with basal values in both groups and compared with placebo. There was also a significant decrease in partial thromboplastin time (but not prothrombin time) and significant increases in factor VIII and its components, von Willebrand factor and ristocetin cofactor activity, but not in factors VII, IX, X, XI, or XII. Increased fibrinolysis could be blocked by concomitant administration of tranexamic acid. No important side effects were seen. The multimer pattern of von Willebrand factor was studied for the first time in chronic liver disease. It was normal, but after administration of desmopressin acetate the percentage of multimers of higher molecular weight increased significantly. This may be an important mechanism in the shortening of the bleeding time in cirrhosis, as has been shown in uraemia and other conditions after administration of desmopressin acetate. Desmopressin acetate may be useful in correcting defects in primary haemostasis in chronic liver disease.     

Developing rDNA products for treatment of hemophilia A.

Current therapy for hemophilia A requires frequent infusion of plasma-derived human factor VIII with the associated drawbacks of potential viral contamination, high cost and limited plasma availability. Factor VIII replacement therapy has been improved through increased knowledge of molecular mechanisms regulating blood coagulation, derived largely from the isolation of the factor VIII gene and its expression in mammalian cells. Homogeneous pure preparations of factor VIII--the largest, most complex protein pharmaceutical produced to date through recombinant DNA technology--can now be produced for successful treatment of hemophilia A.     

Paradoxical bleeding as a complication of the treatment of hemophilia with factor VIII and factor IX preparations]

Paradoxical bleedings are complications occurring under replacement therapy in haemophiliacs by disturbancies of the primary haemostasis. They have been observed during treatment with factor-VIII- and prothrombin-complex concentrates of long duration and in high dosage. Clinical complications, for example delayed wound healing as well as spontaneous bleedings into the skin and from the mucous membranes, have been observed in one quarter of haemophiliacs under substitution therapy. In one third of these patients pathological parameters of primary haemostasis (prolonged bleeding time, reduced retention, retraction, ADP- and collagen-induced aggregation and the platelet factor 3 release) were found out. The following mechanisms or substances may be the cause for these disturbancies: 1. fibrinogen and factor-VIII split products 2. high content of proteins predominantly fibrinogen and factor-VIII-related antigen 3. antigen-antibody reactions 4. development of inhibitors against the Willebrand factor. For treatment of the paradoxical bleedings freshly prepared cryoprecipitate, prednison and Etamsylatum have been used.     

Controlled substitution with blood products in hemostatic disorders

It is essential for an efficient substitution to define the nature of the defect as good as possible. Simple screening tests allow a rapid classification. Prophylactic substitution is recommended in potentially reversible defects (bone marrow aplasia in connection with leukaemia treatment) and/or imminent bleeding (eventually complicated by additional risk factors). If bleeding cannot be stopped surgically therapeutic substitution is indicated. In case of bone marrow failure, a substitution may be particularly promising. In presence of an increased peripheral platelet destruction (disseminated intravascular coagulation, antithrombocytic antibodies) treatment of the basic disease is mandatory. Combined hemostatic defects can be influenced by fresh frozen plasma (FFP). Fresh whole blood (not older than 48 hours) may be considered in cases of thrombocytopenia and concomitant anemia. For isolated defects (e.g. hemophilias with or without antibodies, congenital afibrinogenemia, lack of factor XIII) special preparations are at hand. The clinical effect of substitution depends on the specific activity of the preparation, on the volume of expansion in the recipient and on other pharmacokinetic factors. Hepatitis and antibody-production may be considered as particularly grave side-effects.     

Clinical use of blood,blood components and blood products.

The goal of modern transfusion therapy is to provide appropriate replacement therapy with blood components as opposed to whole blood for patients with specific hematologic deficiencies. A prerequisite of component therapy is, therefore, correct identification of the deficiency. Appropriate use of components avoids many of the hazards associated with the use of whole blood, and at the same time makes maximal use of this valuable resource. Blood components separated from whole blood soon after collection and appropriately stored can, in combination, provide all the factors present in fresh whole blood. Red cell concentrates prepared from multiple packs have a hematocrit of approximately 70%. They may be stored for up to 3 weeks at 4 degrees C and are recommended for most situations requiring red cell transfusions. Platelet concentrates, which can be stored for up to 72 hours at 22 degrees C, may be used for thrombocytopenic patients. Fresh frozen plasma, stored plasma, cryoprecipitated factor VIII, factor VIII concentrate and factor IX complex concentrate are available for the proper treatment of patients with hemorrhagic disorders due to coagulation factor deficiencies. Similarly, albumin and immune serum globulin are available for their oncotic and antibody properties respectively. Thus, the availability and appropriate use of the various blood products allows not only optimal transfusion therapy for each patient, but also fuller utilization of national blood resources.     

Safety of human blood products: inactivation of retroviruses by heat treatment at 60 degrees C

Acquired immune deficiency syndrome (AIDS) can be transferred to patients by blood transfusions or human blood preparations, such as cryoprecipitates or factor VIII concentrates. Retroviruses have been discussed as infectious AIDS agents and more recently human T-lymphotropic retroviruses designated as HTLV type III and LAV (lymphadenopathy-associated virus) have been isolated from AIDS patients. Whether heat treatment at 60 degrees C (pasteurization) of liquid human plasma protein preparations inactivates retroviruses was therefore investigated. Pasteurization had already been included in the routine manufacturing process of human plasma protein preparations in order to guarantee safety with regard to hepatitis B. Since high titer preparations of human retroviruses were not available, heat inactivation was studied using Rous sarcoma virus added to the various plasma protein preparations tested. This retrovirus which was obtained in preparations of 6.0 log10 FFU/ml was shown to be at least as heat stable as two mammalian retroviruses studied, i.e., feline and simian sarcoma virus. In all of eight different plasma protein preparations tested, Rous sarcoma virus was completely inactivated after a heat treatment lasting no longer than 4 hr. It is thus concluded that pasteurization of liquid plasma protein preparations at 60 degrees C over a period of 10 hr must confer safety to these products with respect to AIDS, provided that the AIDS agents are retroviruses of comparable heat stability as Rous sarcoma virus and the mammalian retroviruses tested.     

Spontaneous postpartum factor VIII inhibitor development with bleeding into the face and neck

The coagulopathy caused by factor VIII inhibitor development can cause life-threatening bleeding that may be difficult to manage. Control of the bleeding, medically and surgically, and normalization of factor VIII and inhibitor levels are the goals of treatment; several modes of therapy are available. A case of spontaneous post-partum development of factor VIII inhibitors was presented and discussed.     

Possibilities to elevate the factor VIII yield

By referring initially to remarks about the structure and function of the coagulation factor VIII and about the manufacture and demand for preparations for the substitution treatment in patients affected with haemophilia A, possibilities are presented how to increase the collection of factor VIII by applying intensive measures. These involve the impact on the basic material (including donors) as well as process variables within the range of plasma collection and process technique. On the basis of own research results and data from literature the following measures can be introduced and evaluated as far as their effect on the collection of factor VIII is concerned: Donor testing, selection: increased by 25% approximately Plasmapheresis, blood bags: (prerequisite for certain technological measures) Thawing technique: increase by 20-30% approximately (thaw siphon) Citrate-free anticoagulant: increase by 30% approximately (e.g. heparin) Donor conditioning: increased by 200-400% approximately (DDAVP) The establishment of possible and reasonable combinations of measures can contribute to intensify the collection of factor VIII. The advantages to be expected are mentioned. The level of gene-technological collection of factor VIII is dealt with prospectively.     

Problems of multiple transfusions

The use of blood infusion in large amounts is increasing sharply. Increased knowledge of blood group antigens has alerted physicians to the possible hazards of hemolytic reactions to subgroups that must be eliminated by proper cross-matching techniques. Multiple transfusions of preserved blood often defeat their purpose in control of bleeding, for thrombocytopenia is enhanced. Careful selection of blood or preparations of plasma concentrates offer increased protection to the recipient.Plastic bag equipment increases the yield of viable platelets and keeps blood in usable condition for longer periods of storage. The use of multiple transfusions has complicated the selection of preserved blood to control pigment metabolism.     

Improved monoclonal antibody tumor/background ratios with exchange transfusions

Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both ¹³¹I-5G6.4 specific and ¹²⁵I-UPC-10 non-specific MoAb. Twenty-four hours later 65–80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P < 0.05) decreased normal tissue activities of ¹³¹I (except for muscle) by 63–85%. while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1–0.8 to 2.3–6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.     

Platelet adhesiveness and aggregation in combined factor V and factor VIII deficiency and in combined factor VII and factor VIII deficiency.

Platelet aggregation and adhesiveness were studied in 3 patients with combined factor V and factor VIII deficiency and in 3 patients with combined factor VII and factor VIII deficiency. The first three patients belonged to three different kindreds whereas the second group belonged to the same kindred. Serotonin C14 uptake and release was also found to be normal in these patients. These studies indicate that platelet function is normal in combined defects of factor VIII. These findings were in agreement with the presence of a normal bleeding time and a normal factor VIII antigen level in all these patients.     

Gene therapy strategies for hemophilia: benefits versus risks

Hemophilia is an inherited bleeding disorder caused by a deficiency of functional clotting factors VIII or IX in the blood plasma. The drawbacks of the classical protein substitution therapy fueled interest in alternative treatments by gene therapy. Hemophilia has been recognized as an ideal target disease for gene therapy because a relatively modest increase in clotting factor levels can result in a significant therapeutic benefit. Consequently, introducing a functional FVIII or FIX gene copy into the appropriate target cells could ultimately provide a cure for hemophilic patients. Several cell types have been explored for hemophilia gene therapy, including hepatocytes, muscle, endothelial and hematopoietic cells. Both nonviral and viral vectors have been considered for the development of hemophilia gene therapy, including transposons, γ‐retroviral, lentiviral, adenoviral and adeno‐associated viral vectors. Several of these strategies have resulted in stable correction of the bleeding diathesis in hemophilia A and B murine as well as canine models, paving the way towards clinical trials. Although clotting factor expression has been detected in hemophilic patients treated by gene therapy, the challenge now lies in obtaining prolonged therapeutic FVIII or FIX levels in these patients. This review highlights the benefits and potential risks of the different gene therapy strategies for hemophilia that have been developed. Copyright © 2010 John Wiley & Sons, Ltd.     

Lower inhibitor development in hemophilia A mice following administration of recombinant factor VIII-O-phospho-L-serine complex

Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations.