Preparation Of Human Thrombin Research Articles

Cross Reactivity of Rabbit Anti-Recothrom (Human Recombinant Thrombin) Antibodies with Molecular Variants of Human Thrombin, and Various Bovine Thrombin Preparations

AbstractAbstract 4418Bovine thrombin preparations are widely used as topical hemostatic agents. While earlier bovine thrombin preparations were reported to produce immunologic responses upon repeated exposure, the refined bovine thrombin and recombinant human thrombin preparations (Zymogenetics, Seattle, WA, USA) are claimed to be less immunogenic. It was hypothesized that recombinant human thrombin despite its reduced immunogenic nature, upon repeated administration may result in the generation of antibodies and that may cross react with bovine and human thrombin. To test this hypothesis, groups of rabbits were challenged repeatedly with human recombinant thrombin over a 6 month period. Purified IgG preparations were obtained using protein G columns.To generate specific antisera in rabbits, the Recothrom (Human recombinant thrombin) conjugated with keyhole lymphocyte hemocyanin(KLH) as a carrier were administered intravenously to individual groups of rabbits (n=3) at a dosage of 100 μ g using standard immunologic methods. Preimmune blood and antiserum were collected from each rabbit in 0,30,50,90,120,150,180 days. In order to determine their cross reactivity with 3 preparations of bovine thrombin with differing purity, namely thrombin 4A, thrombin 4B, and bovine prothrombin were evaluated by immunoblotting techniques using the rabbit IgG fractions. In addition, to determine the relative cross reactivity of anti-Recothrom antibodies with human-α and γ- thrombin, one batch of each was evaluated by immunoblotting techniques using the rabbit IgG fractions. Gel electrophoresis and Western blotting were carried out. In brief, samples were subjected to electrophoresis through 4% to 20% gradient Tris-4-(2-hydro-xyethyl)-1-piperazineethanesulfonic acid (HEPES)-sodium dodecyl sulfate (SDS) polyacrylamide mini gels and then electrotransferred onto nitrocellulose membranes. The membranes were blocked with 5% milk and probed with anti-Recothrom IgG. The membranes were then washed and incubated with horseradish peroxidase conjugated donkey anti-rabbit IgG (1:20000 dilutions).After extensively washing; immunoreactive bands were detected with SuperSignal West Pico Chemiluminescent Substrate followed by film exposure for 1- to 5 minutes. The results from these studies suggested that anti-Recothrom antibodies cross react with human thrombins (α and γ), Evithrom brand of human thrombin, and bovine thrombin in a time dependent fashion. Although the Recothrom was not immunogenic in rabbit up to 90 days, antibodies harvested on day 150 cross-reacted with human α and γ thrombin as well as Evithrom. Visible cross reactivity, immunoblots around 36 KDa with human thrombins, bovine crude thrombin, and bovine thrombin 4A and 4B were observed with anti-Recothrom IgG collected from day 180. Bovine prothrombin did not exhibit any cross reactivity with anti-recothrombin antibodies at any time period. Thus, this data suggest that it is likely that antibodies against human recombinant thrombin may also cross-react with human thrombin and bovine thrombin. Therefore, the use of Recothrom may not be recommended in patients who have been previously exposed to human thrombin and bovine thrombin preparations. Disclosures:Fareed:King Pharmaceutical: Research Funding.

Comparison of Recombinant Human Thrombin and Plasma-Derived Human α-Thrombin

Bleeding can be a serious complication of surgery, and topical thrombin is widely used as an adjunct to hemostasis in diverse surgical settings. The potent hemostatic properties of thrombin derive from its ability to activate platelets directly to aggregate and adhere to damaged vessels and to catalyze the formation simultaneously of a fibrin matrix. Application of exogenous thrombin bypasses the physiological process of generating a thrombin burst by directly initiating the terminal reactions of blood clot formation. Currently, thrombin used to control surgical bleeding is primarily from bovine plasma, with a small percentage from human plasma. Human thrombin isolated from pooled plasma carries the risk of transmitting plasma-borne pathogens or prion diseases. The bovine preparations have been associated with protein and preparative contaminants that pose potential risks of developing cross-reacting antibodies. There is a need for a pure therapeutic preparation of human thrombin. Recombinant human thrombin (rhThrombin) has been efficiently produced from a prethrombin-1 precursor obtained from Chinese hamster ovary cell culture. This rhThrombin is substantially free of process-derived contaminants and has been characterized extensively in terms of composition, primary, secondary, and tertiary structure, enzymatic activity; and in vivo pharmacology. In vivo studies of topically applied rhThrombin have shown it is effective in achieving hemostasis in a rabbit liver excisional wound model. Clinical studies are ongoing to evaluate the safety and efficacy of rhThrombin as an adjunct to hemostasis in patients undergoing surgery.

Purification of salmon thrombin and its potential as an alternative to mammalian thrombins in fibrin sealants

A method to produce highly purified thrombin from salmon blood is described, and a series of biochemical, cell biologic, and biophysical assays demonstrate the functional similarities and some differences between salmon and human thrombins. Salmon thrombin with specific activity greater than 1000 units/mg total protein can be prepared by modifications of the methods used for purification of human thrombin. Using a synthetic substrate based on the human fibrinogen A-alpha polypeptide sequence as an indicator of enzymatic activity, salmon and human thrombin preparations contain similar specific activities per mass of purified protein. Salmon thrombin activates human fibrinogen and initiates the formation of fibrin clots whose structure and rheologic properties are indistinguishable from those of human fibrin clotted by human thrombin. Salmon thrombin also activates human platelets. Approximately 10 times higher activities are needed for the same rate of platelet aggregation compared to human thrombin, and some aspects of platelet activation, most notably phosphatidylserine exposure, are diminished relative to the effects of human thrombin. This latter finding suggests that salmon thrombin may not activate all of the receptors that are targets of human thrombin, although it does appear to activate signals that are sufficient to produce normal rates of activation and aggregation as measured by conventional aggregometry. Together with the recent purification of salmon fibrinogen and its application in mammalian wound healing, the availability of salmon thrombin allows the formulation of biological sealants devoid of any exogenous mammalian proteins and so may aid the design of materials with increased safety from infectious disease transmission.

Large-Scale Preparation of Human Thrombin: Polyethylene Glycol Potentiates the Factor Xa-Mediated Activation of Prothrombin

We investigated the ability of polyethylene glycol 4000 to accelerate thrombin generation in a mixture of prothrombin and factor X at concentrations of 1–30%. In the presence of 5 mM of CaCl 2, polyethylene glycol 4000 promoted prothrombin activation at concentrations above 1%. The peak of activation was seen at levels of 14 and 20% of polyethylene glycol 4000. The effect of the polyethylene glycol was remarkably dependent on its molecular weight; molecular weights greater than 2000 were required for accelerating thrombin generation. Under optimal conditions, polyethylene glycol 4000, in the presence of CaCl 2, promoted conversion of all of the prothrombin into thrombin and its derivatives. We conclude that polyethylene glycol 4000, at concentrations ranging from 14 to 20%, effectively accelerates thrombin generation in the presence of 5 mM of CaCl 2. This new method for preparing thrombin is based on the use of polyethylene glycol 4000 and CaCl 2 and is applicable to the manufacture of thrombin.

Transmission of serum hepatitis. 1970

There is evidence that transmission of serum hepatitis is associated with transmission of virus-like particles, approximately, 20 millimicron in diameter, containing the Australia or serum hepatitis (SH) antigen, which is currently referred to as the hepatitis associated antigen (HAA). Virus-like particles containing HAA were in the following materials, inoculation of which produced serum hepatitis: (1) a pool of human plasma, (2) serum obtained during the acute phase of hepatitis from a recipient of the plasma pool, (3) a preparation of human thrombin, and (4) serum from a proved hepatitis carrier. The HAA appeared in the serum samples of 61 individuals inoculated with these materials; serum hepatitis developed in 38 of them. Inoculation of dilutions of the plasma pool showed that serum hepatitis can be transmitted by materials containing HAA in amounts too low to be detected by current techniques.

Transmission of Serum Hepatitis

There is evidence that transmission of serum hepatitis is associated with transmission of virus-like particles, approximately, 20 millimicron in diameter, containing the Australia or serum hepatitis (SH) antigen, which is currently referred to as the hepatitis associated antigen (HAA). Virus-like particles containing HAA were in the following materials, inoculation of which produced serum hepatitis: (1) a pool of human plasma, (2) serum obtained during the acute phase of hepatitis from a recipient of the plasma pool, (3) a preparation of human thrombin, and (4) serum from a proved hepatitis carrier. The HAA appeared in the serum samples of 61 individuals inoculated with these materials; serum hepatitis developed in 38 of them. Inoculation of dilutions of the plasma pool showed that serum hepatitis can be transmitted by materials containing HAA in amounts too low to be detected by current techniques.

Isolation and characterization of multiple forms of human thrombin

Established procedures for the isolation of human prothrombin (1) and thrombin (2) have been modified so that large quantities of human thrombin could be isolated in a rapid, yet facile, manner. Thrombins derived from the activation of human prothrombin with thromboplastin or venom from the Australian Taipan (Oxyuranus Scutellatus Scutellatus) have been isolated and characterized with respect to their molecular weights and isoelectric points. Multiple active human thrombin components were found. Molecular weights of proteins found in these preparations conformed to the molecular weights reported for bovine thrombins. Activity studies with the synthetic substrate Benzoyl-Phe-Val-Arg-p-nitro-anilide and inhibitors fibrinopeptides A and B, have been performed. The majority of coagulant and esterase activity of the human thrombin preparations was associated with a protein component with an isoelectric point of 7,1. Coagulant, but no esterase activity, was found at isoelectric point 6, 7. Additional minor peaks of both activities had isoelectric points of 5,6 and 6,2, which are the sole isoelectric points reported previously for bovine thrombin. Results of the characterization studies show that thromboplastin and taipan venom activation produce very similar thrombins.

Vasodilator response in the hindlimb (dog) to various thrombin preparations.

ARTICLESVasodilator response in the hindlimb (dog) to various thrombin preparationsWL Joyner, LR Yonce, and PG IatridisWL Joyner, LR Yonce, and PG IatridisPublished Online:01 Aug 1973https://doi.org/10.1152/ajplegacy.1973.225.2.487MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByPleiotropic actions of factor Xa inhibition in cardiovascular prevention: mechanistic insights and implications for anti-thrombotic treatment15 September 2020 | Cardiovascular Research, Vol. 117, No. 9UNCONTROLLED HEMORRHAGE DIFFERS FROM VOLUME- OR PRESSURE-MATCHED CONTROLLED HEMORRHAGE IN SWINEShock, Vol. 28, No. 4Effects of Intraarterial Thrombin in the Swine ModelJournal of Vascular and Interventional Radiology, Vol. 12, No. 2Different Hypotensive Responses to Intravenous Bovine and Human Thrombin Preparations in SwineThe Journal of Trauma: Injury, Infection, and Critical Care, Vol. 50, No. 1 More from this issue > Volume 225Issue 2August 1973Pages 487-492 Copyright & PermissionsCopyright © 1973 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1973.225.2.487PubMed4722413History Published online 1 August 1973 Published in print 1 August 1973 Metrics

The Effects of Salts and of Ionic Strength on the Hydrolysis of TAME (p-toluenesulfonyl-L-arginine methyl ester) by Thrombin and Thrombokinase

SummaryThe effects of varying concentrations of alkali chlorides (LiCl, NaCl, KC1, RbCl and CsCl), alkaline earth chlorides (BeCl2, MgCl2, CaCl2, SrCl2, and BaCl2), choline chloride and Tris,HCl (pH 8.0 at 37°) on the rates of hydrolysis of TAME by a purified preparation of human thrombin and of bovine thrombokinase were determined in 0.25 M Tris.HCl buffer, pH 8.0 at 37°. Each salt had its own individual effects on the reactions and these effects were completely different when thrombin instead of thrombokinase was used. Each salt, however, had the same qualitative effects on TAME hydrolysis by crude bovine thrombin as by purified human thrombin, but only minor effects on the hydrolysis of TAME by trypsin.With the exception of LiCl, low concentrations of alkali chlorides had inhibitory and high concentrations had acceleratory effects on both the thrombin-TAME and the thrombokinase-TAME reactions. LiCl had no accelerating effects on either reaction and it was a stronger inhibitor of thrombokinase than of thrombin. In contrast to the alkali chlorides, the alkaline earth chlorides had no acceleratory effects but had inhibitory effects on both reactions. MgCl2, however, was an exception. It weakly accelerated the thrombokinase -TAME and weakly inhibited the thrombin -TAME reaction. When comparing 0.15 M concentrations of the salts (with the exception of BeCl2 which was the strongest inhibitor of both reactions), NaCl was the strongest inhibitor of the thrombin - TAME reaction, followed by CaCl2, but BaCl2 was the strongest inhibitor of the thrombokinase -TAME reaction, followed by KC1 and LiCl.Choline chloride and Tris.HCl in concentrations up to 3 M had no significant effects on the rate of hydrolysis of TAME by either human or bovine thrombin. Increasing the ionic strength above 0.16 (lower values were not tested), therefore, may have no effect on this reaction, and all of the inhibitions and accelerations found in the presence of the alkali chlorides or the alkaline earth chlorides may be entirely due to the individual cations. On the other hand, increasing the ionic strength may produce a small inhibitory effect, as found in the presence of LiCl or MgCl2, and the lack of any effect in presence of even large concentrations of Tris.HCl or choline chloride may be due to a fortuitous balancing of the weak inhibitory effect of ionic strength by the weak acceleratory effect of the choline and Tris cations.Choline chloride and Tris.HCl had no inhibitory effects but accelerated the thrombokinase -TAME reaction. Choline chloride was the strongest accelerator tested. Rates in the presence of 0.15 and 3 M choline chloride were respectively 204 and 601 % of the controls. Although the effects of ionic strength changes on this reaction could not be established, the data indicate that increasing the ionic strength has at most only a small effect, and the inhibitions and accelerations found are due primarily to the specific cations present.

Transmission of Serum Hepatitis

There is evidence that transmission of serum hepatitis is associated with transmission of virus-like particles, approximately 20 mμ in diameter, containing the Australia or serum hepatitis (SH) antigen, which is currently referred to as the hepatitis associated antigen (HAA). Virus-like particles containing HAA were in the following materials, inoculation of which produced serum hepatitis: (1) a pool of human plasma, (2) serum obtained during the acute phase of hepatitis from a recipient of the plasma pool, (3) a preparation of human thrombin, and (4) serum from a proved hepatitis carrier. The HAA appeared in the serum samples of 61 individuals inoculated with these materials; serum hepatitis developed in 38 of them. Inoculation of dilutions of the plasma pool showed that serum hepatitis can be transmitted by materials containing HAA in amounts too low to be detected by current techniques.

A Comparison of the Esterase (TAMe) and Clotting Activities of Human and Bovine Thrombin Preparations*

A comparison has been made between the clotting activity (C) and TAMe esterase activity (E) during prothrombin activation and subsequent purification of human thrombin. A limited number of studies were made using bovine thrombin for comparison purposes. Under certain circumstances, the ratio of C to E for human thrombin is not constant, particularly upon gel-filtration at low ionic strength and during prolonged contact with 25% sodium citrate. Under other circumstances, however, these enzymic parameters are stable and human thrombin appears to have a ratio of C to E of approximately 6 while bovine thrombin has a ratio of 3. This finding may confirm the observations of others concerning the higher specific activity of human thrombin than the bovine enzyme against bovine fibrinogen. Only a single component having C and E activity has been isolated from human prothrombin preparations activated to thrombin by sodium citrate or biologically and purified by either gel filtration or ion exchange chromatography. Kinetic studies on human thrombin preparations indicate that in all other respects they are similar in enzymic properties to those reported for the purified bovine enzyme.

Fibrinolysis in Peptone and Anaphylactic Shock in the Dog

FIBRINOLYSIS in peptone and anaphylactic shock can be demonstrated either directly, by estimation of flbrinogen in samples taken after the injection of the respective agent, or by adding protamine in order to counteract the effect of the heparin which is discharged into the blood stream. We have shown1 that even the addition of an excess of a powerful preparation of human thrombin is unable to correct the decrease in fibrinogen, which can be estimated by the method of Cullen and Van Slyke2. Since thrombin was able to correct the inhibitory effect of heparin added in amounts such as those appearing in anaphylactic shock, we conclude that a drop in fibrinogen actually occurs during anaphylactic and peptone shock. Moreover, in the case of peptone shock, fibrinolysis can be directly observed, since in several cases the blood clots very quickly and the coagulum redissolves after a few hours. Apparently, Nolf3 was the first to describe this flbrinolytic effect as occurring after peptone injection, in animals in which the liver had been removed or the circulation reduced to the upper part of the body, by ligaturing the thoracic aorta and inferior vena cava, just above the diaphragm. It is now clear that in Nolf's experiments, the removal of the liver prevented the discharge of heparin, since it has been proved that heparin comes from the liver in peptone and anaphylactic shock4.