Removal from Bovine Prothrombin of the Substrate for Russell’s Viper Venom

1969 ◽  
Vol 21 (02) ◽  
pp. 203-216 ◽  
Author(s):  
J. H Milstone ◽  
N Oulianoff

SummaryBovine prothrombin was prepared by adsorption on barium sulfate. After elution, it was passed through thick filter-cakes of Standard Super-Cel, which removed some venom substrate (factor X). Almost all the remaining venom substrate was removed by repeated passage through columns of DEAE-cellulose. Finally, the ratio of venom substrate to prothrombin was considerably less than 1/1,000 that of plasma. The prothrombin was also poor in factor V. It yielded very little thrombin upon incubation with Russell’s viper venom, factor V, phospholipid and calcium chloride. However, inclusion of bovine plasma at a final dilution of 1/10,000 caused the mixture to produce thrombin rapidly. This system offers promise for the assay of venom substrate in plasma.Thrombokinase derived from bovine plasma was able, at 0.000071 mg/ml, to substitute for both the venom and its substrate in thrombin-producing systems. However, with this small amount of thrombokinase, phospholipid was indispensable. The system was sensitive to 0.00001 mg phospholipid/ml.With 1,000 times as much thrombokinase, prothrombin was activated without addition of accessory factors, in the presence of oxalate. Removal of venom substrate did not affect this response of prothrombin. Nor did removal of venom substrate from the prothrombin prevent its activation by crystallized trypsin in the presence of oxalate.

1961 ◽  
Vol 06 (03) ◽  
pp. 435-444 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers

SummaryAn attempt was made to obtain Ac-globulin from bovine plasma. The concentrates contain mostly protein, and phosphorus is also present. The stability characteristics vary from one preparation to another, but in general there was no loss before 1 month in a deep freeze or before 1 week in an icebox, or before 5 hours at room temperature. Reducing agents destroy the activity rapidly. S-acetylmercaptosuccinic anhydride is an effective stabilizing agent. Greatest stability was at pH 6.0.In the purification bovine plasma is adsorbed with barium carbonate and diluted 6-fold with water. Protein is removed at pH 6.0 and the Ac-globulin is precipitated at pH 5.0. Rivanol and alcohol fractionation is followed by chromatography on Amberlite IRC-50 or DEAE-cellulose. The final product is obtained by isoelectric precipitation.


1977 ◽  
Author(s):  
M.C. Guillin ◽  
A. Bezeaud ◽  
J.P. Freeman ◽  
C.M. Jackson

It is known that prior to bind bovine prothrombin and to become fully functional, bovine Factor V must itself be “activated” by either thrombin or an enzyme isolable from Russell’s viper venom. The purpose of this work was to determine if Factor V activation is also required in order for it to bind bovine Factor Xa.This has been investigated by measuring the binding of both “native” (unactivated) Factor V and Factor V activated by the Russell’s viper venom activating enzyme, to a column of agarose-bound Factor Xa. The experiments were also performed using diisopropylfluorophosphate (DFP) inhibited Factor Xa covalently bound to agarose. Both purified bovine Factor V (Va) and bovine plasma were used and gave the same results. In order to prevent initiation of clotting in bovine plasma, heparin wad added to the plasma to promote inactivation of Factor Xa by antithrombin III.The results indicate that Factor V activation is a prerequisite for it to bind Factor Xa ; Factor Va binds both Factor Xa and DFP inhibited Factor Xa, unmodified Factor V does not.These experiments suggest that Factor V may not participate in prothrombin activation at all, until after some thrombin has been formed. If this is so, an alternate pathway by which the first thrombin is generated must be considered and may be proposed to be simply that involving Factor Xa, phospholipid and Ca2+ alone.


1981 ◽  
Author(s):  
D L Aronson ◽  
J Bagley

The in vitro correction of the prolonged APTT of hemophilic plasma has been ascribed to an uncharacterized entity “Factor VIII Bypassing Activity.” Such products also correct the prolonged APTT plasma deficient in Factor IX, Factor X and Factor XII, but not of Factor V deficient plasma. Correction of the APTT in Factor VIII deficient plasma by early stage coagulants such as Factor XIIa, Kallikrein and Factor IXa is minimal. These results indicate that this in vitro activity acts at the level of either the activation of Factor X or the activation of prothrombin.A coagulant has been prepared from serum by barium precipitation, heparin-agarose, DEAE cellulose and high pressure liquid chromatography (HPLC). The in vitro coagulant properties are similar to “activated” prothrombin complex (Autoplex) and the biologic and chemical properties are identical to activated Factor X.Infusion of the partially purified serum coagulant into normal dogs was well tolerated and, in contrast to Factor IX concentrates, gave no signs of DIC. Infusion into bleeding hemophilic dogs had no hemostatic effect. It is concluded that a major portion of the in vitro potency of activated prothrombin concentrates is due to activated Factor X, a material which when infused has no in vivo hemostatic effect.Acknowledgments - The authors gratefully acknowledge the studies of Dr. Henry Kingdon in hemophilic dogs.


1977 ◽  
Author(s):  
H. Vinazzer

The exact action of factor VIII inhibitor bypassing activity (FEIBA) is still unclear. For this reason, a series of experimental studies was carried out. Procoagulant activities were examined by standard one-stage methods while factor Xa and thrombin were measured by chromogenic substrates. Activities of factors II, VII, IX, and X were similar to PPSB fractions. In addition, low factor V activity and a phospholipid were detected. No activated factor X was present in FEIBA but there was a trace amount of 2.1 NIH units of thrombin per 100 FEIBA units. On addition of calcium chloride slow thrombin formation could be observed which however, reached 1100 NIH units of thrombin per 100 FEIBA units within an incubation time of 10 min. The velocity of thrombin formation was greatly enhanced by addition of a PTT reagent and of thromboplastin respectively. Factor Xa on the other hand, was neither formed after addition of calcium chloride nor by a PTT reagent. Tissue thromboplastin however, activated Xa from FEIBA in the same manner as a PTT reagent plus barium sulfate plasma. From these results, the conclusion could be drawn that thrombin could readily be made available from FEIBA while activation of Xa either needed the complete endogenous pathway or the presence of tissue thromboplastin. The procoagulant activity of FEIBA therefore, could be attributed to direct thrombin formation. By this process, an activation of the clotting mechanism in plasmas deficient in endogenous coagulation factors, and a complete independence from the presence or absence of a specific antibody could be explained.


Biochemistry ◽  
1969 ◽  
Vol 8 (4) ◽  
pp. 1397-1405 ◽  
Author(s):  
Sandra Schiffman ◽  
Ida Theodor ◽  
Samuel I. Rapaport
Keyword(s):  
Factor V ◽  
Factor X ◽  

1987 ◽  
Author(s):  
Than Than ◽  
Khin Ei Han ◽  
Hutton RA ◽  
Mvint Lwin ◽  
Tin Nu Swe ◽  
...  

Amongst its many actions, Russell's viper (RV) venom activates factors X and V and enhances fibrinolysis, leading to defibrination which contributes to the clinical sequelae of RV bite. Early administration of antivenom may be life-saving, but not all of those bitten become sufficiently envenomed to require treatment. In an attempt to predict at an early stage those subjects who will progress to defibrination, we have serially monitored the haemostatic changes in 20 bite victims using the PT, APTT, thrombin time, platelet count, assays for factors X and V and fibrinogen and fibrin(ogen) degradation products (FDP).In five patients, no evidence of defibrination was seen at any time and none of these developed obvious clinical symptoms. In a further six subjects, slight prolongation of the PT (16-21/14s), APTT (39-51/38s) and thrombin time (16-25/14s) occurred concomitantly with a moderate fall in factor X (20-80%), factor V (30-66%) and fibrinogen (0.6-2.Og/1), but FDP never exceeded 40ug/ml. In the remaining nine subjects who all eventually defibrinated completely, moderate coagulation factor deficiency and thrombocytopenia developed as early as 1-2h after bite. The most pronounced and consistent changes were a rise in FDP to above 80ug/ml (80-640ug/ml) and a fall in factor V (2-50%), these results being obtained on admission, 1-12h after bite. We conclude that an FDP level of 80ug/ml or more is highly suggestive of impending defibrination and could be regarded as a criterion for commencing antivenom therapy.


Blood ◽  
1963 ◽  
Vol 21 (6) ◽  
pp. 745-754 ◽  
Author(s):  
THEODORE H. SPAET ◽  
JOSÉ CINTRON

Abstract The basic reagent used was an eluate obtained from barium sulfate used to adsorb various sera. When this eluate was prepared from normal rabbit serum, it responded to treatment with coagulants from adsorbed plasma, with Stypven, or with 25 per cent sodium citrate to give products with similar if not identical properties. With each preparation a stable complex formed with cephalin which withstood washing, was relatively heat-stable, was inactivated by adsorbed serum, and which required factor V for optimal prothrombin conversion. In eluates prepared from human serum, normal activation occurred in the absence of factor IX, but was defective in the absence of factor X. A preparation of factor X purified by DEAE cellulose chromatography was activated by 25 per cent sodium citrate. It is suggested that product I, the product of Stypven activation, and autoprothrombin C represent similar or identical reagents; it is further suggested that factor X is their common precursor.


1973 ◽  
Vol 131 (4) ◽  
pp. 791-797 ◽  
Author(s):  
Jolyon Jesty ◽  
M. Peter Esnouf

The preparation of activated Factor X from reaction mixtures of bovine Factor X and Russell's-viper venom is described. The molecular weight of purified protein varies about a mean value of 40000; this variation is the result of at least two forms of Factor Xa. The action of activated Factor X, together with purified Factor V, was studied on purified prothrombin and the reaction products were isolated. In addition to thrombin, two other polypeptides with molecular weights of 16000 and 19500 were recovered.


1974 ◽  
Vol 32 (01) ◽  
pp. 057-064 ◽  
Author(s):  
Y Nemerson ◽  
S.A Silverberg ◽  
J Jesty

SummaryTwo reactions of the extrinsic pathway of coagulation, the activations of Factor X and prothrombin, have been studied in purified systems and shown to be self-damping. Factor X was activated by the tissue factor - Factor VII complex, and prothrombin by two systems: the coagulant protein of Taipan venom, and the physiological complex of activated Factor X, Factor V, lipid, and calcium ions. In each case the yield of enzyme, activated Factor X or thrombin, is a function of the concentration of activator. These and other observations are considered as a basis for a control mechanism in coagulation.


1994 ◽  
Vol 72 (06) ◽  
pp. 862-868 ◽  
Author(s):  
Frederick A Ofosu ◽  
J C Lormeau ◽  
Sharon Craven ◽  
Lori Dewar ◽  
Noorildan Anvari

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.


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