Comparative biochemical studies of fresh frozen plasma and pooled solvent/detergent-treated plasma (octaplasLG® ) with focus on protein S and its impact in different thrombin generation assay set-ups.

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.

Purification and biochemical characterization of functional complement factor H from human plasma fractions.

BACKGROUND AND OBJECTIVES: Complement factor H (CFH) acts as major regulator of the alternative pathway of complement and its mutations and polymorphisms predispose to various human diseases. We aimed to develop a scalable purification process of CFH from human plasma fractions to supply a pathogen-safe and functional CFH concentrate. MATERIALS AND METHODS: Starting with intermediates of cold ethanol fractionation of plasma, CFH purification was performed with three chromatographic steps including solvent detergent treatment and nanofiltration. CFH functionality was tested by a haemolysis assay using sheep erythrocytes, by determining decay acceleration activity on C3 convertases and cofactor activity of C3b cleavage. CFH identity was confirmed by Western blot and mass spectrometry. RESULTS: Three scalable chromatographic steps highly purified full-length and native CFH from human plasma fractions. The purification process enabled the removal of truncated and dysfunctional CFH species, yielding a native CFH concentrate as demonstrated in sensitive functional in vitro assays. CONCLUSION: This novel process provides a pathogen-safe and functional CFH concentrate that can be produced on an industrial scale and is suitable for pre-/clinical studies.

Removal of prion infectivity by affinity ligand chromatography during OctaplasLG® manufacturing--results from animal bioassay studies.

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 log(10)) 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 log(10) ID(50)/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.

Biochemical quality of the pharmaceutically licensed plasma OctaplasLG after implementation of a novel prion protein (PrPSc) removal technology and reduction of the solvent/detergent (S/D) process time.

BACKGROUND AND OBJECTIVES: A new chromatographic step for the selective binding of pathological prion proteins (PrP(Sc)) to an affinity ligand, developed and optimized for PrP(Sc) 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 PrP(Sc), without hampering the proven quality of this product.

Prion removal effect of a specific affinity ligand introduced into the manufacturing process of the pharmaceutical quality solvent/detergent (S/D)-treated plasma OctaplasLG.

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.

Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.

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.

The crystal structure and ion channel activity of human annexin II, a peripheral membrane protein.

Annexin II binds in a calcium-dependent manner to acidic phospholipids and is a substrate of some protein kinases. An N-terminally shortened form of human annexin II was crystallized and its molecular structure determined. It is very similar to two previously described members of this protein family, annexin I and annexin V. The protein structure is nearly completely alpha-helical organized as four compact domains which consist of five alpha-helices each. The domains surround a hydrophilic pore. The calcium binding sites are located at the convex side of the structure as in annexin V. Recombinant and natural porcine annexin II are active as ion channel with characteristics similar to annexin V, while N-terminally shortened annexin II and the heterotetramer (annexin II-p11)2 are inactive. Two cysteine residues, Cys133 and Cys262, form a disulphide bridge connecting domains II and III, adding further weight to the notion that ion channel activity does not require major structural rearrangements.

Inhibition of human skin phospholipase A2 by "lipocortins" is an indirect effect of substrate/lipocortin interaction.

Proteins of the annexin/lipocortin family have been claimed to mediate the anti-inflammatory action of glucocorticosteroids by the inhibition of phospholipases A2. This hypothesis has been challenged by the finding that annexins do not directly interact with the enzyme in a classical enzyme/inhibitor behavior, but more likely block the access of the phospholipase A2 to its substrate by binding to phospholipids. Because former studies with skin phospholipase A2 suggested a specific regulation by annexin-1, we investigated the substrate dependence of this effect. For this purpose phospholipase A2 activities in human epidermis and dermis homogenates were measured in the presence of various amounts of annexins-1, -2, or -5. The respective annexin was preincubated in separate series either with the substrate or with the enzyme. We found a partial inhibition of both epidermal and dermal phospholipase A2 activities with all annexins tested (annexin-5 >> annexin-2 > annexin-1). The inhibitory effect was absolutely dependent on the annexin/phospholipid ratio and occurred only at very high annexin concentrations relative to the amount of substrate. Our data demonstrate that the inhibition of human skin phospholipase A2 by annexins depends on the substrate concentrations, as has been shown for phospholipases A2 of other origins as well. All observations can be explained by the current "substrate depletion model" characterizing the indirect effects of annexins on phospholipase A2 activities. It is therefore rather unlikely that annexins are directly involved in the regulation of phospholipase A2 activity of human skin under physiologic conditions.

The calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution. Implications for membrane binding and calcium channel activity.

Crystal structure analysis and refinement at 2.0 A resolution of a rhombohedral crystal form of human annexin V at high calcium concentration revealed a domain motion compared to the previously analysed hexagonal crystal form. Five calcium ions were located on the convex face of the molecule. Three strongly bound calciums are liganded at protruding interhelical loops and Asp or Glu residues in homologous positions in repeats I, II and IV. Five proteinaceous oxygens and one solvent molecule form the coordination polyhedron in each case. The unoccupied seventh site is suggested as the phospholipid headgroup binding site. Two more weakly bound sites were identified by lanthanum labelling. The structural features suggest that annexin V attaches with its convex face to membranes by specific calcium mediated interactions with at least three phospholipids. The adjacent membrane bilayer may thus become locally disordered and permeable to allow calcium inflow through the central polar channel of the molecule.

Inhibition of in vitro clot growth by r-hirudin is more effective and longer sustained than by an analogous peptide.

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.

Localization of annexins in normal and diseased human skin.

Annexins (AX) or lipocortins are a family of calcium and phospholipid binding proteins that have been implicated to play a role in the regulation of inflammation and cellular differentiation. To investigate a potential role of AX in skin disorders we studied the distribution of six different AX in normal human skin (NHS) and several inflammatory and hyperproliferative skin diseases. A distinct staining pattern could only be shown for AX-1 and AX-2. In NHS AX-1-antibody (Ab) displayed a very strong reactivity with eccrine sweat ducts. In the diseases investigated we found a highly increased expression of AX-1 in keratinocytes (KCs) in the vicinity of inflammatory processes such as psoriasis. Furthermore, the AX-1 expression was increased in differentiated squamous cell carcinoma (SCC) whereas undifferentiated SCC and basal cell carcinoma were negative. AX-3, -4, -5, and -6 showed no distinctive expression pattern. Our data demonstrate an abnormal distribution of AX-1 in association with proliferating KCs under inflammatory and neoplastic conditions. Its pattern of reactivity shows similarities to the known distribution of the EGF-receptor kinase, which has been demonstrated to phosphorylate AX-1 with high activity in various cellular systems. These results support the concept that the appearance of AX-1 is linked to a certain level of KC differentiation.

Autoantibodies to annexins: a diagnostic marker for cutaneous disorders?

Annexins/lipocortins are a group of structurally related calcium and lipid binding proteins which have been implicated as mediators of the anti-inflammatory action of corticosteroids. Autoantibodies against annexin-1 have been reported in association with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis and their presence has been hypothesized as the reason for the steroid resistance phenomenon. In this study we investigated IgG- and IgM-autoantibodies against annexin-1,-2,-3,-4,-5 and -6 in sera of 221 patients with skin disorders and 114 healthy blood donors with newly established ELISAs. Patients were clustered into 5 groups according to their diagnosis: autoimmune diseases, psoriasis, leg ulcer, malignant melanoma, and miscellaneous diseases. Autoantibodies directed against each annexin were detectable in all investigated groups, in the control group as well as in the disease groups, without displaying any significant correlation to any of the disease states. The homogenous distribution of annexin-autoantibodies throughout the control group and all the disease groups studied, do not support the implication of annexin-autoantibodies in pathophysiological states and make them an unlikely candidate for use as a diagnostic marker.

Cloning and expression of a cDNA encoding human placental protein 11, a putative serine protease with diagnostic significance as a tumor marker.

The placental protein 11 (PP11) can act as a tumor marker because of its specific association with various forms of cancer. A lambda gt11 cDNA library prepared from human placenta was screened with a polyclonal anti-PP11 antiserum. Out of 10(6) independent clones, only one clone reacted with the anti-PP11 antiserum. The isolated cDNA coded only for the carboxy-terminal part of PP11 and was subsequently used to rescreen a lambda gt10 placental cDNA library. Two cDNA clones out of 10(6) screened were identified encoding the entire protein of 369 amino acids, including a typical hydrophobic signal sequence of 18 amino acids. Expression of the PP11 cDNA coding sequence in Escherichia coli resulted in the synthesis of a protein with the expected size which can be specifically immunoprecipitated with anti-PP11 antiserum. Fractionation experiments revealed that two forms of the protein are present in the bacterial cell: a higher-molecular-weight form of approximately 42 kD in the cytoplasm and a smaller-molecular-weight form of approximately 42 kD in the periplasm. This result indicates that PP11 can be synthesized in E. coli and is process by removal of the hydrophobic signal sequence. Both the placental and the processed recombinant PP11 protein exhibit a protease activity.

Comparative study on the in vitro effectiveness of antithrombotic agents.

The synthetic low molecular weight inhibitors MD 805, FUT-175 and FOY as well as heparin and r-Hirudin were compared for their in vitro antithrombotic potencies and protease specificities. The amidolytic activity of thrombin and the plasma coagulation were effectively inhibited by MD 805 and, in particular, by r-Hirudin. FUT-175 and FOY revealed only weak inhibition. None of the synthetic substances discriminated between alpha-, beta- and gamma-thrombin. Beside r-Hirudin only MD 805 revealed a relatively good specificity for thrombin. On the contrary, FUT-175 and FOY are unspecific and can not be classified as thrombin inhibitors.

In-vivo antithrombotic potency of placenta protein 4 (annexin V).

The antithrombotic properties of Placenta Protein 4 (PP4) were investigated in laser or photochemically induced thrombus formation models in rats. In both in-vivo test-systems PP4 displayed a significant antithrombotic effect at dose levels as low as 0.3 and 1.0 mg/kg body weight. Bleeding times, surprisingly, were not prolonged significantly at these dose regimens. Maximal inhibition of thrombus formation in the laser-model was observed 15 min after intravenous administration of PP4, but was not recognizable in a clear-cut reaction in the second model. Determination of PP4 plasma levels in two monkeys revealed a half-life of 11.5 and 14.9 min, respectively. The maximal anticoagulant effect was observed between 15 and 30 min after administration of PP4 as determined functionally by means of thrombelastography.

Anticoagulant properties of placenta protein 4 (annexin V).

A placenta protein, originally termed PP4, was found to inhibit the aPTT in a concentration-dependent manner. PP4 which turned out to be identical with a vascular anticoagulant of the annexin type, inhibits the blood clotting process by binding of the essential lipids in a reaction which is dependent on calcium ions. Also in the presence of calcium PP4 combines with platelet membranes neutralizing their procoagulant effect. By fluorescence-microscopy binding of PP4 to stimulated macrophages is shown. The antithrombotic effect of PP4 is demonstrated by means of thrombelastography of human blood. Coagulation triggered by the addition of thromboplastin/lipid-mixtures is extinguished by PP4.

Determination of metalloproteinases, plasminogen-activators and their inhibitors in the synovial fluids of patients with rheumatoid arthritis during chemical synoviorthesis.

The concentrations of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-8 (MMP-8), matrix metalloproteinase-9 (MMP-9), lactoferrin and urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and the inhibitors, tissue inhibitor of metalloproteinase-1 (TIMP-1), plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor (PAI-2), and alpha2-macroglobulin in the synovial fluids of patients with rheumatoid arthritis was determined before and during chemical synoviorthesis with a sodium salt of the fatty acids from cod-liver oil (Varicocid). Synovial fluids were obtained before treatment from 37 patients with rheumatoid arthritis and, in most cases, at 8 and 24 h after injection of the agent. Well-established ELISAs were used to determine the amounts of all proteins. All patients with rheumatoid arthritis revealed very high levels of metalloproteinases (about 1-15 mu g/ml) in their synovial fluids. During the inflammation inducing treatment the granulocyte enzymes increased. In contrast to this, the level of MMP-1 decreased. All granulocyte-derived enzymes were strongly correlated with each other, whereas their dependence on the granulocyte count was only weak. uPA and PAI-2 showed good correlations with the granulocytes-derived enzymes, but were also only weakly correlating with the cell counts. t-PA was not detected by the ELISA used. The proteases, MMP-8, MMP-9 and uPA were increased 8 h after the treatment, whereas the specific inhibitors TIMP-1, PAI-1 and PAI-2 showed significant changes only 24 h after the injection. Matrix metalloproteinases are important factors in the pathogenesis of rheumatoid arthritis. The inflammatory activity in the joint could be better correlated to the granulocyte enzymes than to the granulocyte counts. The levels of uPA and PAI-2 are also parallel to the granulocyte enzyme levels and might underly the same regulatory mechanism.

Separation of antithrombin III variants by micellar electrokinetic chromatography.

The characterisation of proteins is still one of the most challenging analytical tasks in modern bioanalysis. Due to the complex structure of proteins, several analytical techniques are often required to get sufficient information. Antithrombin III (AT III), a high-molecular-mass plasma glycoprotein which is an important protease inhibitor and the main modulator of thrombin activity, circulates in plasma in two isoforms, the so-called AT III-alpha (90-95%) and -beta (5-10%). Micellar electrokinetic chromatography was used to analytically separate these AT III variants, which differ in their affinity to the polysaccharide heparin.

Factor V Leiden-like behaviour of animal plasma and its use for calibration of activated protein C-dependent assays.

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.

Thrombin-hirudin complex stability: a comparison with the thrombin-antithrombin III complex.

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.