Therapeutic factor XIII preparations and perspectives for recombinant factor XIII.

With the increasing availability of human plasma this source was used to substitute for the production of factor XIII concentrate from placenta. Prior to changing the source material, the virus safety in accordance with the Committee for Proprietary Medicinal Products (CPMP) guidelines and the half-life were investigated. Concerning the virus safety, the following cumulative log 10 clearance factors were obtained: human immunodeficiency virus (HIV) > or = 18.9, herpes simplex virus (HSV-1) > or = 21.5, polio > or = 19.1, bovine viral diarrhea virus (BVDV) > or = 13.3. Half-life studies of factor XIII from plasma in comparison with factor XIII from placenta were done in rabbits by determination of the antigen and in patients with congenital factor XIII deficiency by determination of the activity and antigen. In rabbits, the terminal half-life of the antigen was 78.2 hours for factor XIII from placenta and 87.0 hours for factor XIII from plasma. In patients the half-lives were similar: 9.2 days for activity and antigen of factor XIII from plasma and 8.5 days (activity) versus 9.4 days (antigen) for the placenta-derived factor XIII. Some further clinical studies with factor XIII concentrates are also reviewed. Newer developments concerning recombinant factor XIII, its expression, characterization, and properties are summarized. Concerning the physicochemical data, the behavior in plasma was characterized by the formation of high-molecular-weight complexes, and first in vivo results, the recombinant factor XIII product was comparable to the naturally occurring material.

Activity of coagulation and fibrinolysis parameters in animals.

Using diagnostics for the determination of clotting factors and fibrinolytic parameters in human plasma, samples from rat, guinea pig, rabbit, cat, dog, sheep, cattle, horse, pig, and monkey were analysed. The human system was employed even for standard curves and controls. Results obtained in this way are relative values in relation to pooled fresh human plasma of healthy donors which is defined to contain 100% of the norm or 1 unit of each factor per 1 ml. Under these conditions, marked differences between the human clotting system and those of different animal species appear. Thus, rabbits have an about 50 times higher activity of factor V than humans and guinea pigs only 6% of the factor VII activity of human plasma. Plasma levels of fibrinogen, alpha 2-antiplasmin and antithrombin III in plasma samples of the investigated animal species are in the range of human plasma. Differences in the plasma level of single factors as compared to human plasma are reflected by clotting times of the screening tests. For the determination of plasminogen, streptokinase was used as activator of the fibrinolytic system. Hence, the results obtained by this method merely reflect the activatability of the animal plasminogen in comparison to the human system, however, do not allow statements concerning the real plasminogen content of the plasma sample from the animal species. For pharmacological investigations a proper selection of the animal species is important to prevent wrong conclusions.

Polymorphonuclear leucocyte elastase in Plasmodium falciparum malaria.

Sixty-one patients with falciparum malaria were studied prospectively to determine the plasma concentrations of the lysosomal proteinase, polymorphonuclear leucocyte elastase (PMN-elastase) and their relationship to disease severity. The patients were divided into 3 groups; severe (parasitaemia > 5%) or vital organ dysfunction (n = 23), moderate (parasitaemia 1%-5% without complications) (n = 15), and mild (parasitaemia < 1%) (n = 23). The mean plasma PMN-elastase level in 10 healthy Thai volunteers was 49.5 (SD = 21.6) ng/ml (range 33-65 ng/ml). Plasma PMN-elastase concentrations on admission were elevated (> 2 x SD above normal) in all patients with severe malaria and were above 100 ng/ml in 86.6% and 65% of the moderately severe and mild patients respectively. PMN-elastase levels during the first 3 hospital days were significantly higher in severe malaria compared with the other 2 groups (P = < 0.001-0.013). The levels decreased as the patients became afebrile and aparasitaemic. Admission plasma concentrations of PMN-elastase correlated directly with bilirubin (rs = 0.50, P < 0.001), serum glutamic oxalacetic transaminase (rs = 0.54, P0.001), parasite count (rs = 0.62, P < 0.001), blood urea nitrogen (rs = 0.54, P < 0.001) and inversely with antithrombin III activity (rs = 0.54, P < 0.001) and the platelet count (rs = 0.58, P < 0.001). Polymorphonuclear leucocyte activation may contribute to the pathogenesis of severe malaria.

Biochemical and histological findings on the effect of fibronectin in rabbits with experimental corneal disorders.

The effects of purified plasma fibronectin (FN) in corneal disorders were investigated. Histological findings, the levels of ascorbic acid (AA) and glutathione (GSH) in tear fluids were observed sequentially in rabbits with experimental corneal damage and the degree of corneal lesions was observed. The results indicated that healing of the tissue damage was more promoted in the FN group than in the controls and it was suggested that FN promoted the healing of corneal disorders.

Crystallization of blood coagulation factor XIII by an automated procedure.

Both recombinant blood coagulation factor XIII alpha-chain and factor XIII isolated from human placenta have been crystallized using a novel robotic system for the automatic screening of crystallization conditions. The monoclinic and orthorhombic crystals obtained are suitable for X-ray analysis.

Characterization of recombinant factor XIIIa produced in Saccharomyces cerevisiae.

Recombinant factor XIIIa (FXIIIa), produced in Saccharomyces cerevisiae, was recovered as a fully active cytosolic component and rigorously compared to natural F XIIIa from human placenta with respect to physicochemical and functional properties. Identical parameters were found in SDS polyacrylamide gel electrophoresis, analytical ultracentrifugation and HPLC gel filtration, and all spectral characteristics including derivative UV absorbance, fluorescence and circular dichroism were identical. Similarly, the interaction of both proteins with polyclonal antibodies directed against the entire FXIIIa or its N-terminal 4 kD activation peptide were identical. Furthermore, thrombin cleavage and fibrin cross-linking showed indistinguishable patterns. The only difference we observed was with respect to endgroup analysis. The recombinant protein is homogeneous, whereas placental FXIIIa shows multiple electrophoretic bands caused by microheterogeneity in the C-terminal part of the protein.

Characterization of recombinant human antithrombin III synthesized in Chinese hamster ovary cells.

Biochemical and physiochemical properties of recombinant human antithrombin III were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human plasma when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Comparison of the NH2-terminal sequences of recombinant and human plasma-derived antithrombin III showed that on synthesis and secretion of the recombinant protein from Chinese hamster ovary cells the signal peptide is correctly cleaved by the corresponding endoplasmic signal peptidase. The recombinant antithrombin III has identical properties in heparin binding and biological activities as determined in vitro by two-dimensional immunoelectrophoresis, progressive inhibitor, and heparin cofactor assays. Analysis of the carbohydrate portion of recombinant antithrombin III synthesized in Chinese hamster ovary cells revealed glycosylation of the complex type. Characterization of the oligosaccharide chains present in the recombinant protein reveals three major fractions, A (20%), B (60%), and C (20%). Fraction A contains tri- and tetraantennary complex-type oligosaccharides, fraction B contains biantennary oligosaccharides, and fraction C partially truncated biantennary structures. Pharmacokinetic studies with recombinant and plasma-derived antithrombin III in rabbits showed that the clearance behavior of both proteins is very similar and can be described by a double exponential decrease with almost identical kinetic parameters.

Activation of the coagulation cascade in falciparum malaria.

The incidence and progression of coagulation abnormalities were studied in 52 patients with acute falciparum malaria. The patients were prospectively divided into 3 groups; severe (parasitaemia greater than or equal to 5% or vital organ dysfunction), 12 patients; moderate (parasitaemia 1%- less than 5% without complications), 16 patients; and mild (parasitaemia less than 1%), 24 patients. No case died or developed clinical evidence of disseminated intravascular coagulation. Conventional indices of coagulation (prothrombin time, partial thromboplastin time, fibrinogen, fibrin degradation products) were usually within the normal range but reduced plasma concentrations of antithrombin III (AT-III) levels were noted in all groups, and the incidence was significantly higher in patients with severe and moderate malaria (83% and 81%) compared with the mild group (37%; P less than 0.005). Depletion of AT-III was associated with thrombocytopenia, decreased AT-III activity and elevated plasma concentrations of thrombin-antithrombin III complexes (P less than 0.01), confirming activation of the coagulation cascade and increased clotting factor consumption. AT-III levels returned to normal coincident with clinical improvement. Activation of coagulation is a common and sensitive measure of disease activity in acute falciparum malaria. It is not a specific feature, nor is there evidence to suggest it has a primary pathological role in severe infections.

Reduced fibrinolytic capacity and its restoration by plasminogen substitution in acute renal failure.

A common finding in acute renal failure, particularly if it is caused by septic shock, consists of fibrin deposits in the intrarenal blood vessels. In a study of fibrinolytic parameters in 82 patients with severe bacterial infections, a significant negative correlation between plasminogen plasma concentration and serum creatinine was found. On admission the plasminogen levels were lower than the alpha 2-antiplasmin concentrations, which means a reduction of the fibrinolytic capacity due to a preponderance of the inhibitor. Preliminary experience with a replacement therapy is here reported. In 9 patients with an acute renal failure due to septicaemia or other serious diseases with shock, a substitution with fresh frozen plasma and antithrombin III concentrate was carried out in order to stop disseminated coagulation. A considerable increase of urine excretion was observed in 5 of these patients in close connection with the additional administration of a plasminogen concentrate.

Factor XIII: enzymatic and clinical aspects.

The present knowledge about clotting F XIII is roughly displayed. It is explained why F XIII is not a clotting factor in the strict sense of the word and that its biological function more general is the crosslinking of proteins via a transamidase reaction, the reduction of the permeability of the microvasculature, the stimulation of connective tissue cells, and finally the steering of connective tissue processes involved in wound healing. These effects are demonstrated as well in cell culture studies, animal experiments as by clinical results. Concerning the clinical results with F XIII, its effects on sclerodermia, subarachnoidal hemorrhage, inflammatory bowel diseases, Purpura Schönlein Henoch, and the wound healing are described in more detail.

[Properties and virus safety of a pasteurized antithrombin III concentrate]. / Eigenschaften und Virussicherheit eines pasteurisierten Antithrombin III-Konzentrates.

In the last decade it appeared more than once that blood donations were contaminated by hitherto unknown viruses. Due to this fact, further steps have to be included into the manufacturing procedure of even up to now safe products from human plasma to eliminate potential new risks. A procedure is described which enables the pasteurization of antithrombin III analogous to albumin without major changes in the properties of the molecule. Thus the new product Kybernin HS/P was obtained. The efficacy of the pasteurization step was tested using relevant human pathogenic viruses. The following titers of important viruses could be totally inactivated within the pasteurization time (h) given in brackets HIV: (human immunodeficiency virus) greater than or equal to 10(6.7) (1), CMV (cytomegaly virus) greater than or equal to 10(4.5) (1), HSV (Herpes simplex virus) greater than or equal to 10(6.8) (4), Polio (poliomyelitis virus) greater than or equal to 10(6.9) (4).

Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Recombinant plasmids were constructed that direct the synthesis of human antithrombin III in baker's yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. The signal sequence of antithrombin III was recognized by both yeast species, and antithrombin III was secreted into the medium. When the signal sequence was replaced by a sequence of ten arbitrary amino acids, the product expressed from such a construct stayed inside the cell. Antithrombin III was glycosylated by the baker's and fission yeast and was immunologically identical to antithrombin III isolated from human plasma. Antithrombin III isolated from the culture media of recombinant yeasts was biologically active, as could be shown by progressive inhibitor activity and heparin cofactor activity.

Virus safety of pasteurized factor VIII and factor IX concentrates: study in virgin patients.

In a long term surveillance study hemophilia A and B patients were treated with a Factor VIII HS and Factor IX HS concentrate respectively, both pasteurized by heating in solution: 10 hours at 60 degrees C. None of 31 virgin hemophiliacs treated with Factor VIII HS Behringwerke developed hepatitis B during a follow up between 6 to 60 months. One patient who received 379.280 IU Factor VIII by 977 applications showed a seroconversion after 961 days of treatment. Passive/active immunisation is suggested. 4 patients had moderate elevations of transaminases (less than 120 U/l) without clinical signs of liver disease. 2 patients suffered a non-icteric NA NB-hepatitis two months after synovectomy in the same hospital. 6 virgin hemophilia B patients who had been treated with Factor IX concentrate HS Behringwerke remained serologically negative and did not develop any symptoms indicative of a hepatic disease during a follow up between 11-29 months. The HTLV-III safety of Factor VIII HS and Factor IX HS Behringwerke is presently under investigation by determination of the corresponding antibody.

Impaired fibrinolysis and protein C increase after cadaver kidney transplantation.

Severe rejection crises occurred in 13 out of 20 cadaver kidney recipients; removal of the graft was inevitable in 10 cases. The plasminogen levels were found to be lower (76.5 +/- 2.9%) in the 13 patients with impaired graft acceptance (group B) than in the 7 patients experiencing only moderate rejection (group A, 93.7 +/- 5.2%, p less than 0.01) or the 41 patients with chronic renal disease (group C, 88.2 +/- 2.3%, p less than 0.01). Since alpha 2-antiplasmin was found to be enhanced, the alpha 2-antiplasmin/plasminogen ratio was high (1.56 +/- 0.07) in group B as compared with group A (1.19 +/- 0.05, p less than 0.002) and group C (1.11 +/- 0.04, p less than 0.001). The fibrinolytic capacity, which was assessed by a "reversed fibrin plate" assay, declined significantly after transplantation, and was lower in group B greater than 20 days after grafting. The neoantigen alpha 2-antiplasmin-plasmin complex was not detected in any patient; thus hyperfibrinolysis is unlikely to be responsible for the plasminogen decrease. Protein C rose above preoperative values (group A 117.1 +/- 8.7%; group B 133.7 +/- 9.0%) and reached higher levels in group A (221 +/- 24.1%) than in group B (157.4 +/- 14.8%, p less than 0.05) between days 11 and 20 after transplantation. No constant alterations were found with respect to antithrombin III and fibrinogen. Fibrin deposits, which reduce perfusion, can be found in the vessels of rejected kidney grafts. An impaired fibrinolytic capacity and a subsequent inability to remove these clots may be significant for the outcome of transplantation.

[Disturbances in coagulation, fibrinolytic and complement systems in septic shock]. / Störungen des Gerinnungs-, Fibrinolyse- und Komplementsystems beim septischen Schock.

Plasma levels of selected proteins of coagulation, fibrinolysis and complement from patients with septic shock have been investigated. All values were corrected on the basis of a plasma protein content of 5.5%. Considerable decreases were found in the plasma levels of the contact factors activity. The antigen content in the case of HMW-kininogen remained in the normal range, whereas in the case of prekallikrein it dropped to about 30% of norm. Considering the proteins of the unspecific defense and the complement system, primarily HRG, alpha 2-macroglobulin and C4 showed remarkable decreases. From the changes of the plasma levels of the considered proteins one can conclude that in septic shock primarily the contact phase of coagulation and the alternative pathway of complement are involved. The consumption of F XIII points to a thrombin generation during shock. The marked reduction of the C4-component could be due to an isolated proteolysis of this component, since the classical pathway of complement activation seems not to be involved in the shock events. Serial controls of the plasma levels of the proteins under investigation showed in some patients considerable individual deviations from the general trend. A considerable increase of the F XIII A level appears to be a prognostically favorable result, whereas constantly low values of F XIII and fibronectin without a tendency to normalization or a constant decrease of these proteins often is connected with a fatal outcome.

The clinical significance of alpha 1-antitrypsin-elastase (alpha 1AT-ELP) and alpha 2-antiplasmin-plasmin (alpha 2AP-PL) complexes for the differentiation of coagulation protein turnover: indications for plasma protein substitution in patients with septicaemia.

In inflammation, particularly in septicaemia, complex coagulation disorders may lead to a dangerous haemorrhagic diathesis. The conventional concept for this syndrome called DIC implicates the occurrence of active thrombin in the circulation, which may be followed by hyperfibrinolysis due to plasmin formation. In this study data are presented suggesting an important role for a third proteolytic system, granulocytic elastase. The complexes of plasmin and elastase with their specific inhibitors, alpha 2-antiplasmin-plasmin (alpha 2AP-PI) and alpha 1-antitrypsin-elastase (alpha 1AT-ELP) were determined immunologically. The alpha 1AT-ELP appears mainly in gram-negative septicaemia, particularly in meningococcal disease. The estimation of alpha 2AP-PI and alpha 1AT-ELP, together with a method for the detection of the antithrombin III--thrombin complex which remains to be established, is a suitable tool for for the differential diagnosis of the consumption of coagulation proteins. The assumption that at least three proteolytic systems participate in the development of the haemorrhagic diathesis during inflammation leads to the concept of a broad, comprehensive substitution therapy with e.g. concentrates of AT III, PPSB, or fresh frozen plasma. The aim of this treatment is to replace not only the consumed procoagulatory factors, but also the lacking inhibitors in order to control this "abnormal proteolysis syndrome".

Impaired fibrinolysis after kidney transplantation.

Impaired fibrinolysis is associated with low plasma plasminogen (PLG) and a predominance of its inhibitor alpha 2-antiplasmin (APL). Plasma PLG and APL were determined in 10 recipients who retained their cadaveric graft (A) and 10 patients who lost it immediately after transplantation (B). In group A only five patients had a PLG less than 70 per cent and/or APL/PLG ratios exceeding 1.8, accompanied by severe albeit reversible acute rejection in three of them. In contrast nine of 10 group B patients had low PLG and/or an elevated APL/PLG ratio. Thus impaired fibrinolysis frequently appears to be associated with deterioration of cadaveric graft function.

[Therapy of patients with severe liver insufficiency using antithrombin III and plasma derivatives]. / Therapie von Patienten mit schwerer Leberinsuffizienz mit Antithrombin III und Plasmaderivaten.

The treatment of three patients suffering from carbon tetrachloride intoxication with antithrombin III and plasma derivatives is reported. In these patients an acute liver failure had been proven, characterized by a disturbed protein synthesis and a severe haemostasis defect. The latter manifested itself by a consumption of platelets, clotting enzymes and the inhibitors antithrombin III and alpha 2-antiplasmin. It was due to an intravascular coagulation leading to disturbances in the microcirculation. This often letal circulus vitiosus could be stopped in these patients by treatment with antithrombin III concentrate combined with low dose heparin and fresh frozen plasma. Clinical improvements are discussed on the basis of the pathophysiology of the liver.

On the aggregation of fibrinogen molecules.

Aggregates of human and bovine fibrinogen were obtained under various conditions, ranging from segment-like precipitates to highly ordered paracrystals and crystals. Their biochemical characterization and the interpretation of their banding pattern by electron optical means was attempted. The observed extra vivum aggregation of fibrinogen without thrombin action, i.e. without cleavage of fibrino-peptides A and B is of particular interest with regard to Copley's theory of the endoendothelial fibrin lining. The in vivo formation of such fibrinogen gels is discussed.