Manufacturing of a prothrombin complex concentrate aiming at low thrombogenicity.

The paper describes the production of a prothrombin complex concentrate (PCC) with high virus safety and a well-balanced content of vitamin K-dependent clotting factors and inhibitors. Solid-phase extraction is followed in a second step by optimized anion exchange chromatography using a radial column. A step for virus removal by nanofiltration is introduced in addition to the solvent/detergent step. By speeding up the chromatographic step, the period of time required for production is reduced considerably. The activities of the four vitamin K-dependent clotting factors II, VII, IX and X are in ratios of about 1:1:1:1. Protein C, Protein S, and Protein Z are also present in therapeutically effective concentrations. The product shows no thrombogenicity, in either in vivo nor in vitro models. Clinical investigations show that the PCC is a safe and efficient preparation for the substitutive treatment of FIX or FVII in patients suffering from the respective deficiencies. All bleeding episodes have been efficiently controlled with relatively low doses of the concentrate. The surgical procedures have been conducted without any problems in severely FIX and FVIII deficient patients.

In vitro toxicity of surfactants in U937 cells: cell membrane integrity and mitochondrial function.

Surface-active agents are components of many drugs and cosmetics. In order to examine their cytotoxic and membrane-toxic potential, various surfactants were examined in U937 cells using the tetrazolium reduction assay EZ4U, a modified XTT test, and the arachidonic acid release test (AART). EZ4U measures the ability of living cells to reduce a colorless tetrazolium salt to an orange water-soluble formazan derivative by mitochondrial dehydrogenases. [3H]arachidonic acid ([3H]AA) is rapidly incorporated into cell membrane phospholipids. Due to membrane disintegration or enzymatic catalysis, it is released into the cell culture medium and can be measured by scintillation technique. The results after 24-hour-exposure are as follow (CC50 microg/ml; RC50 microg/ml): benzalkonium chloride (0.6), Cremophor A25 (1.4; 5.9), sodium cetearyl sulfate (1.6; 19.9), Brij78 (1.6; 7.7), TEGO betaine E (3.0; 19.3), TEGO betaine CKD (4.1; 20.2), TEGO betaine L7 (7.5; 20.0), sodium dodecyl sulfate (7.5; 39.0), Triton X-100 (8.9; 110), polysorbate 80 (48.1; 491), soybean lecithin (7920; 19940).

Pharmacological stimulation of t-PA release.

The acute release of tissue-type plasminogen activator t-PA from the vascular endothelium is of decisive importance for the prevention of intravascular fibrin deposits. A dose-dependent t-PA release from the isolated perfused vascular preparations may be induced by mediators (platelet-activating factor, bradykinin, histamine) adrenergic and cholinergic transmitters (isoprenaline, acetylcholine), thrombin, heparin and analogues, and 1-desamino-8-D-arginine-vasopression (DDAVP). Most of the compounds were shown to enhance the t-PA activity also in animal experiments (rats, rabbits, mini pigs). The pharmacologic stimulation of the t-PA release may be convenient for short-term thrombosis, prophylaxis and partial thrombolysis. Presently, this could only be achieved by unfractionated and low molecular weight heparins which have been shown to release t-PA.

Assessment of membrane toxicity using [(3)H]arachidonic acid release in U937 cells.

The release of arachidonic acid (AA) from membrane phospholipids is part of a signal system with which cells respond to environmental changes of various kinds. Using the promonocytic cell line U937, the kinetics of AA release was studied in [(3)H]AA-labelled U937 cells under the influence of various chemicals. Membrane-toxic agents such as lysolecithin and sodium dodecyl sulfate (SDS) cause pronounced enhancement of [(3)H]AA release in U937 cells. The effect occurred immediately after exposure and proved to be dose-dependent, non-enzymatic and irreversible. With regard to mean [(3)H]AA-releasing rate in untreated U937 cells, the following maximum tolerable concentrations were found: lysolecithin, 0.5 mug/ml; SDS, 2 mug/ml; dimethoate, 31.3 mug/ml; chloramine, 160 mug/ml; 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/ml; formamide, 50 mg/ml and dimethyl sulfoxide, 100 mg/ml. AA release represents a highly sensitive bioindicator for the membrane-toxic effect of a chemical substance.

A natural rubber drainage tube with antithrombogenic lumen surface.

A drainage tube was made by radiation vulcanization of a high polymeric substance based on natural rubber elastomers. Pentosan polysulphate sodium bound to a carrier substance (synthetic type 4A or 13X zeolite) was incorporated in the drainage tube which was then tested for its anticoagulant properties during perfusion with Tris buffer solution, citrated plasma, and blood, resp. The amount of pentosan polysulphate sodium released from the tube walls during perfusion with human citrated plasma in an open circulatory system was sufficient to exert an anticoagulant effect on the streaming plasma. This effect was corroborated by prolonged thrombin times and by unclottability in case of recalcified plasma samples in thrombelastographic studies. The antithrombogenicity test according to Chandler in a closed circulatory system revealed thrombus formation times (TFT) of more than 24 h (control: TFT = 1-3 min in native blood).

HIV-1 gp120 and NMDA induce protein kinase C translocation differentially in rat primary neuronal cultures.

Exposure of rat cortical neurons to the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 in vitro causes a rise in the intracellular Ca2+ level and a subsequent translocation of protein kinase C (PKC) from the cytosol to the membrane. Such a translocation persists for at least 2 h, but only in cultures with media not depleted of endogenous glutamate. Enzymatic degradation of glutamate in the medium by the enzyme glutamate-pyruvate transaminase (GPT) abolishes the long-lasting effect of gp120 on the association state of PKC; under this incubation condition the translocation period is < 1 h. Memantine and the ganglioside GM1 prevent N-methyl D-aspartate receptor-mediated long-term translocation of PKC and gp120-mediated neurotoxicity (in the absence of GPT); they have no effect on short-term translocation of PKC. We suggest that gp120-caused neuronal death involves an indirect sensitization step of the NMDA receptors, which ultimately induces neuronal death.

Characterization of human immunodeficiency virus-1-infected cells of myeloid-monocytic lineage (ML-1, HL-60, THP-1, U-937).

The myeloid-monocytic cells ML-1, HL-60, THP-1, and U-937 were chronically infected (for > 2 years) with the lymphotropic human immunodeficiency virus type 1 (HIV-1) strain HTLV-IIIB. Reinfection experiments revealed that viruses obtained from chronically infected ML-1/HIV-1 and HL-60/HIV-1 cells showed a low infectivity if tested with uninfected ML-1 and HL-60 cells in contrast to virus preparations from chronically infected THP-1/HIV-1 and U-937/HIV-1 with their corresponding uninfected cell lines. Analyses of selected cell surface markers revealed a differential expression of CD4, CD8, CD11c, CD14, CD15, CD20, HLA-DR, and HLA-DQ in non- or chronically infected cells. In chronically infected cells, the steady-state levels for tumor necrosis factor-alpha, interleukin (IL)-1 beta, and granulocyte-macrophage colony-stimulating factor mRNA remained unchanged whereas the one for IL-6 dropped.

Effect of sodium pentosan polysulphate on the thrombogenicity of prothrombin complex concentrates.

The addition of pentosan polysulphate sodium (NaPPS) to thrombogenic prothrombin complex concentrates (PCC) dose-dependently reduces or abolishes thrombus formation in rats in the stasis model acc. to Wessler. However, no reduction of thrombogenicity was found in PCC preparations manufactured in the presence of NaPPS.

Influence of hypersulfated lactobionic acid amides on tissue plasminogen activator release.

Under our experimental conditions, the sulfated bis-lactobionic acid amide, LW 10079, showed the strongest t-PA-releasing effect in the isolated perfused pig ear. In rats the sulfated bis-lactobionic acid amide LW 10121 was the most potent compound in acute t-PA release. In both experiments the bis-lactobionic acid amide LW 10082 did not work as a releaser of t-PA. Therefore in the case of LW 10082, the activation of the fibrinolytic pathway as a possible mechanism of its antithrombotic effect can be excluded.

Spontaneous fatal pulmonary thrombo-embolism in a mini-pig.

Spontaneous thrombo-embolism is an extremely rare disease in swine. We observed such a disease in a mini-pig. The animal attracted attention, because it did not eat its fill and breathed hamperally and shallowly but without stridor. Blood gas analysis and ECG findings indicated changes like in an acute pulmonary infection. 24 h after it had been admitted to our animal pasture the animal was dead. The reason was a pulmonary thrombo-embolism.