Hemostasis disorders after transfusions

Disturbances of haemostasis caused immunologically and non-immunologically were observed after transfusion of blood and blood derivatives. Transfusion of heparin blood increased the bleeding susceptibility only in case of pre-existing high-degree defects of haemostasis or if they were performed as massive or exchange transfusions. Massive transfusions with blood stored for a long time will induce complex defects. Under intensive substitution therapy of haemophilia A the so-called paradoxical bleeding will occur in spite of a high factor VIII level. These bleedings are supposed to be disturbances of the thrombocyte function and are caused by fibrin(ogen) derivatives. Post-transfusional thrombocytopenias may be brought to remission by repeated plasmapheresis. Factor specific inhibitory bodies will appear after substitution in a small percentage of haemophilic patients. 5 to 7 days after the onset of therapy an anamnestic reaction can be observed as a titre increase by leaps. Usually, the inhibitory titre will decrease to a mostly low basal value in the course of three to five months. The therapy with cyclophosphamide simultaneously started with the substitution will more frequently prevent the anamnestic reaction or reduce it. Titres with more than 5 units cannot be overcome at the beginning even by higher concentrations of preparations. The substitution therapy should be preceded by exchange transfusions or plasmapheresis of up to 25 units. With still higher titres only procedures of inhibitor-bypassing are possible with factor VIII preparations of animal origin or better with activated prothrombin complex preparations, such as FEIBA. Recent reports give evidence that permanent substitution with factor VIII concentrates at a highest dosage can eliminate the production of inhibitors completely.     

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.     

Blood coagulation and fibrinolysis in blood donors after plasmapheresis]

Authors were interested in blood coagulation proteins and fibrinolysis in blood donors following several plasmaphereses. This interest was related to the occurrence of thrombo-embolic and hemorrhagic complications in these subjects. Blood morphology, serum protein, blood coagulation and fibrinolysis have been examined in 40 healthy blood donors, aged between 19 and 46 years, who gave blood plasma by plasmapheresis technique for 1-59 times. Results did not show any significant changes in blood morphology and serum protein levels prior to and after consecutive plasmaphereses. No significant decrease in blood coagulation proteins and fibrinolysis has been noted. However, a significant increase in factors VIII and IX activities was noted in several blood donors, who underwent the largest number of plasmaphereses. It may predispose these donors to thrombo-embolic complications.     

Increased factor VIII/vWf levels in patients with reduced platelet number

Factor VIII/von Willebrand factor (VIII/vWf) related properties were studied in twenty six patients with thrombocytopenia. Fifteen patients were affected by idiopathic thrombocytopenic purpura (ITP) and 11 patients by thrombocytopenia of a different nature or non-ITP (n-ITP). All patients showed an enhancement of platelet associated IgG (PAIgG). A significant increase of factor VIII ristocetin cofactor (VIII R: RCoF) and factor VIII related antigen (VIII R:Ag) was found in ITP patients while normal values were observed for factor VIII coagulant (VIII:C). All factor VIII/vWf components, on the contrary, were increased in n-ITP group with a prevalence of VIII R:RCoF as observed in ITP group even though with lower mean values. Multimeric analysis of VIII/vWf demonstrated a higher concentration of all multimeric components, with major representation of higher molecular weight multimers (HMWM) in patients of both groups. Two patients were studied before and after improvement in platelet count. A decrease of vWf related properties (VIII R:RCoF and VIII R:Ag) concomitant with the increase in platelet count was found. In n-ITP patients a statistical correlation between VIII R:RCoF and PAIgG was also observed while no correlation was found between other factor VIII/vWf components and PAIgG both in ITP and n-ITP patients.     

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.     

Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex

Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca(2+) and two Cu(2+) ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.     

Factor VIII activity and factor VIII-associated antigen in healthy newborns and in newborns with stressing perinatal factors

Factor VIII coagulation activity (VIII:C) and factor VIII associated antigen (VIII:AGN) were determined in healthy newborns and in children with charging perinatal factors ("risk children"). VIII:C values of healthy newborns may be compared with those of grown-ups with normal coagulation. Risk children have somewhat higher values than newborns, the difference, however, being statistically not significant. The concentration of VIII:AGN is clearly increased in both groups on the first day of life. Moreover, VIII:AGN is being eliminated more slowly in risk children. The increased VIII:AGN concentrations are considered as a sequel of stress conditions caused by birth, whereas the discrepancy between VIII:C and VIII:AGN is due to a thrombin effect.     

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.     

A novel approach in potential anticoagulants from peptides epitope 558–565 of A2 subunit of factor VIII

Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser⁵⁵⁸–Gln⁵⁶⁵ region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa–FIXa interaction. Based on this, a series of linear peptides, analogs of the 558–565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.     

The relationship of biological and immunological activities of factor VIII

Factor VIII is an essential blood clotting factor which consists of two protein moieties, each with distinct biological functions and antigenic determinants. The immunological markers were originally seen as indicators of the biological activities; however this view has been increasingly challenged. We have investigated the biological and immunological properties of Factor VIII to clarify these relationships. Plasma stored at room temperature for 21 days lost biological activity, but retained immunological activity: The procoagulant activity was reduced to 35% and the ristocetin cofactor activity to 75.4% of their original levels; but the reactivities of both procoagulant antigen and Factor VIII related antigen were maintained. A dissociation of activities was also demonstrated in serum, in which the procoagulant activity was 10% and the procoagulant antigen 72% of corresponding plasma values. These results indicate that the antigenic reactivities are not appropriate markers for Factor VIII biological activity.     

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.     

Comparison of thawing of plasma by microwave or water bath: preliminary longitudinal biological study of hemostatic parameters

Exploration of haemostasis was performed on plasmas thawed in an experimental microwave oven comparatively to a 37 degrees C water bath. Factor VIII:R:Ag, procoagulant and antigenic fibrinogen, and Fg:C/Fg:Ag ratio were found to be significantly, slightly decreased with microwave thawing. Factor VIII:C and VIII:C/VIII:R:Ag ratio were found to be increased with microwaves. Antigenic fractions were decreased because of partial precipitation. In addition, Fibrinogen slightly lost its activity; on the contrary, factor VIIIC was activated by micro-waves. All this allows to select parameters for new experimental microwave ovens development.     

Factor VIII and factor IX in a twin population. Evidence for a major effect of ABO locus on factor VIII level.

In order to establish the relative importance of genetic factors on the variation in plasma concentration of coagulation factors VIII and IX, these parameters were determined in 74 monozygotic and 84 like-sexed dizygotic twin pairs. The twins belonged to two age groups: 33-39 years and 57-62 years. Factor VIII was determined as factor VIII coagulant antigen (VIIICAg) and as factor VIII-related antigen (VIIIRAg). Factor IX was determined as factor IX antigen (IXAg). A higher value for each coagulation factor was found in the older-age group compared to the younger group, whereas no difference was found between the sexes. A significant correlation was found between values for VIIIRAg and VIIICAg (r = .56). For VIIICAg, it could be demonstrated that the age effect was secondary to the age effect on VIIIRAg. The concentration of VIIICAg and VIIIRAg varied among ABO blood types, being lowest in type O individuals, higher in A2 individuals, and highest in A1 and B individuals. The effect of the ABO locus on VIIICAg was secondary to an effect on VIIIRAg. Analysis of variance revealed a significant genetic influence on the variance of VIIICAg and VIIIRAg with a heritability estimate of .57 for VIIICAg and .66 for VIIIRAg. This is in agreement with a previous hypothesis of an effect of several autosomal genes on factor VIII concentration. Thirty percent of the genetic variance of VIIIRAg was due to the effect of ABO blood type. The ABO locus is therefore a major locus for the determination of factor VIII concentration. No significant genetic effect on the variation in plasma concentration of IXAg could be detected.     

Exercise-induced changes of Factor VIII complex in healthy subjects and in type-I diabetics: relation between growth hormone and Von Willebrand Factor increments

This work aims to study the exercise-induced changes of Factor VIII Complex both in healthy subjects and in type I diabetics without vascular complications, and to investigate the possible relations between growth hormone and Von Willebrand's Factor response to exercise. Results show that maximal exercise performed by cycle ergometer causes a significant increment of the procoagulant subunit (VIII:C) and of Von Willebrand Factor (VIII:RiCoF) both in healthy controls and in type I diabetics, whereas a slight increment of Factor VIII-Related antigen (VIIIR:Ag) is observed only in diabetics. The shape of the mean GH response to exercise parallels the one of Von Willebrand's Factor: however, the presence of VIII:RiCoF increments also in GH non-responders supports the conclusion that growth hormone is not the only factor involved in the regulation of Von Willebrand's Factor exercise-induced increase.     

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.     

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.     

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.     

Expression of blood clotting factor VIII:C gene in capillary endothelial cells.

The essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.     

Intranasal desmopressin (DDAVP) by spray in mild hemophilia A and von Willebrand's disease type I

Desmopressin acetate (1-desamino-8-D-arginine vasopressin, DDAVP) has mostly been given by the parenteral route for the treatment of mild hemophilia A and von Willebrand's disease type I. In the present study the hemostatic effects of desmopressin acetate administered intranasally by spray in a dose of 300 micrograms and intravenously 0.3-0.4 micrograms/kg were assessed and compared in 8 patients with hemophilia A and 22 patients with von Willebrand's disease type I. A bioequivalent response to intravenous and intranasal desmopressin acetate was found in Factor VIII coagulant activity (VIII:C) in the hemophilia patients. In the von Willebrand patients, an equivalent shortening of the bleeding time was seen after the two modes of administration, even though intravenous injection gave a higher increase in plasma levels of VIII:C and vWF:Ag. In five patients with von Willebrand's disease the duration of the spray effect on VIII:C and vWF:Ag was followed for 24 h. After 12 h the mean level of VIII:C was 1.4, and of vWF:Ag 1.5, times the basal level. The findings suggest that the spray can be recommended for home or prophylactic treatment of patients with mild hemophilia A and von Willebrand's disease.