The thrombolytic therapy is usually associated with bleeding risks due to a systemic fibrinogenolysis. We investigated whether it is possible to block fibrinogenolysis without impairing the lytic activity of fibrin-bound plasmin molecules. The present data show that at increased a2-antiplasmin levels as high as 6-fold the normal concentration fibrinolysis was not impaired, while the haemostatic parameters as aPTT, TT and the fibrinogen concentration remained normal. Essentially similar results were observed with all plasminogen activators (streptokinase, urokinase and a t-PA mutant) assayed. The use of a2-macroglobulin leads to identical observations. With aprotinin, however, a complete inhibition of the lysis was observed. The data presented here show that it is possible with the combined use of a plasminogen activator and a2-antiplasmin to reach at least in vitro a normal fibrinolysis without impairment of the haemostatic parameters.

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 (>2xSD 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 (r(s)=0.50, P<0.001), serum glutamic oxalacetic transaminase (r(s)=0.54, P0.001), parasite count (r(s)=0.62, P<0.001), blood urea nitrogen (r(s)=0.54, P<0.001) and inversely with antithrombin III activity (r(s)=0.54, P<0.001) and the platelet count (r(s)=0.58, P<0.001). Polymorphonuclear leucocyte activation may contribute to the pathogenesis of severe malaria.

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.

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.

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.

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.

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.

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.

Erste Hinweise auf einen Zusammenhang zwischen der Reaktionsfähigkeit der reaktiven Fibrinolyse und der Nierenfunktion ergaben sich aus einer früheren Untersuchung an nierentransplantierten Patienten. Dabei hatten sich ein Absinken des Plasminogenspiegels und ein ungünstig hoher Quotient α2-Antiplasmin/Plasmi-nogen bei Transplantatabstoßung aller Schweregrade gefunden. Da bei schweren Infektionen eine akute Niereninsuffizienz bis hin zur kompletten Anurie auftreten kann, wurden 82 Patienten mit erheblichen bakteriellen Infektionen untersucht. Es zeigte sich eine signifikante inverse Beziehung zwischen Plasminogen und Serumkra-tinin während des gesamten Krankheitsverlaufes. Daraufhin wurde bei 11 Patienten mit akutem Nierenversagen in Ergänzung zur Substitution von Antithrombin III und fresh frozen plasma ein Plasminogenkonzentrat verabreicht. Bei 7 dieser Patienten war ein signifikanter Anstieg der Diurese in engem zeitlichen Zusammenhang mit dem Beginn der Plasminogensubstitution zu verzeichnen.

The possible occurance of up to now unknown human pathogenic viruses makes it necessary to reconsider thestrategies for the preparation of each plasma protein concentrate used for substitution therapy. Even up to now safe products may be contaminated by infectious particles in the near future. Hence, each procedure for the preparation of such proteins should critically be checked for the elimination of contaminating proteins and should contain an inactivation step for pathogenic agents. Since about 4 decades the pasteurization of albumin in presence of stabilizers has been found to be a safe and mild method to inactivate infectious agents. Using the stabilizersglycine and sucrose, we have now been able to pasteurize antithrombin III without the usual changes in molecular properties. The product is more than 98 % pure and contains only traces of contaminating proteins. The pasteurization does not alter the electrophoretic behavior of the molecule in membrane electrophoresis (ME) and polyacrylamidgel electrophoresis (PAGE). The reactivity with heparin is nearly unchanged as tested by heparin cofactor activity and twodimensional immunoelectrophoresis. No neoantigen formation due to the pasteurization could be detected. Using model viruses the efficacy of the pasteurization step has been tested. The following results have been obtained: HIV ≥106.7 (1), CMV ≥ 104.5 (2), HSV ≥106.8 (4), Poliomyelitis virus ≥106.9 (4); the number in brackets represent the time in hours necessary to totally inactivate the given virus titer.

Fibrinogen is available for substitution in afibrinogenaemic patientssince about 4 decades. However, it soon turned out that those concentrates bear a high risk of transmitting serum hepatitis. Over many years it was not possible to produce safe concentrates of fibrinogen. Hence, the therapy with this protein was limited to vital indications. We have now succeeded to stabilize fibrinogen inaqueous solution for pasteurization over 10 to 20 hours at 60°C. The efficacy of the virus inactivation was tested using various animal viruses. Following results were obtained. Tests in chimpanzees for hepatitis B safety revealed that this procedure inactivates and eliminates 105.2 CID50 of hepatitis B virus; HIV experiments are going on. By immunizing rabbits with the pasteurized fibrinogen and absorption of the antiserum obtained with the unpasteurized product, an exposition of neoantigens during heating in aqueous solution could be excluded. This result could be further confirmed using passive cutaneous anaphylaxis. The coagulability of the pasteurized fibrinogen is unchanged, compared to not pasteurized material. It iseasily soluble and can be used for both, i. v. infusion and as a tissue adhesive. The clinical tolerability is very good.

Isolation and purification of antithrombin III (AT III) by affinity chromatography on immobilized heparin is a standard method for the large scale preparation of this protein from human or animal plasma. Hence, after AT III became available by gentechnological methods, we tried to adapt this procedure for the isolation of AT III from supernatants of mammalian- and yeast-cells. Indeed, it was possible to use this method also for the isolation of the recombinant gene products. Since, however, the cell growth media contain heterologous protein or peptide mixtures like fetal calf serum, the method had to be improved to avoid the adsorption of non human proteins or peptides. We are now able to purify AT III from CHO-cell-superna-tants to more than 95 % purity. The characterization of this AT III-product by double immuno diffusion revealed that it is immunologically totally identical with the authentic material from plasma. AT III antigen content, progressive inhibitor activity and heparin cofactor activity compare very well in the final product; hence, it is totally active compared to AT III from plasma. In polyacrylamidegel electrophoresis most of the material migrated differently to the authentic material showing 9 bands in equal distance to each other, instead four in the At III from plasma. After degradation with sialinidase from both AT III preparations identical cleavage products were obtained migrating predominantly as a single band. Hence, the electrophoretic heterogeneity seems to be due to a different degree of sialinyla-tion of the products.

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.