Eirik Bjørklid

The protein component of human brain thromboplastin

Abstract The protein component of human brain tissue thromboplastin (factor III) has been purified by deoxycholate (DOC) extraction, ultracentrifugation, gel filtration and finally repeated preparative polyacrylamide gel electrophoresis (PGE) in the presence of sodium dodecylsulphate (SDS). The final preparations gave one band in analytical PGE. Reduced and alkylated protein appeared as a band of molecular weight about 53 000 in SDS-PGE. The protein had a low solubility in aqueous solutions in the absence of detergents. When recombined with an optimal amount of the phospholipid fraction of tissue thromboplastin (fraction B) the procoagulant thromboplastin activity was regained. Neither alone nor after recombination with phospholipid did the protein catalyze the hydrolysis of aminoacyl-β-naphthylamides or casein.

Human factor VII associated with endotoxin stimulated monocytes in whole blood

Abstract Evidence is provided for a calcium dependent binding of factor VII to stimulated monocytes in whole blood. The binding of factor VII to the monocytes in this study was directly related to the exposure of tissue thromboplastin on the surface of the endotoxin stimulated monocytes. In such monocytes synthesis of tissue thromboplastin is well known to be enhanced. Antibodies against the apoprotein of tissue thromboplastin prevented the appearance of factor VII activity in monocyte suspensions isolated from blood incubated with endotoxin. This could be explained by the failure of factor α-VIIa generation in blood containing thromboplastin antibodies. Factor α-VIIa is probably the form of factor VII being bound to the monocytes.

What is blood borne tissue factor

In a recent letter to the Editors-in-Chief of Thrombosis Research we reported on blood borne TF [1]. Microparticles and monocytes were isolated fromnon-stimulated and LPSand LPS+PMA-stimulatedwhole blood and their TF antigen and activity quantified using an immunoblot technique and a specific two stage assay, respectively. This approach indicated that circulating microparticles had quite a high content of TF antigen, even though their TF activity turned out extremely low. Thus their specific TF activity was found to be very low compared to that of resting as well as stimulated monocytes of whole blood. Considering the many contradictory reports on blood borne TF, it is with regret that we have to inform you that the letter mentioned above, perpetuates rather than resolves the confusion, due to an inherent flaw that we have identified in the immunoblotting system used for quantifying TF antigen. Our rationale for choosing immunoblotting,was tofind awayaround the problemsof non-specific binding to cells experiencedwhen using commercial antibodies in conjunction with the FACS system. However, very recently we discovered that the Sigma anti-mouse IgG secondary antibody developed in goat affinity isolated antigen specific antibody (Saint Louis, Missouri, USA) that we had used unfortunately cross-reacted with human IgG. Thus, omission of the primary anti-TF antibody in the assay did not noticeably affect the blotting pattern obtained. Similar results were observed when using polyclonal rabbit antibodies to human TF in conjunction with a secondary Chemicon anti-rabbit IgG antibody, developed in goat and affinity isolated antigen specific antibody adsorbedwith human IgG, in the system. Obviously, our candidate band for TF was actually human IgG heavy chain detected by the secondary antibody, the same dominating band as seen when using a standard TF preparation as antigen sample. Since similar immuno-blotting results for TF have been presented also by others, we find reporting on this issue all the more urgent. Particularly the microparticles of unstimulated blood appeared to have anunaccounted for a quite high content of TFantigen, considering their apparently non-extant TF activity, as reported in our previous letter. When repeating the study using a commercial ELISA (IMUBIND Tissue Factor ELISA Kit, American Diagnostica Inc) for the quantification of TF antigen, quite different results were obtained. As can be seen in Table 1, in contrast to that of monocytes, the antigen content of microparticles turned out lower than the detection limit (0.3 pM) of this assay. The TF activity of microparticles from non-stimulated blood was hardly measurable, as found also in our previous report [1]. Althoughmicroparticles from LPS-stimulated bloodhad trace amounts of TF-activity, it was less than 0.5% of that in monocytes from similarly stimulated blood. Correspondingly, the TF activity of microparticles from LPS+PMA stimulated blood was only 0.7% of that of monocytes from similarly stimulated blood. These findings are in accordancewith the notion that in healthy individuals TF is hardly present in circulating microparticles, suggesting that what is measured as TF antigen in plasma must either be associated with even smaller particles (exosomes) or represent some soluble form(s), e.g. alternatively

An immunoradiometric assay for factor III (tissue thromboplastin).

A solid‐phase immunoradiometric assay for tissue thromboplastin (factor III) has been established based on its displacing effect on the binding of 125I‐labelled factor III‐antibodies to polyvinyl tubes coated with the purified protein component of factor III (apoprotein III). By this method circulating tissue thromboplastin can be detected in experimental animals receiving infusions of crude or purified tissue thromboplastin and in patients undergoing major orthopaedic surgery.

The isoelectric point of phospholipase C from Bacillus cereus

The exact value of the isoelectric point of phospholipase C (phosphatidylcholine cholinephosphohydrolase , EC 3, I .4.3) from &zcillus cereus is a matter of some dispute. Based on observations of the enzyme’s behaviour on ion exchange columns, Zwaal et al. [l] concluded that the isoelectric point was between 7.2 and 8.5. However, using isoelectric focusing Otnaess et al. [2] demonstrated a pI value of 6.5. More recently, Ikezawa et al. [3] found two forms of this enzyme in isoelectric focusing with pX values of 6.8 and 7.5. Whilst carrying out isoelectric focusing studies on phospholipase C (B. cereus) we observed that even recrystallized enzyme appeared heterogeneous and have investigated this phenomenon further and now present pf values for 3 different forms of this enzyme.

The effect of phospholipase C on DNA synthesis, morphology and phospholipid content of isolated HeLa cell nuclei

Isolated HeLa cell nuclei have been treated with purified phospholipase C (Bacillus cereus) and sphingomyelinase (Staphylococcus aureus). The phospholipids of untreated nuclei consisted of about 67% phosphatidylcholine, 23% phosphatidylethanolamine, 7% sphingomyelin, 2% phosphatidylserine and 1% phosphatidylinositol. Phospholipase C degraded 80-90% of the total phospholipids of the nuclei. Such nuclei seemed ultrastructurally intact, and had an average diameter and a protein loss during incubation which were not significantly different from those of controls. Their rate of DNA synthesis was only slightly reduced after treatment with phospholipase C alone and slightly more reduced when phospholipase C was used in combination with sphingomyelinase. This suggests that the polar head-groups of the nuclear phospholipids are of very limited importance in DNA synthesis. Since it has been reported that phospholipase C treatment releases nascent DNA from a membrane complex, the absence of a concommitant reduction in DNA synthesis may suggest that this complex is not necessary for the replication of DNA. Phospholipase C did not significantly influence the stability of the DNA product and gave only a slight inhibition of cytosol and nuclear DNA polymerases when tested with exogenous template.

THE DEVELOPMENT OF MONOSPECIFIC ANTIBODIES AGAINST HUMAN THROMBO- PLASTIN APOPROTEIN (APOPROTEIN III) AND THEIR APPLICATION IN THE IMMUNOCYTOCHEMICAL DETECTION OF THE ANTIGEN IN BLOOD CELLS

Human thromboplastin apoprotein (apoprotein III) purified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was purified a further 2-4 fold by PAGE in the presence of digitonin. Subsequent line immunoelectrophoresis of the protein revealed several lines, only one of which contained inhibitory antibodies. New inhibitory antibodies which were raised by using this particular line to immunize rabbits produced only a single line in immunoelectrophoresis of apoprotein III, with precipitated inhibitory antibodies being present only in the line. When these antibodies were used in electroblot immunobinding studies of crude thromboplastin after SDS-PAGE staining was found mainly in a single band of MW about 50,000, but also to some extent in immunologically related higher MW material. Prior deglycosylation of the thromboplastin using trinitrobenzenesulfonic acid resulted in a shift of the bulk of the main band representing an apparent MW reduction of 16%, and a corresponding shift in the position of protein with the capacity to bind inhibitory antibodies. Besides being a good criterion of specificity of the antibodies this also suggests that non-carbohydrate parts of apoprotein III may be involved in the interaction with Factor VII. Immunoperoxidase staining of unstimulated or endotoxin stimulated blood cells using the antibodies revealed the presence of significant amounts of apoprotein III only in stimulated monocytes, apparently available on the surface of the cells since it was detectable also by preembedding staining of fixed cells in suspension. The result is strong evidence that apoprotein III is synthesized de novo in monocytes upon endotoxin stimulation.

DNA synthesis in HeLa cell nuclei isolated in a non-aqueous medium

Nuclei were isolated from synchronized HeLa cells in the S-phase by a modification of the non-aqueous method described by Kirsch et al. (Science (1970) 168, 1592-1595). The method involved lyophilization of the cells, homogenization in non-aqueous glycerol and centrifugation in a gradient of 0-35% (w/w) 3-chloro-1,2-propanediol in glycerol. Such nucleic incorporated deoxyribonucleotides into DNA when incubated in an aqueous buffer containing Mg2+, ATP, dATP, dGTP, dCTP and dTTP. The product was sensitive to DNAase and banded with bulk DNA in isopycnic centrifugation. Sedimentation of the product in alkaline sucrose gradients after labelling of the nuclei for 2 min revealed labelled material in the 5 S peak and in the 18 S area. The material in the 5 S peak moved into the 12 S area after a 13 min chase.