SEPARATION OF AUTOPROTHROMBIN III FROM BOVINE PROTHROMBIN PREPARATIONS

1964 ◽  
Vol 42 (2) ◽  
pp. 229-233 ◽  
Author(s):  
Walter H. Seegers ◽  
Edmond R. Cole ◽  
Nobuo Aoki ◽  
Charles R. Harmison
Keyword(s):  
Calcium Ions ◽  
Blood Clotting ◽  
Sodium Citrate ◽  
Single Component ◽  
Normal Blood ◽  
Citrate Solution ◽  
Tissue Extracts ◽  
Autoprothrombin C

Purified prothrombin was activated by means of purified thrombin, Ac-globulin, calcium ions, and crude "cephalin." Thrombin and autoprothrombin III generated. The latter was isolated as a single component by the same methods found suitable for the isolation of autoprothrombin C. It contained no autoprothrombin C activity, but some generated spontaneously, and also in 25% sodium citrate solution. It may be that autoprothrombin C generally does not form in normal blood clotting unless tissue extracts are involved. This implies the possibility that the most potent procoagulant power in the genesis of thrombosis is derived from tissues.

AUTOPROTHROMBIN C: A SECOND ENZYME FROM PROTHROMBIN

1962 ◽  
Vol 40 (1) ◽  
pp. 597-605 ◽  
Author(s):  
Ewa Marciniak ◽  
Walter H. Seegers
Keyword(s):  
Calcium Ions ◽  
Esterase Activity ◽  
Sodium Citrate ◽  
Citrate Solution ◽  
Tissue Extracts ◽  
End Products ◽  
Ph Range ◽  
Autoprothrombin C ◽  
Prothrombin Activation ◽  
Rapid Conversion

In addition to thrombin, there is another derivative of prothrombin which is an end product of prothrombin activation. It is an accelerator of prothrombin activation, and is called autoprothrombin C. The activity develops from purified bovine prothrombin in 25% sodium citrate solution simultaneously with thrombin. It has been separated from thrombin by chromatography on Amberlite IRC-50 under the conditions previously used for the isolation of thrombin. The fraction which separates from thrombin has esterase activity and very likely this esterase activity is associated with the autoprothrombin C molecule. Since the autoprothrombin C and the thrombin are both derived from prothrombin, at least two enzymes are the end products of prothrombin activation. Autoprothrombin C catalyzed the activation of purified prothrombin in 25% sodium citrate solution, and this function was easily inhibited with p-toluenesulphonyl-L-arginine methyl ester. Autoprothrombin C preparations were mixed with platelets, Ac-globulin, and calcium ions to obtain rapid conversion of purified prothrombin to thrombin. This activation mixture did not generate autoprothrombin C and some unspecified substance most likely needs to be added in order to obtain the autoprothrombin C activity. The activity developed together with thrombin when tissue extracts, Ac-globulin, and calcium ions were used for the activation of prothrombin. Autoprothrombin C is relatively stable over the pH range 5.5 to 8.5. It is stable up to 56 °C for 30 minutes. Plasma contains a substance that inactivates autoprothrombin C.

AUTOPROTHROMBIN C: A SECOND ENZYME FROM PROTHROMBIN

1962 ◽  
Vol 40 (5) ◽  
pp. 597-605 ◽  
Author(s):  
Ewa Marciniak ◽  
Walter H. Seegers
Keyword(s):  
Calcium Ions ◽  
Esterase Activity ◽  
Sodium Citrate ◽  
Citrate Solution ◽  
Tissue Extracts ◽  
End Products ◽  
Ph Range ◽  
Autoprothrombin C ◽  
Prothrombin Activation ◽  
Rapid Conversion

In addition to thrombin, there is another derivative of prothrombin which is an end product of prothrombin activation. It is an accelerator of prothrombin activation, and is called autoprothrombin C. The activity develops from purified bovine prothrombin in 25% sodium citrate solution simultaneously with thrombin. It has been separated from thrombin by chromatography on Amberlite IRC-50 under the conditions previously used for the isolation of thrombin. The fraction which separates from thrombin has esterase activity and very likely this esterase activity is associated with the autoprothrombin C molecule. Since the autoprothrombin C and the thrombin are both derived from prothrombin, at least two enzymes are the end products of prothrombin activation. Autoprothrombin C catalyzed the activation of purified prothrombin in 25% sodium citrate solution, and this function was easily inhibited with p-toluenesulphonyl-L-arginine methyl ester. Autoprothrombin C preparations were mixed with platelets, Ac-globulin, and calcium ions to obtain rapid conversion of purified prothrombin to thrombin. This activation mixture did not generate autoprothrombin C and some unspecified substance most likely needs to be added in order to obtain the autoprothrombin C activity. The activity developed together with thrombin when tissue extracts, Ac-globulin, and calcium ions were used for the activation of prothrombin. Autoprothrombin C is relatively stable over the pH range 5.5 to 8.5. It is stable up to 56 °C for 30 minutes. Plasma contains a substance that inactivates autoprothrombin C.

Autocatalysis in prothrombin activation

1962 ◽  
Vol 203 (3) ◽  
pp. 397-400 ◽  
Author(s):  
Walter H. Seegers ◽  
Ewa Marciniak ◽  
Edmond R. Cole
Keyword(s):  
Methyl Ester ◽  
Calcium Ions ◽  
Thrombin Generation ◽  
Sodium Citrate ◽  
Citrate Solution ◽  
Intermediate Products ◽  
End Products ◽  
Autoprothrombin C ◽  
Autocatalytic Activation ◽  
Prothrombin Activation

Two enzymes, thrombin and autoprothrombin C, are derived from purified prothrombin by autocatalytic activation in 25% sodium citrate solution. The thrombin but not the autoprothrombin C activity is destroyed by diisopropylfluorophosphate. Autoprothrombin C is a procoagulant, which catalyzes the conversion of prothrombin to thrombin in a prothrombin-activating mixture consisting of calcium ions, Ac-globulin, and crude cephalin. Depending upon the amount of p-toluenesulphonyl-l-arginine methyl ester added to the prothrombin-activation mixture the thrombin generation may be retarded or inhibited completely. The view is expressed that all prothrombin activations are fundamentally autocatalytic. The end products of prothrombin activation involved are autoprothrombin C and thrombin, while the intermediate products of prothrombin activation are the autoprothrombins.

Autoprothrombin C Activity from Prothrombin with Snake Venom or Trypsin

1962 ◽  
Vol 08 (03) ◽  
pp. 425-433 ◽  
Author(s):  
Ewa Marciniak ◽  
Edmond R Cole ◽  
Walter H Seegers
Keyword(s):  
Snake Venom ◽  
Thrombolytic Agent ◽  
Sodium Citrate ◽  
Specific Activity ◽  
High Specific Activity ◽  
Citrate Solution ◽  
Clotting Factors ◽  
Activation Products ◽  
Russell’S Viper ◽  
Autoprothrombin C

SummarySuitable conditions were found for the generation of autoprothrombin C from purified prothrombin with the use of Russell’s viper venom or trypsin. DEAE chromatographed prothrombin is structurally altered and has never been found to yield autoprothrombin C and also did not yield it when Russell’s viper venom or trypsin were used. Autoprothrombin C is derived from prothrombin with tissue extract thromboplastin, but not in large amounts with the intrinsic clotting factors. With the latter thrombin and autoprothrombin III are the chief activation products. Autoprothrombin III concentrates were prepared from serum and upon activation with 25% sodium citrate solution or with Russell’s viper venom large amounts of autoprothrombin C were obtained, and this was of high specific activity. Theoretically trypsin is not a thrombolytic agent, but on the contrary should lead to intravascular clotting.

Conversion of Prothrombin to Autoprothrombin II (Platelet Cofactor II) and Its Relation to the Blood Clotting Mechanisms

1956 ◽  
Vol 184 (2) ◽  
pp. 259-264 ◽  
Author(s):  
Walter H. Seegers ◽  
Shirley A. Johnson
Keyword(s):  
Blood Clotting ◽  
Sodium Citrate ◽  
Hemophilia A ◽  
The Other ◽  
Citrate Solution ◽  
Serum Samples ◽  
Platelet Factor ◽  
Healthy Persons

Under certain restricted conditions purified thrombin can be added to purified prothrombin and the latter transforms to a derivative called autoprothrombin II. Except in 25% sodium citrate solution, it has not been converted to thrombin. It functions as a cofactor with purified platelet factor 3 in the conversion of prothrombin to thrombin. Serum from healthy persons and in hemophilia A contains autoprothrombin II activity that can be adsorbed on BaCO3. Autoprothrombin II, prepared in the laboratory, can be added to the two types of adsorbed serum and the autoprothrombin II activity is restored to the serum. Serum from PTA patient, and a patient receiving dicumarol was found to contain very little autoprothrombin II activity. By adding an appropriate quantity of autoprothrombin II, prepared in the laboratory, the autoprothrombin II activity of the PTC serum was not completely restored whereas it was with the other two serum samples.

Blood-Clotting Enzymology

1968 ◽  
Vol 14 (2) ◽  
pp. 97-115 ◽  
Author(s):  
Walter H Seegers ◽  
Lowell McCoy ◽  
Ewa Marciniak
Keyword(s):  
Vitamin K ◽  
Calcium Ions ◽  
Blood Clotting ◽  
Tissue Injury ◽  
Accessory Protein ◽  
Plasma Component ◽  
Protein Aggregate ◽  
Quarter Century ◽  
Thrombin Activity ◽  
Autoprothrombin C

Abstract During the past quarter century perhaps no single phenomenon has attracted so much attention in biology and medicine as blood clotting. It plays a role in numerous clinical conditions where it was not taken into account previously. Knowledge of the basic mechanisms recently passed through a tumulous stage of growth, but is now delineated in terms of simple enzymology. Most of the components have been obtained in purified form. There are three main events: the formation of autoprothrombin C (F-Xa, thrombokinase), the formation of thrombin, and the formation of fibrin. Prothrombin itself is a more marvelous protein than was predicted. It is a protein aggregate that contains the precursor of thrombin, the precursor of autoprothrombin C, as well as accessory protein. When blood coagulation is initiated by tissue injury, large amounts of autoprothrombin C activity arise in the presence of calcium ions and tissue thromboplastin. When there is no tissue injury, small amounts of autoprothrombin C are produced by platelet cofactor I (F-VIII, AHF) which functions with the lipids of platelets. Autoprothrombin C is the enzyme which produces thrombin activity, and being a part of the prothrombin aggregate it is in close proximity to the specific bond(s) that must be split to get thrombin activity. Although autoprothrombin C alone is sufficient to produce thrombin, the reaction is greatly accelerated by plasma Ac-globulin (F-V), lipids from platelets, and calcium ions. The acceleration produced by components functioning in pairs and relays makes possible the essential integration with the anatomic location of resources. Tissue materials pair with plasma resources to produce autoprothrombin C, and then they pair with another plasma component (Ac-globulin) to make autoprothrombin C function. In the absence of tissue injury, platelet materials pair with plasma resources to produce autoprothrombin C, and then platelets again work with another plasma component (Ac-globulin) to support the function of autoprothrombin C. Platelets are essential because their lipids are needed for the formation of autoprothrombin C, as well as for accelerating its function. Whatever involves platelets can be the stimulus to accelerate blood clotting; this includes Hageman factor. Platelets are the true point of beginning for the first step in blood clotting and are involved in the second and third. Vitamin K deficiency, or counteraction of Vitamin K by Dicumarol reduces prothrombin synthesis by the liver. As a consequence the precursor of thrombin, the precursor of autoprothrombin C, and the accessory protein of the molecular aggregate which constitutes prothrombin is reduced in concentration. It has been found possible to produce abnormal conditinos experimentally and have relatively more of the precursor of thrombin released to the blood than is normally associated with the precursor of autoprothrombin C. Up to the point of incorporation to form a single prothrombin molecule, the synthesis of subunits proceeds by separately operating mechanisms.

Transformation of Autoprothrombin III to Autoprothrombin C in Sodium Citrate Solution

1968 ◽  
Vol 19 (01/02) ◽  
pp. 204-212 ◽  
Author(s):  
R Kipfer ◽  
W. H Seegers
Keyword(s):  
Trypsin Inhibitor ◽  
Sodium Citrate ◽  
Competitive Inhibitor ◽  
Soybean Trypsin Inhibitor ◽  
Citrate Solution ◽  
Phenyl Sulfone ◽  
Autoprothrombin C ◽  
Zero Time

SummaryAll reactions studied occurred in 25% sodium citrate solution. The conversion of prethrombin to thrombin with autoprothrombin C was retarded by 3,4,4’-triaminodi-phenyl sulfone. The compound functioned as a competitive inhibitor. Purified autoprothrombin III converted to autoprothrombin C more rapidly when autoprothrombin C was added at zero time. Soybean trypsin inhibitor, which neutralizes autopro-thrombin C activity, blocked the conversion of autoprothrombin III to autoprothrom-bin C, and 3,4,4’-triaminodiphenylsulf one inhibited the development of autoprothrombin C activity. The activation of prothrombin in 25% sodium citrate solution consists of three main events; namely, 1. the dissociation of prothrombin into subunits, 2. the formation of autoprothrombin C, and 3. the formation of thrombin.

Enzymes and Blood Clotting. II. Autoprothrombin C, Russell’s Viper Venom (Stypven), and Trypsin

1963 ◽  
Vol 09 (01) ◽  
pp. 062-073 ◽  
Author(s):  
John H Ferguson ◽  
Ella Gray Wilson Ennis
Keyword(s):  
Human Blood ◽  
Calcium Ions ◽  
Present Status ◽  
Blood Clotting ◽  
Blood Platelets ◽  
Accessory Factor ◽  
Russell’S Viper ◽  
Autoprothrombin C ◽  
Human Blood Platelets ◽  
Lipid Requirement

SummaryIn its dependence upon prothromboplastic phosphatide and calcium ions, in the conversion of prothrombin to thrombin, Autoprothrombin C is a thromboplastic enzyme. The only other demonstrated accessory factor is V, and any needs for VII, X, XI, or XII are ruled out by the present experiments, as were VIII or IX formerly. The lipid requirement may be satisfied by either phosphatidylserine (PS) or phosphatidylethanolamine (PE), purified from human blood platelets. Comparisons are made with Russell’s viper venom (“stypven”), and with trypsin. The trypsin potentiation of factors VII and X is prevented by pancreatic inhibitor (PI). However, when PI is added after this maximal potentiation, it is no longer inhibitory. The present status of knowledge concerning enzymes in relation to blood coagulation is discussed.

On the Purification and Conversion of Human Prothrombin

1959 ◽  
Vol 03 (02) ◽  
pp. 194-213 ◽  
Author(s):  
F Streuli
Keyword(s):  
Calcium Ions ◽  
Sodium Citrate ◽  
Citrate Solution ◽  
Isoelectric Precipitation ◽  
Clotting System ◽  
Immunological Method

SummaryA combined chemical and immunological method for the purification of human prothrombin is described. After isoelectric precipitation a purified prothrombin is obtained. Its activity per mg of protein is 210 folds higher than that of plasma. This prothrombin and the prothrombin purified on a BaSO4 column are not converted into thrombin in a 25% sodium-citrate solution. No thrombin is formed by the addition of calcium ions and brain thromboplastin. For its conversion, a complete clotting system (extrinsic or intrinsic) is necessary. Our experiments present no evidence for the hypothesis that Factors VII, IX, PPA and Stuart-Prower are prothrombin derivatives.The conversion of prothrombin to thrombin by means of isolated blood thromboplastin is studied. The results still do not permit one to decide whether the reaction is stoichiometric or enzymatic.

Activation of Prothrombin

1958 ◽  
Vol 193 (1) ◽  
pp. 169-180 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers
Keyword(s):  
Calcium Ions ◽  
Esterase Activity ◽  
Sodium Citrate ◽  
Strong Solutions ◽  
Sulfate Solution ◽  
Protamine Sulfate ◽  
Citrate Solution ◽  
Platelet Factor ◽  
Autocatalytic Activation ◽  
Prothrombin Activation

In experiments with purified biothrombin, it was found that strong solutions of sodium citrate or protamine sulfate (0.1% w/v) or purified platelet factor 3 depress the esterase activity and leave the clotting power unaltered. Apparently a depression of esterase activity is beneficial for the autocatalytic activation of purified prothrombin. In protamine sulfate solution, prothrombin gradually becomes thrombin and the yield of thrombin is even higher than in 25% sodium citrate solution. Prothrombin also depresses the esterase activity of biothrombin, and itself serves as a substrate for the enzyme thrombin. When prothrombin becomes an inactive derivative or a substance refractory to being converted to thrombin in the presence of Ac-globulin, thromboplastin and calcium ions, it can nevertheless be changed to thrombin with the use of thrombin as a catalyst, just as was previously accomplished with the use of 25% sodium citrate solutions. Theoretically, a prothrombin derivative(s) can serve as substrate competitor for thrombin and thus be an accelerator of prothrombin activation, or the derivative, under appropriate conditions can itself give rise to thrombin. Thrombin as activator of prothrombin can account for all observed conditions of prothrombin activation. The discovery of thrombin as activator of prothrombin offers a simplified view of the entire blood coagulation mechanisms. Two equations can describe the basic events: Prothrombin(See PDF for Equation)Thrombin; Fibrinogen(See PDF for Equation)Fibrin. Other factors support the production and enzymic function of thrombin and these are called procoagulants. Opposed to these, and normally in exact balance, are those factors that hinder the production or function of thrombin and these are called anticoagulants. In the presence of thrombin prothrombin can change to thrombin without Ac-globulin. Plasma Ac-globulin changes to serum Ac-globulin in the presence of thrombin but not with esterase thrombin. Consequently, the depression of esterase activity does not impair the capacity of thrombin to make the beneficial alteration in Ac-globulin.

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