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: 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.

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.

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.

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.

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 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.

Substitution of Lipids with Bile Salts in the Formation of Thrombin

1969 ◽  
Vol 22 (01) ◽  
pp. 013-027 ◽  
Author(s):  
Monika Barthels ◽  
W. H Seegers
Keyword(s):  
Calcium Ions ◽  
Bile Salts ◽  
Thrombin Generation ◽  
Sodium Deoxycholate ◽  
Sodium Cholate ◽  
Phosphatidyl Serine ◽  
Molar Ratio ◽  
Platelet Factor ◽  
Analytical Reagents ◽  
Autoprothrombin C

SummaryThe generation of thrombin was studied in an activating system consisting of purified thrombin zymogens, purified autoprothrombin C, purified Ac-globulin, lipid or bile salts, and calcium chloride. With the concentration of calcium ions and pH fixed, the effect of varying the other three procoagulants was studied. Bile salts were effective substitutes for lipids in a concentration where micelles form. The approximate effectiveness from highest to lowest was: conjugated sodium salt of taurocholic acid, sodium cholate, sodium deoxycholate. Sodium dehydrocholate was ineffective. Autoprothrombin C is the enzyme for thrombin formation. For accelerating its activity best results were obtained with the simultaneous presence of optimal concentrations of calcium ions, Ac-globulin and lipids or bile salts. Reducing any one of the three to zero concentration decreased the rate and yield of thrombin generation.The form in which the zymogen is used was found to be important. Prothrombin complex, DE AE-prothrombin and prethrombin were studied. Each substrate has its peculiar requirements for yielding thrombin. Prothrombin complex and DEAE-pro-thrombin activated far more rapidly and required 10 times less autoprothrombin C than prethrombin. The yield of thrombin from these substrates was also higher than from prethrombin. DE AE-prothrombin required the least amount of lipid. For the bile salts the required concentrations were nearly always the same from one substrate to another. To a certain extent Benadryl could also be substituted for lipids. In association with rapid thrombin generation from DE AE-prothrombin the Ac-globulin and autoprothrombin C Avere represented in approximately a 6:1 molar ratio. As compared with the weight of the enzyme large amounts of Ac-globulin and cholate were required.DE AE-prothrombin was readily made refractory to the two-stage analytical reagents with purified platelet factor 3 and calcium ions. Combinations of sodium cholate and phosphatidyl serine were also effective, but either one alone was ineffective.

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.

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.

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