Human plasma lipoproteins as accelerators of prothrombin activation.

The activation rate of bovine prothrombin by Factor Xa and Ca2+ has long been known to be greatly enhanced by addition of phospholipid. Upon substitution of human plasma lipoproteins for phospholipid (cephalin) in this activation system, only very low density lipoprotein enhances prothrombin activation. Low density lipoprotein and high density lipoprotein have no stimulatory effect on prothrombin activation. On the other hand, the sonicated lipid extracts from very low, low, and high density lipoproteins all can substitute for phospholipid in potentiating prothrombin activation. The efficiency of each lipid extract, in this regard, depends upon its source of extraction, and is greatest for the lipid extract of very low density lipoprotein.     

Decarboxylation of bovine prothrombin fragment 1 and prothrombin

Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.     

History of hadron therapy accelerators

In the last 60 years, hadron therapy has made great advances passing from a stage of pure research to a well-established treatment modality for solid tumours. In this paper the history of hadron therapy accelerators is reviewed, starting from the first cyclotrons used in the thirties for neutron therapy and passing to more modern and flexible machines used nowadays. The technical developments have been accompanied by clinical studies that allowed the selection of the tumours which are more sensitive to this type of radiotherapy. This paper aims at giving a review of the origin and the present status of hadron therapy accelerators, describing the technological basis and the continuous development of this application to medicine of instruments developed for fundamental science. At the end the present challenges are reviewed.     

Isolation and partial characterization of a human prothrombin variant: prothrombin Barcelona

An abnormal prothrombin variant, Prothrombin Barcelona, has been isolated by chromatography on DEAE Sephadex, from several members of the same family. In the absence of any normal component, it was eluted in two unequal peaks. The second peak was homogeneous. This component had the same molecular weight as normal prothrombin but migrated slightly faster on disc gel electrophoresis. The first peak, the smaller one, was heterogeneous: in addition to a minor band similar to that of the second peak, a major one with less anodic mobility and with a molecular weight of 32,000 was found. A possible chromatographic artefact has been eliminated. The family study gave good arguments for an heterozygote state of both parents, the siblings being homozygote.     

An MCNP-based model for the evaluation of the photoneutron dose in high energy medical electron accelerators

The development of a computational model for the treatment head of a medical electron accelerator (Elekta/Philips SL-18) by the Monte Carlo code mcnp-4C2 is discussed. The model includes the major components of the accelerator head and a pmma phantom representing the patient body. Calculations were performed for a 14 MeV electron beam impinging on the accelerator target and a 10 cm×10 cm beam area at the isocentre. The model was used in order to predict the neutron ambient dose equivalent at the isocentre level and moreover the neutron absorbed dose distribution within the phantom. Calculations were validated against experimental measurements performed by gold foil activation detectors. The results of this study indicated that the equivalent dose at tissues or organs adjacent to the treatment field due to photoneutrons could be up to 10% of the total peripheral dose, for the specific accelerator characteristics examined. Therefore, photoneutrons should be taken into account when accurate dose calculations are required to sensitive tissues that are adjacent to the therapeutic X-ray beam. The method described can be extended to other accelerators and collimation configurations as well, upon specification of treatment head component dimensions, composition and nominal accelerating potential.     

Aging of soman-inhibited acetylcholinesterase: inhibitors and accelerators

The influence of 27 possible effectors, mostly bispyridinium salts, upon the dealkylation (aging) of soman-inhibited acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was examined at pH 7.6 and 25 degrees C. In the absence of effectors, the rate constant of the aging process was 4.0. 10(-2) min-1. At 2 mM, the strongest inhibitor reduced the rate to 0.8. 10(-2) min-1, whereas it was raised to 8.2. 10(-2) min-1 by the most potent accelerator.     

Evolution of prothrombin: Isolation and characterization of the cDNAs encoding chicken and hagfish prothrombin

The cDNA sequences of chicken and hagfish prothrombin have been determined. The sequences predict that prothrombin from both species is synthesized as a prepro-protein consisting of a putative Gla domain, two kringle domains, and a two-chain protease domain. Chicken and hagfish prothrombin share 51.6% amino acid sequence identity (313/627 residues). Both chicken and hagfish prothrombin are structurally very similar to human, bovine, rat, and mouse prothrombin and all six species share 41% amino acid sequence identity. Amino acid sequence alignments of human, bovine, rat, mouse, chicken, and hagfish prothrombin suggest that the thrombin B-chain and the propeptide-Gla domain are the regions most constrained for the common function(s) of vertebrate prothrombins.The nucleotide sequences reported in this paper have been submitted to the EMBL/Genbank database under the following secession numbers: M 81391 for Gallus gallus, M 81393 for Eptatretus stouti.Correspondence to: R.T.A. MacGillivray     

Tb3+ binding to bovine prothrombin and bovine prothrombin fragment 1

The binding of Tb3+ to bovine prothrombin and the amino-terminal 156 residues of prothrombin (F-1) was studied. On the basis of various Tb3+ emission properties, three classes of Tb3+-binding sites were described. The first class contained three high affinity sites in the F-1 region. These sites were filled noncooperatively and were saturated with Tb3+ before the other classes of sites started to fill. Ho3+ quenching of Tb3+ emission showed that these sites were in close proximity to one another (estimated distances 6-12 A). The second class of sites contained three lower affinity sites, also in the F-1 region. These sites bound Tb3+ in a stoichiometric manner and saturated prior to metal binding to the final class of sites. The number of protein ligands binding Tb3+ in the high affinity sites decreased as this second set of sites was filled. Ho3+ quenching of Tb3+ emission suggested that these sites were closely spaced and/or close to the first set of sites. The third class of sites contained 4-6 low affinity sites unique to prothrombin (not in the F-1 region). These sites were not studied extensively, but Tb3+ did not appear to bind stoichiometrically and did not saturate these sites in a manner similar to the other two classes of sites. The emission properties of Tb3+ bound to F-1 were different in KCl versus NaCl containing buffer while the emission properties of Tb3+ bound to prothrombin were not. Optimum conditions for studying lanthanide binding to F-1 (i.e. when Tb3+ bound to F-1 showed emission properties similar to Tb3+ bound to prothrombin) were when F-1 experiments were done at low F-1 concentrations in buffer containing 0.1 M KCl.     

Purification and properties of chicken prothrombin

Prothrombin was isolated from citrated chicken plasma. The isolation depends upon the elimination of an interfering substance closely adherent to chicken prothrombin by treatment with SrCO₃. Subsequent to this, the classical adsorption to barium citrate, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-200 was carried out. Prothrombin purified by this method was found to have a specific activity of 1050 Iowa units (850 N.I.H. thrombin units) per mg. Recovery from plasma averaged 40%. Molecular weight by Sephadex G-200 chromatography was 73,000 ± 5,000 and by dodecyl sulfate sodium salt acrylamide gel electrophoresis 70,000 ± 5,000. A stable dimer of M_r 138,000 was observed in some preparations. The isoelectric pH in both acetate and phosphate buffers (μ = 0.1) was 3.95. Rabbit antibody to chicken prothrombin evidenced a single line by immunoelectrophoresis against purified antigen and chicken plasma.     

Purification and characterization of ostrich prothrombin

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu(2+)-chelate Sepharose chromatography. Ostrich prothrombin exhibited a M(r) of 72,800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin beta-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins.