SOME PROPERTIES OF THROMBIN PREPARATIONS

1959 ◽  
Vol 37 (6) ◽  
pp. 775-785 ◽  
Author(s):  
Walter H. Seegers ◽  
Gerardo Casillas ◽  
Robert S. Shepard ◽  
William R. Thomas ◽  
Paul Halick
Keyword(s):  
Esterase Activity ◽  
Sodium Citrate ◽  
Citrate Solution ◽  
Ideal Model ◽  
Terminal Amino Acid ◽  
Two Stage ◽  
Analytical Reagents ◽  
Terminal Amino ◽  
Autocatalytic Activation ◽  
Prothrombin Activation

Bio-resin thrombin preparations were found to contain three weak precipitinogens. The clotting activity was not demonstrably associated with the precipitinogenic systems. Further, work was done on methods for the purification of citrate resin thrombin, and its clotting activity is also not associated with a precipitinogenic system. The N-terminal amino acid of both bio-resin thrombin and citrate resin thrombin was found to be glutamic acid. The two preparations were found to be homogeneous upon ultracentrifugal examination and could not be differentiated on the basis of sedimentation constants. Since "citrate" activation and "bio" activation produce eventually similar thrombin material, the autocatalytic activation of prothrombin in 25% sodium citrate solution can be used as an ideal model of prothrombin activation. The prothrombin first dissociates to form a derivative that does not form thrombin in the two-stage analytical reagents. Then a second alteration occurs in which the derivative again may form thrombin in the two-stage analytical reagents. Then thrombin activity appears as esterase activity, then as clotting activity. Later the clotting activity may be lost and finally also the esterase activity. The original prothrombin is a precipitinogen while the active thrombin is not.

SOME PROPERTIES OF THROMBIN PREPARATIONS

1959 ◽  
Vol 37 (1) ◽  
pp. 775-785 ◽  
Author(s):  
Walter H. Seegers ◽  
Gerardo Casillas ◽  
Robert S. Shepard ◽  
William R. Thomas ◽  
Paul Halick
Keyword(s):  
Esterase Activity ◽  
Sodium Citrate ◽  
Citrate Solution ◽  
Ideal Model ◽  
Terminal Amino Acid ◽  
Two Stage ◽  
Analytical Reagents ◽  
Terminal Amino ◽  
Autocatalytic Activation ◽  
Prothrombin Activation

Bio-resin thrombin preparations were found to contain three weak precipitinogens. The clotting activity was not demonstrably associated with the precipitinogenic systems. Further, work was done on methods for the purification of citrate resin thrombin, and its clotting activity is also not associated with a precipitinogenic system. The N-terminal amino acid of both bio-resin thrombin and citrate resin thrombin was found to be glutamic acid. The two preparations were found to be homogeneous upon ultracentrifugal examination and could not be differentiated on the basis of sedimentation constants. Since "citrate" activation and "bio" activation produce eventually similar thrombin material, the autocatalytic activation of prothrombin in 25% sodium citrate solution can be used as an ideal model of prothrombin activation. The prothrombin first dissociates to form a derivative that does not form thrombin in the two-stage analytical reagents. Then a second alteration occurs in which the derivative again may form thrombin in the two-stage analytical reagents. Then thrombin activity appears as esterase activity, then as clotting activity. Later the clotting activity may be lost and finally also the esterase activity. The original prothrombin is a precipitinogen while the active thrombin is not.

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.

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.

PURIFICATION OF PROTHROMBIN AND THROMBIN BY CHROMATOGRAPHY ON CELLULOSE

1960 ◽  
Vol 38 (1) ◽  
pp. 1405-1410 ◽  
Author(s):  
Walter H. Seegers ◽  
Ricardo H. Landaburu
Keyword(s):  
Amino Acid ◽  
Sodium Citrate ◽  
Fundamental Difference ◽  
High Yield ◽  
Citrate Solution ◽  
Terminal Amino Acid ◽  
Protamine Sulphate ◽  
Bovine Thrombin ◽  
Diethylaminoethyl Cellulose ◽  
Terminal Amino

A method is described for obtaining purified bovine thrombin by chromatography on phosphate cellulose, and purified bovine prothrombin by chromatography on diethylaminoethyl cellulose. Both proteins are obtained as single components, and in high yield. The purified prothrombin does not convert to thrombin in 25% sodium citrate solution or protamine sulphate solution. The N-terminal amino acid is the same as in purified prothrombin obtained by different methods in this laboratory, but instead of tyrosine and glycine the C-terminal amino acid is serine. A fundamental difference in structure is thus associated with altered activation characteristics.

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.

PURIFICATION OF PROTHROMBIN AND THROMBIN BY CHROMATOGRAPHY ON CELLULOSE

1960 ◽  
Vol 38 (12) ◽  
pp. 1405-1410 ◽  
Author(s):  
Walter H. Seegers ◽  
Ricardo H. Landaburu
Keyword(s):  
Amino Acid ◽  
Sodium Citrate ◽  
Fundamental Difference ◽  
High Yield ◽  
Citrate Solution ◽  
Terminal Amino Acid ◽  
Protamine Sulphate ◽  
Bovine Thrombin ◽  
Diethylaminoethyl Cellulose ◽  
Terminal Amino

A method is described for obtaining purified bovine thrombin by chromatography on phosphate cellulose, and purified bovine prothrombin by chromatography on diethylaminoethyl cellulose. Both proteins are obtained as single components, and in high yield. The purified prothrombin does not convert to thrombin in 25% sodium citrate solution or protamine sulphate solution. The N-terminal amino acid is the same as in purified prothrombin obtained by different methods in this laboratory, but instead of tyrosine and glycine the C-terminal amino acid is serine. A fundamental difference in structure is thus associated with altered activation characteristics.

Generation of proteolytic activity during activation of prothrombin

1959 ◽  
Vol 197 (6) ◽  
pp. 1178-1180 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers
Keyword(s):  
Methyl Ester ◽  
Proteolytic Activity ◽  
Esterase Activity ◽  
Proteolytic Enzyme ◽  
Sodium Citrate ◽  
Citrate Solution ◽  
Platelet Factor ◽  
Thrombin Activity ◽  
Hydrolysis Of

In the activation of purified prothrombin with thrombin, with platelet factor 3, or in 25% sodium citrate solution the free thrombin activity which develops is greater at all times when measured by hydrolysis of p-toluenesulfonyl-arginine-methyl ester than when measured by the clotting of fibrinogen. Since the esterase activity appears before clotting power and remains after clotting power is lost, the clotting property must arise from a dissociable complex arising from the unit that has the esterase function. It is postulated that "clotting thrombin" may be a dimer of the lowest subunit of prothrombin required to have the proteolytic enzyme. The latter could have an important function in our physiology quite apart from the clotting of blood.

Concentration of a Prothrombin Conversion Accelerator and Thrombin Precursor From Serum

1956 ◽  
Vol 186 (2) ◽  
pp. 335-338 ◽  
Author(s):  
Robert I. McClaughry
Keyword(s):  
Protein Fraction ◽  
Sodium Citrate ◽  
Citrate Solution ◽  
Two Stage ◽  
Stage Analysis

The method described by Seegers for obtaining purified prothrombin from plasma has been used for isolating a protein fraction from serum. The yields are about 20% as much protein from serum as from plasma. The serum product is a potent accelerator for the conversion of purified prothrombin to thrombin. Although the serum product contained very little prothrombin, as measured by quantitative two-stage analysis, thrombin could be obtained from it by activation in 25% sodium citrate solution. It is believed that the accelerator isolated from serum is a derivative of prothrombin.

PROTEOLYTIC ENZYMES DERIVED FROM PROTHROMBIN

1965 ◽  
Vol 43 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Osvaldo E. Abdala ◽  
Miguel J. Morrone
Keyword(s):  
Amino Acid ◽  
Proteolytic Enzymes ◽  
Kinetic Studies ◽  
Weight Basis ◽  
Proteolytic Degradation ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Autoprothrombin C ◽  
Proteolytic Capacity ◽  
Prothrombin Activation

Prothrombin activation is a proteolytic degradation of the molecule that results in the formation of three enzymes with proteolytic capacity. The enzymes derived (thrombin, autoprothrombin C, and a new enzyme, prothrombin activating derivative B (PAD-B)) can use prothrombin as substrate. The new enzyme has been purified; the N-terminal amino acid is different from those of thrombin and autoprothrombin C. Kinetic studies give different Km values for the interaction between the substrate (prothrombin) and thrombin, autoprothrombin, and PAD-B. PAD-B corrects the hemophiliac plasma defect in the same way as autoprothrombin C, but PAD-B is more active on a weight basis.

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