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.

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.

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.

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.

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.

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.

Phospholipid-Protein Interactions in the Formation of Prothrombin Activator

1965 ◽  
Vol 14 (03/04) ◽  
pp. 431-444 ◽  
Author(s):  
E. R Cole ◽  
J. L Koppel ◽  
J. H Olwin
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Calcium Ions ◽  
Fixed Number ◽  
Factor V ◽  
Clotting Factors ◽  
Number Of Binding Sites ◽  
C Complex ◽  
Autoprothrombin C

SummarySince Ac-globulin (factor V) is involved in the formation of prothrombin activator, its ability to complex with phospholipids was studied. Purified bovine Ac-globulin was complexed to asolectin, there being presumably a fixed number of binding sites on the phospholipid micelle for Ac-globulin. In contrast to the requirement for calcium ions in the formation of complexes between asolectin and autoprothrombin C, calcium ions were not required for complex formation between asolectin and Ac-globulin to occur ; in fact, the presence of calcium prevented complex formation occurring, the degree of inhibition being dependent on the calcium concentration. By treating isolated, pre-formed aso- lectin-Ac-globulin complexes with calcium chloride solutions, Ac-globulin could be recovered in a much higher state of purity and essentially free of asolectin.Complete activators were formed by first preparing the asolectin-calcium- autoprothrombin C complex and then reacting the complex with Ac-globulin. A small amount of this product was very effective as an activator of purified prothrombin without further addition of calcium or any other cofactor. If the autoprothrombin C preparation used to prepare the complex was free of traces of prothrombin, the complete activator was stable for several hours at room temperature. Stable preparations of the complete activator were centrifuged, resulting in the sedimentation of most of the activity. Experimental evidence also indicated that activator activity was highest when autoprothrombin C and Ac-globulin were complexed to the same phospholipid micelle, rather than when the two clotting factors were complexed to separate micelles. These data suggested that the in vivo prothrombin activator may be a sedimentable complex composed of a thromboplastic enzyme, calcium, Ac-globulin and phospholipid.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

In-Canal Assay of High Specific Activity 60Co at the Advanced Test Reactor

2021 ◽  
pp. 1-7
Author(s):  
Michael A. Reichenberger ◽  
Jagoda M. Urban-Klaehn ◽  
Jason V. Brookman ◽  
Joshua L. Peterson-Droogh ◽  
Jorge Navarro ◽  
...  
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Test Reactor
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