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R L Lundblad

Publications and source records attributed to R L Lundblad.

At least 19 recordsLinked to original sources
PubMed2000

Issues with the assay of factor VIII activity in plasma and factor VIII concentrates.

A review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VII concentrates such as Hemofil M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements. it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor VIIIa during the assay process.

Blood Coagulation Tests↗
PubMed1999

Colorimetric protein assay techniques.

There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

Colorimetry↗
PubMed1998

Measurement of active coagulation factors in Autoplex-T with colorimetric active site-specific assay technology.

Autoplex-T is a partially activated prothrombin complex concentrate used primarily for the treatment of patients expressing factor VIII inhibitors. While Autoplex-T has a demonstrated record of clinical effectiveness, the procoagulant composition of this material has not been reported. This absence of composition data is a reflection of the lack of techniques appropriate for accurately measuring an individual protease such as factor IXa in complex mixtures of similar proteases. The development of Colorimetric Active Site-Specific ImmunoAssay technology (CASSIA) has permitted the accurate analysis of the coagulant enzymes present in Autoplex-T. Ten lots of Autoplex-T were reacted with both biotinylated phenylalanylprolylarginine chloromethylketone and biotinylated glutamylglycylarginine chloromethylketone. Only activated forms of the clotting factors present in Autoplex-T react with the peptide chloromethylketones and were thus separated from the other proteins present in Autoplex-T by adsorption onto streptavidin. The individual proteins bound to streptavidin were then detected with specific antibodies. Mean results from the analysis of ten lots of Autoplex-T (mean values) are as follows: factor Xla, 5.9 nM or 0.8 microg/ml; factor Xa, 46.5 nM or 2.1 microg/ml; factor IXa, 177.8 nM or 11.7 microg/ml; factor VIIa, 68.6 nM or 3.3 microg/ml and factor IIa, 5.3 nM or 0.2 microg/ml. These results are discussed with respect to the mechanism of action of Autoplex-T in the treatment of factor VIII inhibitor patients.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Applications of site-specific chemical modification in the manufacture of biopharmaceuticals: I. An overview.

The chemical modification of proteins has long been a useful approach to elucidating structure-function relationships. Recently, these same chemistries have been finding application in the preparation of proteins for both diagnostic and therapeutic use. These applications include alterations to introduce new properties, such as improved stability, or new traits, such as drug binding and transport, that take advantage of both broad and narrow ranges of selectivity. Site-specific chemical modification of proteins, when combined with the powerful advantages of site-specific mutagenesis, can yield protein agents superior to those generated by either approach alone.

Animals↗
PubMed1996

Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.

Characterization of the carbohydrate moiety is a critical measure of manufacturing process consistency of recombinant human Factor VIII (rFVIII) in Chinese-hamster ovary (CHO) cells. FVIII, a large (300 kDa) glycoprotein, is employed therapeutically for the correction of haemophilia A. While N-linked and O-linked oligosaccharides are found in this protein, the current study focuses on the N-linked oligosaccharides. The N-linked oligosaccharides from rFVIII were released using either peptide N-glycosidase F or endoglycosidase H, derivatized with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonate, and analysed by fluorophore-assisted carbohydrate electrophoresis (FACE). The electrophoretically resolved oligosaccharide bands were isolated and individual bands subjected to digestion with defined pools of exoglycosidases and re-electrophoresed on FACE sequencing gels. The resulting gel patterns were interpreted, based on band mobility shifts, to obtain the sequence structure of the oligosaccharides. A total of eight acidic and 12 neutral structures were identified, and the majority of the oligosaccharides (approximately 92%) were found to be sialylated. All of the major oligosaccharide structures found in CHO-cell-derived rFVIII have also been reported to be present in plasma-derived FVIII. Among the most abundant are disialylated, biantennary, core-fucosylated (approximately 40%), followed by trisialylated, triantennary, core-fucosylated and monosialo, biantennary, core-fucosylated structures (each approximately 18%). The Gal alpha 1-3Gal structures reported to be present in baby-hamster-kidney-cell-derived rFVIII were not found in the CHO-cell-derived protein. The glycosylation patterns were consistent in six random lots of rFVIII [coefficient of variation (%) 3-14] based on percentage lane luminance data of bands that represent approximately 98% of all asparagine-linked oligosaccharides.

Animals↗
PubMed1995

Neutralization of heparin activity by neutrophil lactoferrin.

Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin-induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.

Amino Acid Sequence↗
PubMed1993

Hydrophobic affinity chromatography of human thrombin.

Hydrophobic affinity chromatography on p-chlorobenzylamido-agarose (p-CBA-agarose) was used to characterize various modified forms of human thrombin. Native alpha-thrombin bound tightly to the column and was eluted with either acetonitrile or 1,4-dioxane, while the catalytically inactive prethrombin 2 did not bind to the matrix. Site-specific chemical modification with pyridoxal 5'-phosphate resulted in the loss of at least 80% of fibrinogen clotting activity but did not influence the binding of thrombin to p-CBA agarose. Modification of thrombin with pyridoxal 5'-phosphate is thought to occur at the fibrinogen-binding site and the heparin-binding site. In contrast, binding of thrombin to p-CBA agarose was eliminated by modification of the active site histidine using either H-D-phenylalanyl-L-prolyl-L-arginine chloromethylketone or dansyl-L-glutamyl-glycyl-L-arginine chloromethylketone but not with tosyl-L-lysine chloromethylketone. The presence of either hirudin or heparin blocked the binding of thrombin to p-CBA-agarose but dansyl-arginine-N-(3-ethyl-1,5-pentanediyl)amide had no effect. These results indicate that p-CBA agarose binds to thrombin outside of the enzyme active site and its use should be valuable in characterizing site-specific modified thrombins obtained by either protein engineering or chemical modification.

Acetonitriles↗
PubMed1993

Identification of tissue kallikrein messenger RNA in human neutrophils.

Expression of tissue kallikrein in human neutrophils has been suggested by previous studies using enzymatic and immunochemical techniques. Secretion of this potent biological factor by neutrophils would be of marked significance in the inflammatory process. The present study utilized the polymerase chain reaction following reverse transcriptase generation of total neutrophils cDNA to demonstrate the presence of tissue kallikrein mRNA in the human neutrophils. In addition, use of sequence-specific primers demonstrated the presence of mRNA for the hGK-1 gene, but not for the hPK gene product or the gene for prostate-specific antigen. These results confirm that tissue kallikrein is present in neutrophils and may be secreted as part of the inflammatory process.

Animals↗
PubMed1993

Expression of tissue kallikrein in normal and SV40-transfected human endometrial stromal cells.

The polymerase chain reaction with specific tissue kallikrein primers was utilized to demonstrate the presence of tissue kallikrein mRNA in human endometrial stromal cells. Enzymatic analysis measured with a specific tripeptide nitroanilide substrate demonstrated the presence of tissue kallikrein in the conditioned medium obtained from both normal stromal cells and stromal cells transfected with an origin-defective temperature-sensitive SV40 large T antigen. The transfected stromal cell supernatant exhibited approximately twice as much tissue kallikrein activity as normal stromal cells at 60-100% of cell confluence. The release of tissue kallikrein from transfected stromal cells was confirmed by Western blot analysis and [35S]-methionine incorporation into a 35-kD protein which retains tissue kallikrein activity. These results demonstrate for the first time the expression and secretion of tissue kallikrein in human endometrial stromal cells and provide evidence of possible involvement of tissue kallikrein in cell transformation.

Base Sequence↗
PubMed1992

Affinity chromatography of platelets on immobilized thrombin: retention of catalytic activity by platelet-bound thrombin.

Radioactivity from I125-labeled human platelets was measured to estimate the extent of binding of platelet surface proteins to immobilized thrombin. 1-3% of the radioactivity was bound with 10-20% of this amount apparently irreversibly bound to the thrombin matrix. Site-specific chemical modification of thrombin with pyridoxal-5'-phosphate, N-bromosuccinimide or tetranitromethane resulted in a variable reduction of the amount of radiolabel bound. When thrombin modified with H-D-PheProArg-chloromethyl ketone (PPACK) was coupled to the matrix, there was no difference in the binding of platelet membrane proteins when compared to a control thrombin preparation while thrombin modified with tosyl-Lys-chloromethyl ketone (TLCK) coupled to the matrix did not bind radiolabel any more effectively than albumin which served as the control. However, when thrombin was modified with PPACK after coupling to the agarose matrix, ability to bind radiolabel was lost. Thrombin bound to platelets remained catalytically active when assayed with a peptide nitroanilide substrate. These results suggest tight binding between thrombin and platelets that is not only not dependent on active site integrity but leaves the bound thrombin catalytically competent.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Molecular diversity of tissue kallikrein in human saliva.

The present study was conducted to explore the extent of heterogeneity of tissue kallikrein in saliva using immunological analysis and to demonstrate that such heterogeneity resulted from secretory phenomena and not degradation secondary to secretion. Human mixed saliva was collected by paraffin-stimulation and centrifuged at 10,000 rpm for 10 minutes. Special collectors were used to obtain parotid and submandibular/sublingual saliva using citric acid stimulation. Western blot analysis of human mixed saliva demonstrated major immunoreactive species with molecular masses of < 20 KD, 45 KD, 60 KD, 90 KD and > 200 KD. The polyclonal antibody used for these blotting studies was monofunctional with respect to reaction with purified salivary tissue kallikrein. Only the < 29 KD and 45 KD species were active using an enzyme overlay technique. While a similar distribution of molecular weight material was observed in both parotid and submandibular saliva, the amount of immunoreactive material was markedly less in parotid secretion. In addition, the < 20 KD material was essentially absent in parotid saliva. Treatment of the saliva with thermolysin eliminated the immunoreactive band at 60 KD while treatment with various glycosidases also eliminated some heterogeneity. These results demonstrate considerable variation in tissue kallikrein expression in salivary gland secretions.

Amino Acid Sequence↗
PubMed1992

The influence of age, sex and race on salivary kallikrein levels in human mixed saliva.

Variation in the level of salivary kallikrein in human saliva has been reported as a function of systemic conditions such as reduced salt intake and during the menstrual cycle. Higher levels of salivary kallikrein have been observed in subjects with tumors distant from the oral cavity when compared to control subjects. These studies have not evaluated factors, such as age, which might influence the concentration of glandular kallikrein in saliva. The purpose of the present study was to determine the variation of salivary kallikrein concentration as a function of age. Differences attributable to sex or race were also evaluated. Mixed saliva was collected from 114 subjects, ages 5-91, by paraffin stimulation. Samples were centrifuged and stored at -20 degrees C for subsequent analysis. Glandular kallikrein activity was assayed using D-ValylLeucylArginine-p-nitroanilide as the substrate. In a linear regression model which included sex, race, and age, levels only the factor of age had a significant effect on kallikrein levels. The p-value for the reduced model including only the factor of age was 0.0406 and the R-square was 0.038. Further analysis revealed that females did exhibit significantly higher kallikrein in individuals 40 years or older and that the effect of age appeared to be limited to females. It is concluded that both gender and age must be considered when evaluating salivary kallikrein changes in relationship to systemic disease.

Adolescent↗