Prothrombin separation

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

Wu, H. and Bruley, D.F, Homologous Human Blood Protein Separation Using Immobilized Metal Affinity Chromatography Protein C Separation from Prothrombin with Application to the Separation of Factor IX and Prothrombin, Biotechnol. Prog., 15, 928, 1999. 

There are several possible arrangements tolerating the presence of particles during adsorption of proteins to particulate matrices. Batch adsorption in stirred tanks is performed by contacting adsorbent particles with a cell containing suspension. After protein capture the adsorbent is separated from the broth and the protein of interest can be eluted. This procedure has been described for the isolation of antibiotics [12], the purification of ot-amylase from B. amylo-liquefaciens broth [13], and the isolation of the prothrombin complex from... 

At this point there s a little twist to our developing chicken-and-egg scenario. Even activated Stuart factor can t turn on prothrombin. Stuart factor and prothrombin can be mixed in a test tube for longer than it would take a large animal to bleed to death without any noticeable production of thrombin. It turns out that another protein, called accelerin, is needed to increase the activity of Stuart factor. The dynamic duo—accelerin and activated Stuart factor— cleave prothrombin fast enough to do the bleeding animal some good. So in this step we need two separate proteins to activate one proenzyme. 

Prothrombin complex is the combination of Factors E, VE, DC, and X. It is indicated for individuals with hemophdia B. In a typical production process, the supernatant resulting from the separation of cryoprecipitate is filtered to remove particles and applied to an anion-exchange column. The coagulation factors are eluted, ultrafiltered, and exposed to 60°C for 10 h for virus inactivation. This is followed by diafiltration, ultrafiltration, and sterile filtration. 

FIGURE 9.30 Structures of prothrombin and thrombin. Prothrombin is a protein consisting of 581 amino acids, while thrombin consists of 308 amino acids. Prothrombin is cleaved within the bloodstream, at the indicated peptide bonds, by the factor X /factor Vg complex. The intermediary form of thrombin is called meizothrombin. The activation peptide might be considered to be a waste product of the activation scheme. Please note that thrombin consists of two separate polypeptide chains, and that these are held together by a disulfide bond. Studies by researchers have revealed that prothrombin can also be activated by factor X alone, but here the pathway of peptide bond cleavage is somewhat different than that produced by the factor XJfactor complex. 

CHAPTER 36, FIGURE 6 Cofactor proteins, factor V and factor VIII. Factor V and factor VIII coagulant (not the von Willebrand factor carrier of factor VIII) contain six distinct structural domains. The two A domains. A, and Aj at the N-terminal end of the polypeptide chain, are separated from the Aj domain by a highly glycosylated B domain. The two C domains are at the C-terminal end of the molecule. The A domain sequences are homologous to the A domains of ceruloplasmin. Both factor Va and factor Villa act as catalysts in the activation of prothrombin and factor X, respectively. Activation sites are indicated by green arrows inactivation sites by red arrows. In factor Va, complete inactivation requires cleavage of Arg ° . 

Wu H.P. and Bruley D.E. 1999. Homologous human blood protein separation using immobilized metal affinity chromatography protein C separation from prothrombin with apphcation to the separation of factor IX and prothrombin, Biotechnol. Prog., 15, 928-931. 

Metronidazole 250 mg three times daily for a week inereased the half-life of a single 1.5-mg/kg dose of warfarin by about one-third (from 35 to 46 hours) in 8 healthy subjeets, and inereased the prothrombin time from a mean of 100 to 142 seeonds. When the warfarin enantiomers were given separately, the antieoagulant effeets of 5 -warfarin were virtually doubled and the half-life inereased by 60%, but the response to R-warfarin was only alfeeted in one subjeet. In a retrospeetive eohort study, 32 patients taking warfarin had an INR reading before and during eoneurrent metronidazole use. In these patients, the mean INR inereased from 2.2 to a maximum of 4.3 by day 8 of eoneurrent use. Fourteen of the 32 had an INR above 4, but no bleeding events were reeorded. ... 

Similarly, an inerease in the effects of warfarin, with or without bleeding eomplieations, in patients given co-trimoxazole has been described in a number of other ease reports.In one study in healthy subjects, co-tri-moxazole 480 mg four times daily for 8 days increased the prothrombin time after a single dose of warfarin by 1.5-fold, but no change in the half-life of warfarin was seen. However, a later similar study by the same re-seareh group, in whieh warfarin was given as its separate isomers, co-trimoxazole inereased the AUC of -warfarin by 22% and caused a 5% deerease in AUC of i -warfarin. ... 

Comparable results were found in another similar study simultaneous administration of warfarin and eolestyramine reduced the prothrombin time response by 21%, and separation by 3 hours still caused an 11% reduction in the prothrombin time response. Another study using intravenous warfarin has shown that eolestyramine also reduces the effect of warfarin by this route, presumably by redueing enterohepatic recycling. 

In a study in 3 subjects and one patient, phenylbutazone 200 mg three times daily and twice daily, respectively, given for 11 to 19 days before and 11 days after a single dose of warfarin, markedly increased the prothrombin time, but decreased the half-life of warfarin, and the warfarin AUC. In another study that gave the enantiomers of warfarin separately, it was found that phenylbutazone inhibited the clearance of 5-warfarin, but increased the clearance of7 -warfarin. This was confirmed in other studies, were the AUC of7 -warfarin was decreased by 41% and the AUC of 5-warfarin increased by 18%. ... 

Phosphorylated dextran derivatives, called phosphodex-trans, possess a strong affinity for K-vitamin-dependent coagulation factors, like factor II or prothrombin. This property was used to separate them by affinity chromatography on phosphodextrans, which interact in a similar way as phospholipids from the ceU membrane. ... 

This fraction contains virtually all the antibodies of plasma, as well as prothrombin, the isoagglutinins, plasminogen, and the component C, of complement. The separation of these various components is, of course, of major importance, and a systematic procedure has been developed by Oncley, Melin, Cameron, Richert and Gross (161). Briefly, it is based on the following observations ... 

In the low temperature ethanol fractionation at the Harvard IKlot Plant it was found that plasminogen was highly concentrated in Fraction 111-2,3. It was desired to separate it as completely as possible from the prothrombin with which it was closely associated, and which was very similar in its solubility characteristics. In D. A. Richert s method a considerable degree of concentration was achieved by making use of the fact that most of the fibrinogen, not already precipitated in Fraction I, was concentrated in Fraction 111-2,3. On the addition of a moderate... 

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