Prothrombin Derivatives Research Articles

Preparation of Human Plasma Prothrombin and Some of Its Sedimentation Properties

SUMMARY The aggregation with decreasing pH is in contrast with the dissociation of bovine prothrombin, which is reported to give sub- units of 34,000 in phthalate buffers of pH 4.92 to 6.0 (3, 4). It may not be surprising, however, to find aggregation as the pH approaches the solubility minimum (approximately 4.2 to 4.6). In view of the fact that certain other blood coagulation factors (9, 10, 16), prothrombin derivatives (14, 15), or both may affect the activation of the zymogen, and hence the results of the two- stage assay for prothrombin activity, it was considered possible that the results might be explained by a separation of a prothrom- bin accelerator or accelerators during the partition cell experi- ments or the sucrose gradient studies. Such has been shown to by the case when prothrombin preparations are analyzed by starch block electrophoresis (12). However, assays for three of these factors,3 Factors VII, IX, and X, by methods summarized elsewhere (39, 40) gave activities of less than 3% of the total activities of the prothrombin preparations examined. Factors V, VIII, and XI were undetectable. When assayed in the parti- tion cell experiments, the activity of these factors were found to have sedimentation rates that were not significantly different from that of prothrombin. In addition, mixing experiments were performed with material obtained from the top and bottom of the Prothrombin was isolated in 30 to 40% yield from human acid-citrate-dextrose plasma by a modification of the Lewis and Ware procedure further purified by adsorption on diethylaminoethyl cellulose and Sephadex gel filtration. Prod- ucts obtained were of high specific activity, were electro- phoretically homogeneous from pH 7.0 to 9.0, and were devoid of estraneous plasma proteins by immunoelectrophoresis. Sedi- mentation velocity analyses of these preparations were carried out the partition cell, which permitted calculation of sedimentation coefficients from the schlieren pattern as well from the prothrombin activity (two-stage) and protein isolated from cell at end of each run. The results obtained demonstrated that the zymogen polymerizes at low pH and ionic strength and that the prothrombin activity sediments at a higher rate than the majority of protein. This latter finding was confirmed by sedimentation in a stabilizing sucrose gradient in the bucket rotor of the ultracentrifuge. In addition, sedimen- tation coefficients for prothrombin activity in native plasma have demonstrated similar results, which, however, include a dependence of sedimentation rate on the anticoagulant used. These findings indicate a possible pH-dependent polymerization of human prothrombin and suggest that the specific activity the zymogen may be greater than previously suspected. REFERENCES 1.

Kinetics of Conversion of Prothrombin to Thrombin by Biological Activators: I. DEMONSTRATION OF A PROTHROMBIN DERIVATIVE WHICH REACTS WITH PROTEOLYTIC ENZYME INHIBITORS

Kinetics of Conversion of Prothrombin to Thrombin by Biological Activators: I. DEMONSTRATION OF A PROTHROMBIN DERIVATIVE WHICH REACTS WITH PROTEOLYTIC ENZYME INHIBITORS

Conditions determining end products of prothrombin activation in serum.

When blood clots normally such a small amount of prothrombin and small quantity of accelerators remain in the hour-old serum that the prothrombin consumption test is prolonged. When blood from a severe coagulation abnormality clots, prothrombin and fibrinogen are both found in serum. When the blood from milder coagulation abnormalities or from transfused severe coagulation abnormalities clots no prothrombin remains in the serum but quantities of the prothrombin-like derivative appear. It is suggested that this component may be a derivative of prothrombin, and a comparison with prothrombin has been undertaken. Studies carried out using disulfide-linkage inhibitors, known to inhibit prothrombin activation, also inhibit the coagulation system supported by this new component. The prothrombin-like derivative is not stable at –25 C for more than a few days whereas prothrombin stored under similar conditions is stable indefinitely.

AUTOPROTHROMBIN C: A SECOND ENZYME FROM PROTHROMBIN

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.

Severe Isolated Prothrombin Deficiency : an Acquired State with Complete Recovery

SummaryA 62 year old man acquired a temporary, virtually complete, deficiency of prothrombin as measured by both the Iowa and Oxford 2-stage methods. The etiology agent (or agents) is not known, but one of the drugs listed in Table 1 or an interaction between several was probably responsible. There was no evidence of an inhibitor, and factors I (fibrinogen), V (AcG), VII (SPCA), VIII (AHF), IX (PTC), X (Stuart), and XII (Hageman) were demonstrated to be normal. There were other evidences of mild liver damage and the prothrombin deficiency did not respond immediately to injections of natural vitamin K. Greatly prolonged clotting times but only moderately prolonged prothrombin times and partial thromboplastin times were observed. Bleeding was very severe into many tissues. Thromboplastin generation proceeded normally in the patient’s pro-thrombin-deficient plasma, despite the appearance of only a trace of thrombin. This patient’s history and clotting profile argue against the concept that factor VII (SPCA) and IX (PTC) are derivatives of prothrombin.

AUTOPROTHROMBIN C: A SECOND ENZYME FROM PROTHROMBIN

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 in Irregular Blood Clotting

SummaryIn this study autoprothrombin C was considered in relationship to the hemorrhagic diseases. Concentrates of autoprothrombin C were made from purified prothrombin known to be homogeneous by several criteria. These autoprothrombin C preparations were free of thrombin, and were almost a single component when analyzed by centrifugation. Autoprothrombin C corrected the partial thromboplastin time and produced prothrombin consumption in all the plasmas obtained from patients with hemorrhagic diseases except parahemophilia. In the case of hemophilia B prothrombin consumption was obtained with the addition of purified prothrombin or purified autoprothrombin II. In the case of Stuart plasma prothrombin consumption was rapid after the addition of purified prothrombin or purified prothrombin chromatographed on Amberlite ICR-50, but not after the addition of purified prothrombin that was chromatographed on DEAE cellulose. The latter prothrombin is an abnormal prothrombin molecule and does not readily yield autoprothrombin C. It is concluded that Stuart prothrombin is abnormal and that this is a molecular disease. There is no need to postulate the existence of factor X to account for the irregular prothrombin activation in Stuart plasma. The most important question considered was how can autoprothrombin C be generated from prothrombin to promote the autocatalytic activation of prothrombin. Certain prothrombin molecules do not yield this enzyme, but normal prothrombin does. However, it does so only under certain conditions of activation. By applying the new knowledge of prothrombin chemistry to blood clotting irregularities in hemorrhagic diseases the following main considerations highlight the common deviations: 1. Abnormal prothrombin molecule, 2. Changes related to the derivatives of prothrombin, 3. Accessories needed for the generation of autoprothrombin C so it can function in auto-catalysis are irregular, and 4. Accessories needed for the function of autoprothrombin C after it is out of the prothrombin molecule are irregular. In the first group is Stuart Plasma. In the second group falls hemophilia B (autoprothrombin II) and the so-called factor VII deficiency (autoprothrombin I). In the third group is hemophilia A. In the fourth group is parahemophilia, and the platelet abnormalities.

Antithrombin II Anticoagulant (Autoprothrombin II-A)

SummaryAutoprothrombin II-A activity frequently develops when the autoprothrombin II fraction of prothrombin is dried from the frozen state. When the anticoagulant activity develops, the N-terminal amino acid arginine is found, but not when the anticoagulant activity does not develop. The anticoagulant autoprothrombin II-A retarded the conversion of prothrombin to thrombin when three different procoagulants were used; namely, platelet cofactor I, autoprothrombin II, and lung extract. The procoagulant power of the platelet cofactor I is most easily depressed. It is postulated that autoprothrombin II-A activity develops when prothrombin activates and that platelet cofactor I is a receptor of the anticoagulant. In that way the other procoagulants are not ordinarily inhibited by the autoprothrombin II-A. As a further extension of this speculation, attention is brought to the idea that autoprothrombin II-A could be present in certain hemorrhagic diseases. In the PTA patient there is no autoprothrombin II and most likely no autoprothrombin II-A. The close chemical relationship of these two prothrombin derivatives thus may account for the multiple deficiency.

Partial activation of prothrombin and generation of prothrombin-R with cathepsin B.

Maximal yields of prothrombin-R were readily produced from a number of prothrombin derivatives when these were mixed with purified cathepsin B at pH 7.9. Thrombin was not a product when substrates prepared with either mitochondria or cathepsin B were used in the regeneration system. Conditions were found where the formation of prothrombin-R exhibited characteristics of either zero or first order kinetics with respect to the enzyme, cathepsin B. Iodoacetate partially or completely inhibited the formation of prothrombin-R. Various mechanisms which may account for the production of prothrombin-R are discussed.

Studies on prothrombin derivatives in vitamin K deficiency.

Certain of the blood-clotting have a number of properties in common. These properties include affinity for the insoluble salts of heavy metals (i. e., barium sulfate), relative heat lability and storage stability, and lack of utilization during the course of clotting. The last-named property, whereby contains these in undiminished titer, has led some investigators to use the term serum factors. The presently accepted serum factors are Factor VII, plasma thromboplastin component (PTC), and Stuart factor. 1 Except for its utilization during clotting, prothrombin shares the above-mentioned properties of the serum factors. These similarities have suggested the serum factors are derivatives of or share a common synthetic pathway with prothrombin. Evidence for this view has been provided by the finding that all are depressed following administration of bishydroxycoumarin (Dicumarol) and related drugs, 2-5 suggesting that vitamin K is necessary for their synthesis. Moreover, it has been possible to

Activation of prothrombin.

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.

Prothrombin activation cycle.

SummaryThe prothrombin activation cycle consists of a sequence described by the following: Prothrombin (sensitive to calcium + Ac-globulin + lung thromboplastin) → Prothrombin derivative I (not sensitive to calcium + Ac-globulin + lung thromboplastin) → Prothrombin derivative II (sensitive to calcium + Ac-globulin + lung thromboplastin) → Thrombin. Solutions of prothrombin standing at 4°C go through most of this cycle, the prothrombin molecule alone possessing the peculiar properties that enables it to do this.

Interrelationship of prothrombin and autoprothrombin I in thrombocytopenia following aminopterin administration.

The poor prothrombin consumption test supported by thrombocytopenic serum has been shown in a previous publication to be due to some activity other than prothrombin—a thrombocytopenic activity. This thrombocytopenic factor or activity can be readily adsorbed on inorganic agents and eluted. The autoprothrombin I values were found to be lower in thrombocytopenic serum than in normal serum. When the patient with thrombocytopenia was treated with aminopterin, the prothrombin consumption test remained short while the autoprothrombin I values increased to that of normal serum and then fell again as the treatment was withdrawn. The prothrombin in the serum remained uniformly low throughout the treatment. The relationship of the thrombocytopenic activity and autoprothrombin I as possible derivatives of prothrombin is considered. Submitted on June 12, 1957

Further studies of prothrombin derivatives.

SummaryPurified bovine prothrombin preparations may lose the property of clotting fibrinogen but retain esterase activity. It is likely that a single molecule possesses the 2 activities. When biothrombin has lost clotting activity and only esterase activity remains, it is called esterase thrombin.

The blood coagulation mechanisms in thrombocytopenic blood

SummaryThrombocytopenic serum supports a short prothrombin consumption time, however, the same serum contains as little prothrombin as normal serum. The prothrombin consumption test of thrombocytopenic serum can be prolonged by adsorption on BaCO3 or reduced again by the addition of the sodium citrate eluate. The antihemophilic activity of thrombocytopenic blood disappears when the blood clots as in normal blood and autoprothrombin I is present in much smaller amounts than in normal serum. The authors suggest that a new factor, a possible derivative of prothrombin, is responsible for the short prothrombin consumption value in thrombocytopenic blood.

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.

STUDIES ON THE NATURE AND THE PURIFICATION OF THE COAGULASE-REACTING FACTOR AND ITS RELATION TO PROTHROMBIN

The coagulase-reacting factor (CRF) of human plasma has been purified, concentrated, and largely freed of prothrombin activity by Seitz filtration, absorbtion on hyflo-amphogel columns, controlled phosphate buffer elution, and fractional ammonium sulfate precipitation. The purified CRF preparations localized between the beta and the gamma globulin position on paper electrophoresis, and the principal of two components observed on analytical ultracentrifugation gave sedimentation rates between 2.75 and 3.07. Highly purified prothrombin preparations of Seegers effectively react with staphylocoagulase to lead to the coagulation of plasma. The electrophoretic and ultracentrifugal properties of the human prothrombin preparations examined differ significantly from those of the purified CRF. Derivatives of prothrombin, involving a loss of prothrombin activity, such as thrombin and "autoprothrombin," do not correspondingly lose in CRF function. The hypothesis is proposed that CRF activity is resident in some component or components of the prothrombin molecule not involved in prothrombin function per se, and that therefore prothrombin and CRF activity may either parallel each other when the molecule is intact or diverge when the smaller units only are involved.

Activation of prothrombin.

In the activation of a prothrombin solution, only part of the prothrombin becomes biothrombin, some becomes autoprothrombin I and some becomes autoprothrombin II. Thrombin-thrombin is also obtained, and perhaps other derivatives of prothrombin are formed. Prothrombin thus has multiple potentialities and these can be developed in diverse ways. Ac-globulin, calcium ions and platelet factor 3 can be used to obtain autoprothrombin I. Thrombin and Ac-globulin transforms prothrombin to autoprothrombin II, while platelet factor 3 or calcium ions tend to inhibit the transformation. Since calcium ions and platelet factor 3 also function as activators in one of the prothrombin to biothrombin reactions they are either activators or inhibitors of prothrombin activation depending upon the specified conditions.

Concentration of a prothrombin conversion accelerator and thrombin precursor from serum.

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.

The preparation of prothrombin derivatives and an indication of their properties

Several derivatives of prothrombin have been prepared. Biothrombin may be obtained by activating prothrombin in several different ways with activators obtained from tissues. During the transformation of prothrombin to biothrombin there is very little reduction in molecular weight and most likely no change in solubility in 7% ( w v ) trichloroacetic acid solution. If there are any degradation products during the interaction, their quantity must be small. Biothrombin can be transformed to citrate thrombin by dissolving it in 25% sodium citrate solution. Prothrombin itself can also be transformed to citrate thrombin in the same manner; however, an intermediate forms which is called citrate autoprothrombin. The latter is an accelerator in the activation of prothrombin with activators obtained from tissues. It loses its accelerator property when it transforms to thrombin. Prothrombin can be converted to autoprothrombin in solutions containing a small amount of Ac-globulin, calcium ions, and purified platelet factor 3. Autoprothrombin has accelerator properties under certain conditions of prothrombin activation and is a degradation product of prothrombin. During its formation a considerable quantity of carbohydrate and nitrogeneous material become soluble in trichloroacetic acid. Its sedimentation constant is smaller than that of prothrombin. It can be transformed to citrate thrombin by dissolving it in 25% sodium citrate solution. Methods are described for obtaining concentrates of citrate thrombin, autoprothrombin, and biothrombin.