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Background and Objectives The solvent/detergent treatment enables effective and robust inactivation of all lipid-enveloped viruses, but also inactivates partly sensitive plasma proteins such as protein S. The aim of this study was to investigate the thrombin generation capacity of octaplasLG®, in particular focusing on the function of protein S in thrombin generation assay and the impact of assay settings. Materials and Methods Sixteen octaplasLG® batches and 32 units of single donor fresh frozen plasma (FFP) were investigated. For protein S, both functional activity and free antigen levels were measured. Thrombin generation assay was performed using two fluorogenic tests with different triggers. Finally, rotational thromboelastometry was performed. Results Mean protein S levels were lower in octaplasLG®, but a wider range of values was found for FFP. Clotting parameters and thrombin generation capacities overlapped between the two plasma groups as demonstrated using both thrombin generation assays and different triggers. Spiking studies with protein S-depleted plasma, human purified protein S or antibodies against protein S confirmed a correlation between protein S and thrombin generation capacity under specific assay conditions, especially in an assay with low tissue factor concentration. Conclusion Correlation between protein S and thrombin generation capacity was demonstrated in the TGA. Due to higher variability in protein S content in the FFP group, overlapping haemostatic potentials of the two plasma groups were found.

Background OctaplasLG® is a 2nd-generation virus inactivated pooled plasma for infusion. Prions are removed by the principle of chromatography, utilizing an affinity ligand gel (LG) developed for binding of prion proteins and their infectivity. The goal of this study was to verify, using the gold standard animal bioassay system, whether or not prion infectivity can be removed by the LG affinity step under conditions used in the routine manufacturing process. Materials and methods Aliquots of pooled plasma were spiked with a microsomal/cytosolic (MIC) fraction of brain-derived hamster-adapted scrapie 263K and subjected to the OctaplasLG® manufacturing process. Validated Western blot tests and animal bioassays studies were performed to determine the logarithmic reduction factors (RF) and the prion infectivity binding capacity. Results Bioassay studies demonstrated different logarithmic RFs (i.e. 1·73 and 0·76 log10) at two different plasma-to-resin ratios, the latter one representing the actual manufacturing ratio of 100:1, which can be explained by the differences in the study design. However, both bioassay studies showed a reproducible and high prion infectivity binding capacity of ≥5·64 log10 ID50/ml gel. Conclusion Bioassay studies confirmed the capacity of the LG to bind brain-derived MIC prion proteins spiked into plasma. Even through infectivity was still detected following passage over the LG, this can be attributed to the high loads used in the study design, and the binding capacity of the LG still ensures a significant safety margin – binding the prion agents at the levels of prion infectivity that might be present in plasma and beyond.

BACKGROUND AND OBJECTIVES A new chromatographic step for the selective binding of abnormal prion protein (PrP(Sc)) was developed, and optimization for PrP(Sc) capture was achieved by binding to an affinity ligand attached to synthetic resin particles. This step was implemented into the manufacturing process of the solvent/detergent (S/D)-treated biopharmaceutical quality plasma Octaplas to further improve the safety margin in terms of risk for variant Creutzfeldt-Jakob disease (vCJD) transmission. MATERIALS AND METHODS Intermediates and Octaplas final container material, spiked with hamster brain-derived PrP(Sc)-containing fractions, were used for experiments to establish the feasibility of introducing this novel chromatography step. The binding capacity per millilitre of ligand gel was determined under the selected manufacturing conditions. In addition, the specificity of the ligand gel to bind PrP(Sc) from human sources was investigated. A validated Western blot test was used for the identification and quantification of PrP(Sc). RESULTS A reduction factor of > or = 3.0 log(10) could be demonstrated by Western blotting, utilizing the relevant Octaplas matrix from manufacturing. In this particular cell-free plasma solution, the PrP(Sc) binding capacity of the selected gel was very high (> or = 6 log(10) ID(50)/ml, equivalent to roughly 10 log(10) ID(50)/column at manufacturing scale). The gel binds specifically PrP(Sc) from both animal (hamster and mouse) and human (sporadic and variant CJD) sources. CONCLUSION This new single-use, disposable PrP(Sc)-harvesting gel ensures a very high capacity in terms of removing the pathogenic agent causing vCJD from the new generation OctaplasLG, in the event that prions can be found in plasma from donors incubating the disease and thereby contaminating the raw material plasma used for manufacturing.

Background and Objectives A new chromatographic step for the selective binding of pathological prion proteins (PrPSc) to an affinity ligand, developed and optimized for PrPSc capture and attached to synthetic resin particles (PRDT, USA; ProMetic BioSciences Ltd, Isle of Man, UK) was implemented into the manufacturing process of the solvent/detergent (S/D) treated biopharmaceutical quality plasma Octaplas®. Materials and Methods Pilot batches of Octaplas® with the implemented chromatographic step [labelled as OctaplasLG® (ligand gel)] were manufactured by Octapharma PPGmbH, Vienna, Austria. The biochemical quality was compared directly after manufacturing as well as after 18 months storage. All samples were tested on global coagulation parameters, fibrinogen levels, activities of coagulation factors and protease inhibitors, ADAMTS13 levels, as well as markers of activated coagulation and fibrinolysis. In addition, von Willebrand factor multimeric analysis was performed. Results The incorporation of this novel chromatography into the large-scale routine manufacturing process was shown to be technically feasible and the performance of the column was assessed to be excellent. The biochemical studies showed that Octaplas® and OctaplasLG® produced without and with the new column, respectively, demonstrate an identical biochemical quality. OctaplasLG® remained stable over a period of 18 months stored frozen. A parallel reduction of the S/D virus inactivation step from 4–4·5 to 1–1·5 h led to significantly higher activities of plasmin inhibitor. Conclusion The studies confirmed that the affinity ligand chromatography under the developed conditions can be introduced into the Octaplas® manufacturing process, as a mean to reduce potentially present PrPSc, without hampering the proven quality of this product.

The solvent/detergent treatment enables effective and robust inactivation of all lipid-enveloped viruses, but also inactivates partly sensitive plasma proteins such as protein S. The aim of this study was to investigate the thrombin generation capacity of octaplasLGSixteen octaplasLGMean protein S levels were lower in octaplasLGCorrelation between protein S and thrombin generation capacity was demonstrated in the TGA. Due to higher variability in protein S content in the FFP group, overlapping haemostatic potentials of the two plasma groups were found.

Background and Objectives The pathomechanism of thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS) is associated with a severe deficiency of ADAMTS13 and factor H. The aim of this study was to quantify the levels of ADAMTS13 and factor H in the pharmaceutically licensed plasma for transfusion, Octaplas®, and the universally applicable plasma, Uniplas (development product, working title). Furthermore, Octaplas® batches of blood groups A, B, O, AB, and plasmas derived from different sources were compared. Materials and Methods Twenty-four Octaplas® and three Uniplas batches were selected for the study. ADAMTS13 activities were measured by fluorescence resonance energy transfer assay, ADAMTS13 antigen levels were quantified using enzyme-linked immunosorbent assay test kit, while factor H antigen levels were detected using radial immunodiffusion (RID) methods. In addition, von Willebrand factor (vWF) multimeric analyses were performed. Results Both Octaplas®, produced from US and European plasma of different blood groups, and Uniplas contain ADAMTS13 antigen and activity levels as well as factor H concentrations at normal levels without significant differences. In addition, Octaplas® and Uniplas show a vWF multimeric pattern comparable to normal plasma. Conclusion The study revealed that Octaplas® and Uniplas contain normal levels of ADAMTS13 at low batch-to-batch variations. Therefore, both products can substitute the missing or neutralized protease activity in TTP patients and thereby limit vWF-dependent (platelet-related) thrombosis. In addition, both plasma products contain factor H at a physiological level, and, thus can be used efficiently in the treatment of aHUS patients, which have been shown to benefit from plasma administration.

SummaryA recent clinical sepsis trial reported a significant reduction in 90-day mortality by antithrombin (AT) exclusively in the subgroup of patients without simultaneous heparin prophylaxis. Patients additionally receiving heparin did not benefit from AT treatment. Herein, we studied the microhemodynamic and cellular mechanisms of this adverse effect of heparin on AT actions by the use of intravital microscopy and granulocyte culturing. In Syrian golden hamsters normotensive endotoxemia was induced by 2 mg/kg endotoxin (LPS, E. coli) i.v. In a first group of animals, AT (AT, 250 IU/kg i.v., n = 6) was given 5 min before LPS administration. A second group of animals (Heparin+AT, n = 5) received AT (250 IU/kg i.v.) combined with unfractionated heparin (sodium heparin, 100 IU/kg/24 h, i.v.). Additional animals (LMWH+AT, n = 5) received AT (250 IU/kg i.v.) combined with LMWH (nadroparin 47.5 IU anti-Xa/kg, s.c., 2 h before LPS). LPStreated animals, which received only saline, served as controls (control, n = 6). Using dorsal skinfold fold preparations, LPS-induced microvascular leukocyte-endothelial cell interaction (LE) and alteration of functional capillary density (FCD) were studied by intravital video fluorescence microscopy. In controls, LPS induced a massive increase in LE with a maximum at 8 h and an impressive decrease in FCD over a 24-hour period. Both LPS effects were effectively prevented by AT treatment (p

Plasma samples of 189 healthy subjects were investigated for antigen levels of the recently reported factor VII- and single-chain plasminogen activator-activating protease (FSAP) and the corresponding pro-urokinase activating potencies. While the age of donors had no significant effect on the investigated parameters, female plasmas revealed a trend to higher antigen contents and activity levels. Surprisingly, as much as 9% of all samples contained significantly reduced single-chain urinary plasminogen activator activating potential, whereas antigen concentrations were normal. Additionally, 1% of the plasmas was found to decrease in both FSAP antigen and activity contents. FSAP of three subjects displaying reduced activities throughout a follow-up period of 6 months were purified from plasmas and were characterized. As compared with pool plasma derived FSAP, investigation of the individual preparations confirmed their reduced potency to activate pro-urokinase. However, factor VII activation was not affected. It is speculated that the FSAP binding site for single-chain plasminogen activators is affected, potentially by as yet unknown polymorphism(s) or mutation(s).

An activity detected in a prothrombin complex concentrate, termed 'thrombin-like' due to its amidolytic properties, was recently reported by another working group. This serine-protease revealed partial structural homology with a 'hepatocyte growth factor activator'. An impact of this protease on coagulation has not yet been described. The protease was isolated from plasma fractions by ion exchange chromatography and adsorption to immobilized heparin and/or aprotinin. Clotting tests including the FVIIa-rTF assay were performed employing coagulometry. A monoclonal antibody-derived F(ab')2 to FVIII was used to investigate the FVIII bypassing activity (FEIBA). The identity of the-protease with the so-called 'thrombin-like' protease was supported by sequencing of the amino-termini. Its amidolytic activity was significantly enhanced in the presence of calcium and/or heparin. Incubation with purified FVII revealed the generation of FVIIa, but was prevented by pre-incubation of the protease with aprotinin. In contrast, purified FV and FVIII were inactivated. Studying coagulation parameters, clotting times like plasma recalcification times and the prothrombin times were found to be shortened by addition of the protease. Employing a FVIII-inhibitory F(ab')2 and enhancing clotting times significantly, FEIBA of the protease was found. We demonstrated that the isolated protease activates FVII independent of tissue factor. Net acceleration of coagulation was found in several global clotting assays resulting in an in vitro FEIBA. The physiological relevance of these findings deserves further investigation.

We studied the sensitivity of various animal plasmas to activated human protein C (APC) and found an impaired response in the order monkey < horse < pig < dog < rabbit. We assume that this effect was mainly caused by differences in the APC cleavage region of factor V, as recently described for the factor V Leiden mutation in humans. In APC-dependent assays, which are based on the inactivation of factor Va, 5% (v/v) rabbit plasma added to human plasma mimicked the APC response seen in heterozygous carriers of factor V Leiden; 20% rabbit plasma gave results similar to those seen in homozygotes. We exploited this factor V Leiden-like behaviour to prepare plasma standards for calibration of APC-dependent assays by mixing rabbit and human plasmas. The 0% value was assigned to the clotting time in the absence of APC, while the 100% value was assigned to the clotting time in presence of APC. A normal human plasma pool was used as reference. These standards were used to establish reference curves. On automated coagulation analyzers, the results obtained were automatically reported as 'percent normal'. This not only simplified evaluation, but also improved the comparability of results.

Summary Background, objectives and methods: An accurate, sensitive and precise assay for reliable determination of the ristocetin cofactor activity of von Willebrand factor (VWF:RCo) in plasma and von Willebrand Factor (VWF)-containing concentrates has been evaluated. The assay is based on a commercially available automated protocol with modifications including a combination of adding additional ristocetin and the use of two calibration curves for the high and low measuring ranges. Results: Addition of extra ristocetin resulted in improved measurement of VWF recoveries from various VWF-containing concentrates that were underestimated using the standard automated protocol. The modifications resulted in improved assay performance over an extended measuring range (2.00 - 0.03 IU/mL). Accuracy was tested using VWF deficiency plasma spiked with 1(st) international standard for VWF concentrate. Seven dilutions, ranging between 1.80 and 0.05 IU/mL, were analyzed and resulted in measured concentrations between 80% and 100% of the assigned potency of the standard. Linearity was determined from the regression plot of the same concentrate dilutions and resulted in a correlation coefficient of 0.998. The repeatability, expressed as coefficient of variation, was 2% in the normal range (0.90 IU/mL) and 8% at the level of 0.05 IU/mL. The corresponding reproducibility results were 2% and 15% at the normal and low measuring ranges, respectively. Conclusions: Analysis of patients with von Willebrand disease indicates that the modified automated BCS((R)) protocol has a superior discrimination power compared with the standard protocol. This is especially true in samples with low VWF, as in patients with type 3 von Willebrand disease. (Less)

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.

The pharmacological and pharmacokinetic behaviour of a recombinant tissue-type plasminogen activator (t-PA) mutant with Asn 117 and Asn 184 substituted by Gln (G4) was compared to t-PA wild type expressed in the same cell line. Although the t 1 2 α and β were almost similar for both molecules, the plasma clearance of the partially deglycosylated molecules was 2.3 times slower than that of the native molecule. The relative size of the area under the curve in the β-phase was markedly increased from 27–54.4%. Infusion of less than half of the dose of t-PA wild type (mg/kg) resulted in similar thrombolytic effects. The data presented show that the mutant analysed is more potent than t-PA wild type in vivo. Although the mutant has an increased specific activity and a lowered clearance rate, the thrombolytic data do not demonstrate that these properties are cumulative.

The in vivo antithrombotic and profibrinolytic properties of human plasma protein C were investigated in a metallic coil induced thrombus model in the rat. Protein C, purified by immunoadsorption, was activated by thrombin and thereafter administered to the animals. Half-life of activated protein C (APC) revealed to be 15–16 min for the antigen, but only 7–8 min for the anticoagulant activity. Thrombus formation was reduced by APC in a dose-dependent statistically significant manner. A profibrinolytic effect of APC was observed when administered in combination with human tissue plasminogen activator. APC alone caused a reduction of thrombus weight. However, both effects were statistically not significant.

The human placental proteins PP4 and PP4-X, belonging to the annexin protein family, were expressed in Escherichia coli at high yield. The proteins were purified to homogeneity. The physicochemical parameters of the recombinant proteins were determined and compared with those of their natural placental counterparts. Except for a minor change in the pI, the proteins appeared to be indistinguishable by several criteria. Both recombinant PP4 and recombinant PP4-X were biologically active in a thromboplastin inhibition test and in a phospholipase A2 inhibition test.

Background and Objectives The most common way to thaw frozen coagulation-active plasma products for transfusion is the use of a water bath with good circulation at 30–37 °C. The aim of this study was to perform an extensive biochemical characterization of the pharmaceutically licenced solvent/detergent-treated plasma, Octaplas®, thawed using the SAHARA-III dry tempering system from the company Sarstedt GmbH, Austria. A regular water bath was used in parallel for comparison. Materials and Methods Six batches Octaplas® with different blood groups were thawed in a water bath or using the SAHARA-III dry tempering system in parallel. Thawed plasma was investigated on screening tests for blood coagulation, as well as on the activities of important coagulation factors and protease inhibitors. In addition, markers of activated coagulation and fibrinolysis were tested and von Willebrand factor multimeric analysis was performed. Results There were neither significant differences in the blood coagulation parameters, coagulation factors, protease inhibitors, nor of markers of activated coagulation and fibrinolysis when Octaplas® thawed by the two different methods was tested. The von Willebrand factor analyses showed no influence on the overall profile of the multimeric pattern when using the SAHARA-III dry tempering system. Conclusion Octaplas® can be thawed using the SAHARA-III dry tempering system without any negative influences on the demonstrated quality of this product. The SAHARA-III dry tempering system enables standardized thawing and warming procedure. Furthermore, tempering of Octaplas® in the emergency unit or operating theatre, where no water baths can be utilized, is safe and can be fully endorsed.

The pathomechanism of thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS) is associated with a severe deficiency of ADAMTS13 and factor H. The aim of this study was to quantify the levels of ADAMTS13 and factor H in the pharmaceutically licensed plasma for transfusion, Octaplas, and the universally applicable plasma, Uniplas (development product, working title). Furthermore, Octaplas batches of blood groups A, B, O, AB, and plasmas derived from different sources were compared.Twenty-four Octaplas and three Uniplas batches were selected for the study. ADAMTS13 activities were measured by fluorescence resonance energy transfer assay, ADAMTS13 antigen levels were quantified using enzyme-linked immunosorbent assay test kit, while factor H antigen levels were detected using radial immunodiffusion (RID) methods. In addition, von Willebrand factor (vWF) multimeric analyses were performed.Both Octaplas, produced from US and European plasma of different blood groups, and Uniplas contain ADAMTS13 antigen and activity levels as well as factor H concentrations at normal levels without significant differences. In addition, Octaplas and Uniplas show a vWF multimeric pattern comparable to normal plasma.The study revealed that Octaplas and Uniplas contain normal levels of ADAMTS13 at low batch-to-batch variations. Therefore, both products can substitute the missing or neutralized protease activity in TTP patients and thereby limit vWF-dependent (platelet-related) thrombosis. In addition, both plasma products contain factor H at a physiological level, and, thus can be used efficiently in the treatment of aHUS patients, which have been shown to benefit from plasma administration.

Previous in vitro and in vivo studies have indicated potent antiangiogenic and antitumor activity of latent antithrombin. With the use of intravital fluorescence microscopy, the aim of the present study was to further elucidate the inhibitory action of latent antithrombin on the different steps of angiogenesis, i.e. (i) capillary sprouting, (ii) capillary maturation or (iii) network formation, in melanoma tumor growth. For this purpose, a dorsal skinfold chamber was prepared in 27 Syrian golden hamsters, and 2×105 syngenic A-Mel-3-Fortner melanoma cells were implanted onto the skin muscle surface. The process of tumor angiogenesis was analyzed and quantified at days 3, 5, 7 and 10 after implantation using intravital fluorescence microscopy and computer-assisted image analysis. The analysis included the determination of the size of the microvascular network (in relation to the size of tumor area), microvessel density (defined as the length of red blood cell perfused microvessels in relation to the size of tumor area) as well as capillary diameters, red blood cell velocity and blood perfusion. Animals daily received latent (n = 8) or native (n = 11) antithrombin in a dosage of 25 mg/kg. Nontreated animals (n = 8) served as controls (mean ± SEM; ANOVA, unpaired Student’s t-test). During initial angiogenesis, latent and native antithrombin-treated tumors showed comparable capillary sprouting, but significantly larger capillary diameters compared to controls (day 5: 9.0 ± 0.5 μm and 9.3 ± 0.5 μm versus 7.4 ± 0.4 μm; p < 0.05), indicating a delay in capillary maturation. In contrast, neither latent nor native antithrombin affected microvessel density, which increased from ∼125cm/cm2 to ∼260cm/cm2 between day 3 and day 10. This observation reflects the lack of effect on capillary sprouting. However, analysis of the overall size of the microvascular network indicated a significant reduction at day 10 in latent (89 ± 10 mm2/mm2) and native antithrombin-treated tumors (96 ± 6 mm2/mm2) compared to untreated controls (134 ± 12 mm2/mm2). In conclusion, latent antithrombin does not inhibit capillary sprouting per se, but reduces overall tumor angiogenesis by delaying the maturation of the newly formed capillaries. The comparable effectiveness of native versus latent antithrombin is probably due to the fact that native antithrombin contains substantial amounts of spontaneously formed latent antithrombin.

Leukocyte-endothelial cell interaction and microvascular perfusion failure are characteristic deteriorations of the microcirculation in endotoxaemia and are known to play a crucial role in the development of septic multiple organ dysfunction. Recent studies have indicated that antithrombin III treatment is capable of significantly ameliorating these microcirculatory disorders. Endothelial cells have important anticoagulant systems, including the heparan sulfate-antithrombin system. Antithrombin III stimulates prostacyclin generation in endothelial cells by interacting with heparan sulfate of endothelial cells and inhibits cytokine and tissue factor production in endothelial cells and monocytes. Similar mechanisms may be involved in cellular actions of antithrombin III causing desensitization of chemoattractant receptors of leukocytes by activating the heparan sulfate proteoglycan, syndecan-4. Thus, antithrombin III might be among the useful agents for treating coagulation abnormalities associated with sepsis or other inflammation because it inhibits not only coagulation but also downregulation of anticoagulant activities of endothelial cells and affects leukocyte activation.

The serpin antithrombin III (AT III) is reported to have hemostasis-regulating and anti-inflammatory properties. To determine its ability to influence thrombin-independent leukocyte responses, the direct effects of the AT III concentrate Kybernin P and a monoclonal antibody-purified AT III on neutrophil migration were studied. Chemotactic activity of human neutrophils isolated from the blood of healthy donors was determined in modified Boyden microchemotaxis chambers, and binding studies were performed according to standard experimental protocols. Preincubation in vitro of neutrophils with Kybernin P or immune-adsorbed AT III significantly deactivated migration toward fMet-Leu-Phe, or interleukin-8 (IL-8), in a concentration-dependent manner. In the absence of additional attractants, neutrophils exhibited a migratory response toward gradients of AT III preparations. True chemotaxis was confirmed in checkerboard assays. Analyses revealed that the AT III heparin-binding site interacts with neutrophil membrane–associated heparan sulfate proteoglycan receptors. Mechanisms of intracellular signaling differed; the deactivation of IL-8–induced chemotaxis resulted from tyrphostin-sensitive interactions of AT III-signaling with the IL-8 signal transduction pathway, whereas AT III–induced chemotaxis involved protein kinase C and phosphodiesterases. Signaling similarities between AT III and the proteoglycan syndecan-4 may suggest the binding of AT III to this novel type of membrane receptor. Under physiological conditions, AT III may prevent neutrophils from premature activation. Moreover, the systemic administration of AT III concentrate could have beneficial effects in combating systemic inflammation.

For the manufacture of the PCC Beriplex P/N, nanofiltration was introduced into the production process of Beriplex HS providing an additional means to heat treatment for the clearance/inactivation of viruses. By nanofiltration, large enveloped viruses (HSV-1, HIV-1) were completely eliminated by a factor of more than 7 log10. While medium-sized enveloped viruses (HBV, BVDV) were cleared by a factor of approximately 4 log10, small non-enveloped viruses (poliovirus) were not removed. The product profile remained, no thrombogenic activities were detected.

The specific thrombin inhibitors r-hirudin and a synthetic peptide (I) D-FPRP(G)4-NGDFEEIPEEYL were compared in in vitro tests. r-hirudin proved to be the superior compound with respect to inhibition of amidolytic small substrate turnover that is catalysed by soluble and immobilised thrombin as well as to inhibition of fibrinogen activation. In an in vitro clot model significantly higher molar concentrations of peptide I are needed to achieve fibrin bound thrombin inhibition equivalent to that of r-hirudin. Stable complexes consisting of thrombin and hirudin oppose labile complexes containing the synthetic peptide. The latter leads to a regaining of thrombin activity with subsequent additional fibrin accretion. Analyses of the mixtures of thrombin and peptide I display a time dependent release of amino-terminal D-FPR peptide (III) exhibiting, similar to the residual fragment (peptide II), only weak inhibitory activity. Peptide I and the carboxy-terminal fragment induce, within a certain concentration range, an increase in thrombin activity and clot growth.

Haemostasis is a system of finely adjusted interactions between cells, enzymatic reaction cascades and inhibitors. Disturbances of this balance occur in many disorders, especially in inflammatory processes, septicaemia and cancer. In such cases malignant cells and infectious organisms activate the plasmatic enzyme cascades, especially of the coagulation and fibrinolysis cascades. The resulting consumption and proteolytic degradation of the regulatory proteins contribute to hypercoagulability and secondarily to reactive fibrinolysis, and these may then lead to local thromboses and haemorrhages. These pathogenic events culminate in disseminated intravascular coagulation (DIC), frequently with organ failure and death. Factors of both plasmatic systems are also "misused" by malignant cells for the purposes of growth and metastasis. Prominent examples of this misuse are the formation of a protective fibrin shield against the endogenous defence mechanisms and the local degradation of tissues for tumor proliferation as well as for cell permeation and invasion. In the search for a potential therapy a number of protease inhibitors, predominantly of enzymes of coagulation and fibrinolysis, have been tested in vivo with regard to their efficacy. So far, however, it has not been possible to find a new uniform treatment principle to inhibit the growth and/or metastasis of different types of tumor. The haemorrhagic diathesis and thromboses frequently associated with tumors are generally treated by substitution with plasma components, especially concentrates of coagulation factors and inhibitors.

Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.

Concentrations of annexins I to VI were quantitatively determined in extracts of placenta and different human cell types. They were detectable in all extracts studied, but lymphocytes/monocytes, endothelial cells and fibroblasts had very high annexin contents. The results indicate cell type specific annexin-repertoires. Annexins are intracellular proteins lacking signal sequences but which are detectable in trace amounts in plasma of healthy humans. The majority of plasma samples drawn from 14 patients suffering from myocardial infarction had elevated annexin III, IV and V concentrations. Shortly after infarction increased annexin levels were detected, reaching maximal values 24 to 48 h later. In the course of the following days annexin concentrations returned towards normal plasma levels.

In the present in vitro study the stabilities of the thrombin-hirudin and the thrombin-antithrombin III complexes were investigated. After incubation of the complexes with free inhibitors the thrombin-antithrombin III levels were determined by ELISA. The thrombin-hirudin complex proved to be stable in the presence of antithrombin III or heparin. However, in the presence of heparin and plasma equivalent concentrations of antithrombin III, the thrombin-hirudin complex dissociated and hirudin was displaced. In contrast, both thrombin-antithrombin III and thrombin-antithrombin III/heparin complexes are very stable even in the presence of a large excess of hirudin.

Glucocorticosteroids are among the most useful and most widely prescribed anti-inflammatory drugs. Despite their wide use little is yet known about their mode of action. In the last 10 years a group of proteins called lipocortins or annexins has been characterized. Those proteins exert an inhibitory effect on the synthesis of lipid mediators by way of an important proinflammatory enzyme, phospholipase A2. Phospholipases are known to be involved in cell-signal transduction and generation of inflammatory mediators like prostaglandins, leukotrienes and platelet-activating-factor. The cellular expression of lipocortins is induced by glucocorticosteroids. The inhibition of cellular phospholipases via lipocortins may account for some aspects of the action of glucocorticosteroids.

Das von Bohn et al. (1985) isolierte und charakterisierte Plazentaprotein 4 (PP4) ist ein membranassoziiertes Glykoprotein mit einem Molekulargewicht von 35 kD, welches zunachst in der menschlichen Plazenta nachgewiesen wurde. In unterschiedlichen Konzentrationen ist es auch in anderen Zellen, Geweben und Flussigkeiten des Korpers vorhanden (z.B. Niere, Blase, Amnionflussigkeit, Thrombozyten, Leukozyten u.a.). Studien von Grundmann et al. (1988) ergaben die Zugehorigkeit des PP4 zur Lipocortin/Calpactin-Protein-Familie, die in vitro potente Antikoagulantien sind (Ca2+-vermittelte Phospholipidbindung; „indirekte“Gerinnungshemmung), deren In-vivo-Funktion aber noch unbekannt ist.

Many physiological processes are based on the finely regulated interaction between cells and enzymatic reaction cascades. Mainly proteinases are involved in these processes, which are regulated by inhibitors, principally proteins. If this sensitive balance is disturbed, uncontrolled pathophysiological events can be induced, which are often associated with inflammatory reactions. Characteristic for inflammation are events like contact activation of hemostasis, increasing permeability of blood vessels caused by activation of the Kallikrein-Kinin- and the Complement-system and Plasmin-release induced by activation of fibrinolysis. The following uncontrolled proteolysis, leading to tissue destruction, is mainly associated with the degree of illness. Inflammatory cells excrete besides proteinases also mediators maintaining and increasing these processes. Only when the balance between proteinases and inhibitors is restored, inflammation subsides. Afterwards the controlled course of physiological reactions is possible again.

The aim of this study was to perform an extensive biochemical comparison of the pharmaceutically licensed coagulation active plasma Octaplas with an identical, but universally applicable, development product (Uniplas) and single-donor fresh-frozen plasma (FFP) units subjected to a medical device treatment using a combination of methylene-blue dye and subsequent white-light exposure (MB plasma).

Anti-angiogenic and anti-tumor activities of latent and cleaved antithrombin has been described, and in vitro, the serpin inhibited proliferation of endothelial cells. We have recently observed that direct cellular effects of antithrombin are mediated by syndecan-4, a heparan sulfate proteoglycan known to bind to the heparin-binding site of antithrombin. Syndecan-4 is known to affect proliferation and differentiation of a variety of cell types. We have, therefore, studied direct effects of intact antithrombin on endothelial cells. Human umbilical vein and calf pulmonary artery endothelial cells were studied in the presence or absence of antithrombin concentrate or monoclonal antibody purified antithrombin with and without concomitant presence of synthetic pentasaccharide. Proliferation was assessed in BrDU incorporation and MTT assays. For testing endothelial cell differentiation, capillary tube formation was investigated in matrigel assays. Proliferation of the two types of endothelial was significantly inhibited by 1–10 U/ml of both antithrombin concentrate and antibody-purified antithrombin. Capillary tube formation induced by matrigel was augmented by the presence of 1–10 U/ml of antithrombin concentrate which was partly reversed with pentasaccharide. Results show that in vitro effects of antithrombin on angiogenesis-related endothelial cell functions may be directly exerted by the intact serpine and can be antagonised by pentasaccharide

Sepsis is one of the main causes of death in developed countries. It is commonly associated with disseminated intravascular coagulation (DIC), caused by consumption of coagulation factors and inhibitors such as antithrombin (AT) and the production of interleukin 6 (IL-6) and interleukin 8 (IL-8) have been shown to correlate positively with the severity of disease. Large doses of antithrombin (AT) reduce mortality and morbidity in septic patients and there is increasing evidence to suggest that AT has anti-inflammatory properties in addition to its anticoagulant properties. In our previous in vitro study, we have found that AT inhibits tissue factor and IL-6 production induced by lipopolysaccharides (LPS). In the present investigation, we have studied the effects of AT on LPS induced IL-8 production in three in vitro cellular systems. Citrated whole blood, human umbilical vein endothelial cells (HUVECs) and mononuclear cells (MNCs) were stimulated with LPS for 4–6 hours in the presence and absence of 0–40 IU/ml AT. IL-8 was measured by ELISA. In all three systems, AT dose-dependently inhibited IL-8 production, with greatest inhibition (98.7 ± 5.2% at 40 IU/ml) observed for the whole blood system and the least inhibition seen with MNCs (8.7 ± 21.7% at 40 IU/ml). RNA extraction of time course whole blood experiment followed by the detection of mRNA specific for IL-8 showed that in the absence of AT, mRNA for IL-8 was apparent after 30 min incubation with LPS. However, the level of IL-8 mRNA was found to decrease with increasing concentrations of AT. These results imply the inhibition of IL-8 antigen production by AT is due to the suppression of mRNA and indicated that the anti-inflammatory activity of AT also extends to the inhibition of IL-8, an important cytokine implicated in neutrophil migration.

During disseminated intravascular coagulation (DIC), the extrinsic tissue factor (TF)-dependent pathway has been implicated as the dominant route to thrombin generation and the production of IL-6 has been shown to correlate positively with the severity of sepsis-induced DIC. Pharmacological doses of AT have been shown to reduce mortality and morbidity in patients with DIC and there is increasing evidence to suggest that AT possesses anti-inflammatory properties in addition to its anticoagulant properties. In the present study, we have investigated the effect of AT on LPS-induced TF and IL-6 production in three different in vitro systems. Citrated whole blood. HUVECs and MNCs were stimulated with LPS for 4–6 h in the presence or absence of AT. TF activity was estimated by a TF-dependent clotting or chromogenic assay and IL-6 was measured by ELISA. Our results show a dose-dependent inhibition of TF and IL-6 production by AT, EC50 — ~36 and 20-35 iu/ml respectively in MNCs and HUVECs, but ~14 and

The influence of the insulin concentration on its absorption from the subcutaneous tissues was studied by direct measurement of the appearance of insulin in the circulation after its subcutaneous administration. 10 units of regular insulin of semisynthetic human and porcine origin in strength of 15 units/ml, 40 units/ml, and 100/units/ml were administered as bolus to 9 healthy male volunteers, using switched-off, manually handled insulin pumps. Insulinaemia rose significantly higher (p less than 0.05) and insulin levels peaked earlier after the delivery of the 15 units/ml insulin solutions compared to the 100 units/ml insulin solutions. These findings might be of interest for type-I diabetic patients using insulin pumps that are to be loaded with regular insulin in varying concentrations.