Prothrombin Derivatives Research Articles

The reduction of autoprothrombin. II. Activity with dicumarol.

It is generally considered that the administration of dicumarol is followed by a decrease in prothrombin concentration, and it has been suggested that another plasma factor may also decrease. We have found that there are two derivatives of prothrombin called autoprothrombin and autoprothrombin II. In this paper we present data which show that the concentration of autoprothrombin II can be reduced practically to zero with the use of Dicumarol without observing a bleeding tendency. The decrease in concentration coincides with a decrease in prothrombin concentration but is relatively more rapid.

ELECTROPHORESIS OF AUTOPROTHROMBIN AND BIOTHROMBIN

Concentrates of biothrombin and autoprothrombin were prepared from purified prothrombin and subjected to electrophoresis. The mobility of biothrombin is less than that of prothrombin. The mobility of autoprothrombin is less than that of biothrombin. The biothrombin and autoprothrombin preparations examined were not electrophoretically homogeneous. Prothrombin dissociates during electrophoresis at high voltage gradients. Since this does not occur with biothrombin and autoprothrombin, it seems likely that electrophoretically homogeneous derivatives of prothrombin can be obtained with further purification work.

CONVERSION OF PROTHROMBIN TO AUTOPROTHROMBIN II

With thromboplastin, Ac-globulin, and calcium ions, purified prothrombin converts to biothrombin. This derivative of prothrombin is also obtained with purified platelet factor 3, Ac-globulin, calcium ions, and autoprothrombin II. Need for the latter substance by platelet factor 3 is one marked distinction that can be made between thromboplastin and platelet factor 3. Thromboplastin does not require a cofactor such as platelet cofactor I or autoprothrombin II. Ether extracts of dry brain or of purified platelet factor 3 or of whole platelets yield lipid material. The lipids obtained in this manner are active in a modified Biggs and Douglas thromboplastin generation test; but in contrast to the original thromboplastin or purified platelet factor 3 from which they are obtained do not activate purified prothrombin to thrombin. The lipid material is thus distinctly different from thromboplastin or from platelet factor 3. The lipid is also active in the conversion of prothrombin to autoprothrombin II when purified prothrombin is the substrate or when the prothrombin is in the original plasma or whole blood. Extraction of purified platelet factor 3 leaves a protein residue and it is believed that platelet factor 3 is a lipoprotein. The infrared absorption spectrum of the lipid obtained from purified platelet factor 3 shows strong absorption bands. From the general absorption pattern, we infer that the lipid is not a heterogeneous mixture, but most likely a single substance or consists of several that are nearly alike. Platelet factor 3 lipid(s) is most likely a cephalin-like compound.

ELECTROPHORESIS OF AUTOPROTHROMBIN AND BIOTHROMBIN

Concentrates of biothrombin and autoprothrombin were prepared from purified prothrombin and subjected to electrophoresis. The mobility of biothrombin is less than that of prothrombin. The mobility of autoprothrombin is less than that of biothrombin. The biothrombin and autoprothrombin preparations examined were not electrophoretically homogeneous. Prothrombin dissociates during electrophoresis at high voltage gradients. Since this does not occur with biothrombin and autoprothrombin, it seems likely that electrophoretically homogeneous derivatives of prothrombin can be obtained with further purification work.

Concept of a common protein moiety, containing disulfide bonds, in prothrombin, prothrombin derivative (autoprothrombin) and thrombin.

With a recently devised argentometric amperometric method, disulfide was demonstrated in biothrombin, citrate thrombin and prothrombin derivative (autoprothrombin) in the same quantity (43 µm/100 mg N) as that present in prothrombin. Sulfhydryl groups were not detected in any of these clotting factors. Prolonged storage and denaturation by heating before disulfide could be detected in some samples of thrombin, as well as a difference in sensitivity to the inhibitory effect of sulfhydryl compounds, indicate a distinction between prothrombin and thrombin in the accessibility of their —S—S— bonds. The prothrombin derivative (autoprothrombin) on which the sulfhydryl and disulfide analyses were performed was shown to be deficient in prothrombin and thrombin activity, to exhibit pronounced factor VII activity and to be capable of conversion to thrombin by sodium citrate, but not by biological activators. The inhibition, amperometric and activator studies provide additional support for a monophyletic hypothesis of a single, but complex, glycoprotein, which possesses a protein moiety containing—S—S—bonds, and which by alteration of its side chains or end groups, may exhibit prothrombin, thrombin or accelerator activity. Such studies render the possibility that factor VII is a specific protein entity, increasingly doubtful.

On the nature of the blood coagulation mechanisms in certain clinical states.

Current concepts of deficiency of serum prothrombin conversion accelerator (SPCA) metabolism are all related to prothrombin and its activation. SPCA deficiency is a hemorrhagic diathesis first described by Alexander and associates and subsequently by Owren and others. Patients with hemorrhagic tendencies were helped following administration of serum but this was of short duration, and the use of whole blood was of doubtful value. Plasma of Dicumarol-treated patients as well as plasma of normal persons only partly corrected the function of the coagulation mechanisms. The concentrations of Ac-globulin and prothrombin in the plasma were only slightly below normal. I t has been postulated that the patient with SPCA deficiency is deficient in a precursor of a factor found in serum. This precursor is supposed to be in plasma and to be different from any previously recognized coagulation factor. Owren is especially emphatic concerning its reality, and states that was discovered as a contaminant of prothrombin preparations. .In 1949 proconvertin was separated from prothrombin. .The discovery of patients with a hemorrhagic disease due to a lack of proconvertin has provided a final proof of the existence of this factor. . We have not found it possible to correlate the results of our extensive work with those assertions or with similar points of view. Our data, based on experiments with purified prothrombin, lead us to believe with even more assurance than formerly that the factor found in serum, and variously called factor VII, convertin, or SPCA, is a derivative of prothrombin itself and not of something distinctly different from prothrombin. Even apart from our work on purified prothrombin, it is interesting to note that no one has been able to prepare the hypothetical precursor of the accelerator found in serum and to establish that it is distinctly different from all other clotting factors. It is generally stated that the administration of Dicumarol is followed by a decrease in concentration of prothrombin. That view has been supported for many years and we also uphold it. The hypothetical precursor of SPCA (proSPCA, proconvertin, precursor of factor VII) has recently been reported as reduced in concentration, parallel with reduction of prothrombin. Indeed Biggs and Macfarlane found so great a divergence with Tromexan that they suggested that prothrombin concentration remains at the normal level while factor VII decreases. On the other hand, Owren and Aas, and to a lesser extent, Koller, Loeligpr and Duckert, using a method that is supposed to measure the concentration of the hypothetical precursor (proconvertin, pro-SPCA) in plasma, found

Nature of the blood coagulation mechanisms in SPCA plasma.

Derivatives of purified prothrombin and substances required for the formation of a derivative have been studied in connection with SPCA deficient plasma. The deficiency can be corrected in vitro with prothrombin derivatives. It is concluded that the SPCA patient cannot convert prothrombin to autoprothrombin and thrombin in the same way as a normal individual, and that the precursor of SPCA is prothrombin.

Prothrombin derivatives and the inhibition of thrombin formation with soy bean trypsin inhibitor.

ArticleProthrombin Derivatives and the Inhibition of Thrombin Formation With Soy Bean Trypsin InhibitorNorma Alkjaersig, Erwin Deutsch, and Walter H. SeegersNorma AlkjaersigFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, Michigan, Erwin DeutschFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, Michigan, and Walter H. SeegersFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, MichiganPublished Online:31 Jan 1955https://doi.org/10.1152/ajplegacy.1955.180.2.367MoreSectionsPDF (731 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformationCited ByInhibitors of Blood-Clotting MechanismsSelected Topics On Blood Coagulatio26 September 2008 | CRC Critical Reviews in Biochemistry, Vol. 1, No. 3ROLE OF PLATELETS IN BLOOD CLOTTINGInteraction of Soybean Trypsin Inhibitor with Thrombin and Its Effect on Prothrombin ActivationJournal of Biological Chemistry, Vol. 244, No. 4Action of human thrombokinase on human prothrombin and methyl esterBiochimica et Biophysica Acta (BBA) - Enzymology, Vol. 167, No. 2Physiology of blood coagulation: Advances, problems and present statusPfl�gers Archiv f�r die Gesamte Physiologie des Menschen und der Tiere, Vol. 299, No. 3Isolation and some properties of prethrombin and autoprothrombin IIIArchives of Biochemistry and Biophysics, Vol. 121, No. 2Activation of ProthrombinKinetics of Conversion of Prothrombin to Thrombin by Biological ActivatorsJournal of Biological Chemistry, Vol. 238, No. 1THE ACETYLATION OF PROTHROMBINCanadian Journal of Biochemistry and Physiology, Vol. 38, No. 6THE ACETYLATION OF PROTHROMBINCanadian Journal of Biochemistry and Physiology, Vol. 38, No. 1SOME PROPERTIES OF THROMBIN PREPARATIONSCanadian Journal of Biochemistry and Physiology, Vol. 37, No. 6SOME PROPERTIES OF THROMBIN PREPARATIONSCanadian Journal of Biochemistry and Physiology, Vol. 37, No. 1The preparation of prothrombin derivatives and an indication of their propertiesArchives of Biochemistry and Biophysics, Vol. 61, No. 1 More from this issue > Volume 180Issue 2January 1955Pages 367-370 Copyright & PermissionsCopyright © 1955 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1955.180.2.367PubMed14361742History Received 17 September 1954 Published online 31 January 1955 Published in print 31 January 1955 Metrics

Relationship of certain antihistamine drugs to the activation of purified prothrombin.

ArticleRelationship of Certain Antihistamine Drugs to the Activation of Purified ProthrombinMarvin Murray, Shirley A. Johnson, and Walter H. SeegersMarvin MurrayFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, Michigan, Shirley A. JohnsonFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, Michigan, and Walter H. SeegersFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, MichiganPublished Online:01 Jul 1954https://doi.org/10.1152/ajplegacy.1954.178.1.10MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByBIBLIOGRAPHYMolecular Characteristics of Substances Active in Blood CoagulationHämophilie, Mangelzustand oder Überschuß eines Hemmkörpers?Klinische Wochenschrift, Vol. 44, No. 14INACTIVATION OF PURIFIED AUTOPROTHROMBIN I WITH ANTITHROMBINCanadian Journal of Biochemistry, Vol. 42, No. 10The Detection of platelet defects in patients with mild bleeding disordersThe American Journal of Medicine, Vol. 32, No. 6ThrombocytenfaktorenThe inhibition of prothrombin activation by a lipoïd extract from beef brain26 September 2008 | Archives Internationales de Physiologie et de Biochimie, Vol. 67, No. 1The preparation of prothrombin derivatives and an indication of their propertiesArchives of Biochemistry and Biophysics, Vol. 61, No. 1 More from this issue > Volume 178Issue 1July 1954Pages 10-16 Copyright & PermissionsCopyright © 1954 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1954.178.1.10PubMed13180704History Received 14 January 1954 Published online 1 July 1954 Published in print 1 July 1954 Metrics

Formation of prothrombin derivatives from purified prothrombin.

ArticleFormation of Prothrombin Derivatives From Purified ProthrombinWalter H. Seegers, Edna B. Andrews, and Robert I. McClaughryWalter H. SeegersFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, Michigan, Edna B. AndrewsFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, Michigan, and Robert I. McClaughryFrom the Department of Physiology and Pharmacology, Wayne University College of Medicine, Detroit, MichiganPublished Online:28 Feb 1951https://doi.org/10.1152/ajplegacy.1951.164.3.722MoreSectionsPDF (733 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation More from this issue > Volume 164Issue 3February 1951Pages 722-726 Copyright & PermissionsCopyright © 1951 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1951.164.3.722PubMed14819293History Received 8 October 1950 Published online 28 February 1951 Published in print 28 February 1951 Metrics

SOME PROPERTIES OF PURIFIED PROTHROMBIN AND ITS ACTIVATION WITH SODIUM CITRATE

Abstract Prothrombin preparations were examined by electrophoresis and found to contain approximately 90 per cent of the protein in one component. Such products possess a specific activity of 1,400 units per milligram of dry weight when analyzed with reagents and technics that give a value of 32.9 ± 30 units of prothrombin per cc. of oxalated bovine plasma. The electrophoretic mobility is approximately equivalent to that of α1-globulin. That of thrombin is less. The isoelectric point in 0.1 ionic strength salt solution is near pH 4.2, and for thrombin it is approximately pH 4.8. Analysis of dry preparations indicated 0.96 per cent sulfur, 13.57 per cent nitrogen, 4.58 per cent tyrosine, and 3.33 per cent tryptophane, and no phosphorus was found. The purified material could be activated to thrombin by dissolving the prothrombin in concentrated solutions of sodium citrate, ammonium sulfate, magnesium sulfate, sodium sulfate, potassium citrate, potassium oxalate, and dipotassium hydrogen phosphate. Concentrated solutions of sodium chloride, potassium chloride, ammonium chloride or magnesium chloride do not produce activation. Difficulties were encountered when attempts were made to activate less highly purified prothrombin preparations with sodium citrate. The activation with sodium citrate is autocatalytic, and the yield of thrombin can be increased to a maximum by adding a small amount of 3-methyl-4,6,4'-triaminodiphenyl sulfone to the activation mixture. The sodium citrate concentration must be high, preferably near 25 per cent. A new method for preparing thrombin is based on the use of these principles. During activation the electrophoretic properties of prothrombin change long before appreciable amounts of thrombin activity are found. These changes in electrophoretic properties are associated with a prothrombin derivative which is refractory to the action of the activators calcium plus thromboplastin plus Acglobulin. When activation with sodium citrate is complete almost none of the material which had electrophoretic mobility of prothrombin is found in the electrophoresis boundary patterns. The major portion of the pattern is comprised of two components having mobilities less than prothrombin while a third component with high mobility is especially evident in the descending boundary pattern. Solubility studies indicate that two proteins may possess thrombin activity, but the evidence is not conclusive. An attempt was made to separate the proteins in the thrombin preparation by fractionation with ammonium sulfate, and it was found that their properties are so similar that this procedure is not effective. Thrombin obtained by activation of prothrombin with sodium citrate may be dried from the frozen state and is stable. Prothrombin is not stable after drying in this manner; however, it can be dried with acetone. It is reasoned that since sodium citrate and other salts can produce thrombin from prothrombin, the latter must contain all the structural material needed for the thrombin. For that reason calcium, thromboplastin, and Ac-globulin are regarded as catalysts of prothrombin activation and not as activators which must contribute material substance in prothrombin activation.