NEW ONE-STAGE PROCEDURES FOR THE QUANTITATIVE DETERMINATION OF PROTHROMBIN AND LABILE FACTOR*

Transcription

1 NEW ONE-STAGE PROCEDURES FOR THE QUANTITATIVE DETERMINATION OF PROTHROMBIN AND LABILE FACTOR* MARIO STEFANINI, M.D.f From the Department ofbiochemistry, Marquette University School of Medicine, Milwaukee, Wisconsin It has been demonstrated during the last few years that, besides prothrombin, thromboplastin and calcium, at least one other factor is necessary for the formation of thrombin. This has been variously named component A, 5 labile factor, 7 factor V, 3 and Ac globulin. 14 This observation questions the concept that the prothrombin time, as determined by the ordinary procedures, is directly related to the concentration of prothrombin in plasma, and suggests that a delayed plasma prothrombin time can be due to deficiency not only of prothrombin but also of the labile factor, and possibly, to defective interreaction of the two agents. When, therefore, experimental modifications of the prothrombin activity are studied or when more information is desired on the pathogenesis of a delayed prothrombin time, the separate quantitative determination of plasma prothrombin and labile factor becomes necessary. Technics for ^his purpose are described in the present paper. They are essentially modifications of the one-stage prothrombin determination of Quick 6 and are based on characteristic properties of the two agents. In the development of these procedures a thromboplastin reagent, prepared according to Quick, -has been used which is capable of clotting an equal volume of fresh oxalated plasma in 12 seconds in the presence of an optimal concentration of calcium chloride. For uniform results, this product should be used; it presents no particular difficulties in preparation. If a thromboplastin reagent of different potency is employed, tlie xlilution curves mentioned in the technics described here for the direct calculation of the percentage values from prothrombin times should be reconstructed. The thromboplastin should, of course, be checked periodically to determine if its potency is constant. QUANTITATIVE ONE-STAGE ASSAY OF CONCENTRATION OF LABILE FACTOR IN PLASMA Principle. Oxalated human plasma stored for approximately 15 clays at 4 C. shows a prolonged prothrombin time (usually above 40 seconds) because of the almost complete depletion of the labile factor. If fresh oxalated human plasma containing a normal amount of labile factor is added to the stored plasma in the volumetric proportion of 1 to 10, a prothrombin time of 20 seconds is obtained. 8 The prothrombin time obtained with a pathologic plasma added to * Received for publication, September 9, t Present address: Blood Research Laboratories, Joseph Pratt Diagnostic Hospital, Boston. Massachusetts. 233

2 234 STEPANINI stored plasma is directly related to the concentration in labile factor of the former. Reagents. 1. Stored oxalated human plasma. Blood is collected by venipuncture from a normal subject, mixed with 1/10 volume of sodium oxalate 0.1 M and centrifuged for ten minutes at 2000 r.p.m. The supernatant plasma is transferred O 10 EO '00 Concentration of Labile F&ctor(pa?canl) FIG. 1. Dilution curve for the direct calculation of the concentration of labile factor from the prothrombin time. Average results of several determinations. (Decreasing volumes of fresh normal oxalated plasma were mixed with increasing volumes of sal'vne solution to obtain known concentrations of the labile factor from 100 to 10 per cent. To.09 ml. of stored human oxalated plasma,.01 ml. of each different dilution was added and the prothrombin time of the mixture was determined. The concentration of prothrombin in the mixture varied from 99 to 91 per cent, sufficient to give the shortest possible prothrombin time.) to open glass tubes kept at 4 C. for a minimum of 15 and a maximum of 25 days. This period is specified because there is negligible depletion of the labile factor during the first two weeks of storage while, after 25 days, prothrombin itself begins to be inactivated and fibrinogen denatured. 2. Rabbit brain thromboplastin, prepared according to Quick Calcium chloride 0.02 M. 4. Sodium oxalate 0.1 M.

3 DETERMINATION OF PROTHROMBIN 235 Technic. By venipuncture, 4.5 ml. of the blood to be examined is collected and added to 0.5 ml. of 0.1 M sodium oxalate. The plasma is obtained by centrifugation at 2000 r.p.m. for ten minutes. By means of a pipet graduated to.01 ml.,.09 ml. of the stored plasma is added to.01 ml. of the plasma under examination in an ordinary Pyrex glass tube (130 x 10 mm.) and the prothrombin time of the mixture determined. It is advisable to make sure that normal values are obtained in normal subjects with the reagents employed. Calculation. The percentage of labile factor present in the plasma under examination is calculated directly from the prothrombin time obtained by means of a dilution curve (Fig. 1). The dilution curve is constructed as follows: Decreasing volumes of fresh normal oxalated plasma are mixed with increasing volumes of saline solution to obtain known concentrations of the labile factor, from 100 to 10 per cent;.01 ml. of each dilution is added to.09 ml. of stored plasma and the prothrombin time of the mixture determined. The average analytical values obtained are as follows: Concentration of the labile factor, per SO Prothrombin time, 20.S S At low concentrations of the labile factor, careful attention must be paid to observe the formation of the first thread of fibrin which represents the end point of the reaction. QUANTITATIVE ONE-STAGE DETERMINATION OF ABSOLUTE CONCENTRATION OF PROTHROMBIN IN PLASMA Principle. It has been shown by Quick and Stefanini 9 that prothrombin is completely adsorbed from oxalated plasma by treatment with M Ca 3 (PO^ gel and that the adsorption is prevented by the presence of sodium citrate at a concentration of M or higher. The same authors 10 later showed that prothrombin can be likewise eluted from the gel by treatment with sodium citrate 0.2 M in the volumetric proportion of 1 part to 10 parts of the original volume of plasma. The eluate contains at least 90 per cent of the prothrombin originally present in the treated plasma (concentrated 10 times), a minimal concentration of labile factor and a small amount of fibrinogen. 13 When one volume of the eluate is added to 9 volumes of normal fresh oxalated plasma previously deprothrombinized with M tricalcium phosphate gel, the prothrombin time of the mixture will be directly proportional to the concentration or prothrombin in the original plasma. With this technic, the eluate obtained from a normal plasma will give a prothrombin time of 12 seconds. The procedure offers an accurate technic for the quantitative assay of the concentration of prothrombin in a given plasma. Reagents. 1. Sodium citrate, 0.2 M.

4 236 STEFANINI 2. Sodium oxalate, 0.1 M. 3. Rabbit brain thromboplastin, prepared according.to Quick CaCl 2, 0.02 M. 5. Tricalcium phosphate gel, M. Trisodium phosphate, 158 Gm., is dissolved in 1000 ml. of distilled water. Separately, anhydrous calcium chloride, 66 Gm., is dissolved in 1000 ml. of distilled water. The calcium chloride solution is poured into that of trisodium phosphate while stirring thoroughly. The precipitated tricalcium phosphate is washed repeatedly by decantation with distilled water until most of the sodium chloride has been removed. This procedure may require several days. The volume is brought to 1 liter, thus making a 0.2 M suspension. From this stock, a M suspension is prepared by mixing 4 ml. of the stock suspension with 96 ml. of distilled water. 6. Deprothrombinized normal fresh oxalated plasma. Add 1.8 ml. of venous blood of a healthy subject to 0.2 ml. of 0.1 M sodium oxalate solution. The plasma is obtained by centrifugation at 2000 r.p.m. for ten minutes. One ml. (or a volume equal to that of the plasma to be deprothrombinized) of the M calcium phosphate gel is transferred to a Pyrex glass tube (130 x 10 mm.) and centrifuged at 2000 r.p.m. for five minutes. The supernatant water is poured off and the walls of the tube are carefully dried with filter paper. One ml. of plasma is then transferred to the tube and the packed gel is resuspended by means of a glass rod. The mixture is incubated at room temperature for five minutes and is then centrifuged at 2000 r.p.m. for ten minutes. The supernatant plasma is transferred to a new Pyrex tube and kept in an iced container to prevent any inactivation of the labile factor. This plasma will not clot for an indefinite period after the addition of thromboplastin and calcium chloride, since it has been completely deprived of prothrombin, but it still contains the full amount of labile factor and fibrinogen of the original plasma. For accurate results a fresh deprothrombinized plasma should be prepared every day. Technic. One ml. of the plasma under examination is treated with the packed gel from 1 ml. of M Ca 3 (P0 4 ) 2. After incubation and centrifugation, the supernatant plasma is discarded. The precipitate is washed with distilled water, the walls of the test tube are carefully dried with filter paper and 0.1 ml. of 0.2 M sodium citrate solution is added. The precipitate is suspended in the citrate with a fine glass stirring rod, the mixture incubated at room temperature for five minutes and finally centrifuged at 2000 r.p.m. for ten minutes. The supernatant fluid is collected; it contains all (or almost all) the prothrombin originally present in the plasma, some fibrinogen and labile factor and about 35 to 45 per cent of the Ca 3 (PO,i) 2 gel in solution 12 which, however, does not interfere with the results of the test. To.09 ml. of fresh oxalated normal human plasma,.01 ml. of the eluate is then added and the prothrombin time of the mixture is determined. Calculation. The percentage of prothrombin present is calculated directly by means of a dilution curve (Fig. 2, curve A). The dilution curve is constructed by: (a) preparing a series of dilutions of the eluate obtained from normal plasma in deprothrombinized normal plasma so as to obtain known concentrations of

5 140 DETERMINATION OF PROTHROMBIN 237 prothrombin from 100 to 1 per cent; and (b) by adding.01 ml. of each dilution to.09 ml. of deprothrombinized plasma and determining the prothrombin time of each mixture. The values of Figure 2 show that the hyperbolic curve obtained with the progressive dilution of prothrombin is more pronounced than th&t obtained by mixing decreasing volumes of fresh with increasing volumes of deprothrombinized normal fresh human plasma. This is possibly due to the Concentration of PpoihPombin(per cent) FIG. 2. Dilution curves for the direct calculation of the concentration of prothrombin from the prothrombin time of fresh eluate. Human (curve A) or rabbit (curve B) deprothrombinized plasmas were used as diluents. Average results of several experiments. (Decreasing volumes of normal eluate diluted 1:10 with deprothrombinized plasma were added to deprothrombinized plasma, to obtain known concentrations of prothrombin from 100 to 1 in presence of a constant optimal amount of labile factor and fibrinogen. The prothrombin time of the different mixtures was determined with Quick's one-stage procedure.) activity of an agent, different from labile factor and prothrombin, which is lost during the preparation of the eluate or the deprothrombinization of plasma. Figure 2 also gives the values obtained when deprothrombinized rabbit plasma, rather than human plasma, is used as a"source of fibrinogen and labile factor (curve B). These values are shorter than those obtained with human plasma because of the greater concentration of the available labile factor. In the preparation of deprothrombinized rabbit plasma M Ca 3 (PO.i)2 has to be used for a complete adsorption of prothrombin. Average figures obtained using de- IOO

6 238 STEFANINI prothrombinized human and rabbit plasma as diluent, from which the curves of Figure 2 have been constructed, are given in Table 1. TABLE 1 DILUENT CONCENTRATION OF ELUATE, PER CENT Human Deprothrombinized Plasma Rabbit Deprothrombinized Plasma Prothrombin Time (Seconds) S S DISCUSSION The discovery that factors other than prothrombin, thromboplastin and calcium are necessary for the formation of thrombin modifies, to a considerable extent, the present concept of the significance of the prothrombin time and demands.a revision of the technics currently employed for its determination. A delayed prothrombin time can be due not only to deficiency of prothrombin, but also to deficiency of labile factor or to an abnormal interaction of the two components. Other agents, the existence of which has been suggested by different investigators, may also influence the prothrombin time through mechanisms which require further study. In this paper procedures for the quantitative one-stage assay of prothrombin and labile factor are described. It is not intended that these technics should replace the one-stage method for the determination of the prothrombin time, but rather that they should integrate this procedure. The Quick test expresses the over-all activity of factors, other than thromboplastin and calcium, thai

7 DETERMINATION OF PROTHROMBIN 239 take part in the formation of thrombin. As the over-all "prothrombin activity" and not the absolute concentration of prothrombin determines the speed of formation and the yield of thrombin, the determination of prothrombin time is a reliable index of this mechanism, which is fundamental in assuring efficient hemostasis. The one-stage procedure of Quick remains, therefore, a most useful technic in the study of "prothrombin activity" in clinical conditions. A normal prothrombin time indicates that both prothrombin and labile factor concentrations are normal and makes further investigations unnecessary. Only when the prothrombin time is delayed are other analytical technics imperative. The routine use of the one-stage procedures here described has given interesting information concerning the relative importance of modifications of prothrombin and labile factor in determining a decrease of prothrombin activity. So far we have been unable to find conditions in which the deficiency of labile factor alone could be considered responsible for a delayed prothrombin time. Cases of this type, however, exist and have been described as parahemophilia by Owren 4 and as idiopathic hypoprothrombinemia by Rhoads and Fitz-Hugh 11 and by Giordano. 1 Deficiency of labile factor can be found more frequently in conditions presenting a delayed prothrombin time in which the concentration of prothrombin is also depleted, as may be the case in acute and chronic leukemias and in liver dysfunction. The delay in prothrombin time in most cases is primarily due to deficiency of prothrombin. These observations indicate how to utilize best the procedures described in this paper in the presence of a delayed prothrombin time. The concentration of the labile factor should be determined first, as it is more likely to be normal. If this is the case, deficiency of prothrombin remains the main factor responsible for the delayed prothrombin time and the percentage concentration of prothrombin can be calculated directly from the prothrombin time by means of a dilution curve. If the labile factor is depleted, on the other hand, both its concentration and that of prothrombin should be determined. It may be added here that, in the course of Dicumarol therapy, the determination of the labile factor failed to disclose any depletion of this agent and that of the concentration of prothrombin agreed within ± 5 per cent with the percentage obtained with Quick's original one-stage method. This finding confirms the theory that hypoprothrombinemia may be the main, if not the only, factor in the delayed prothrombin time induced by the drug. It also demonstrates that Quick's method may be safely and accurately used as a guide in Dicumarol therapy. SUMMARY This paper presents new one-stage procedures for the quantitative determination of the plasmatic concentration of prothrombin and labile factor. The development of these procedures has been made necessary by the discovery that other factors, besides those considered in the classical theory of the coagulation of blood, influence the formation of thrombin. An analysis of the principles, technic and applications of the new methods is given.

8 240 STEFANINI Acknowledgment. I am grateful for the inspiration given me by Dr. Armand J. Quick, Professor and Director, Department of Biochemistry, Marquette University School of Medicine, with whom were developed the principles on which the methods presented in this paper are based. REFERENCES 1. GIORDANO, A. S.: Idiopathic hypoprothrombinemia. Am. J. Clin. Path., 13: 2S5-2S7, MUNBO, F. L., AND MUNRO, M. P.: Theinteraction of prothrombin A and B. Am. J. Physiol., 149: 95-99, OWRBN, P. A.: Nve unders0kelsor over blodets koagulasjon. Proc. Norwegian Acad. Sc, 21-23, OWRBN, P. A.: Parahemophilia; hemorrhagic diathesis due to absence of a previously unknown clotting factor. Lancet, 1: , QUICK, A. J.: On the constitution of prothrombin. Am. J. Physiol., 140: , QUICK, A. J.: On the quantitative estimation of prothrombin. Am. J. Clin. Path., 15: , QUICK, A. J.: Components of the prothrombin complex. Am. J. Phvsiol., 151: 63-70, S. QUICK, A. J., AND STEFANINI, M.: The concentration of the labile factor of prothrombin in human, dog and rabbit blood; its significance in the determination of prothrombin activity. J. Lab. and Clin. Med., 33: , 194S. 9. QUICK, A. J., AND STEFANINI, M.: The chemical state of calcium reacting in the coagulation of blood. J. Gen. Physiol., 32: , QUICK, A. J., AND STEFANINI, M.: The concentration of component A in blood, its assay and its relation to the labile factor. J. Lab. and Clin. Med., 34: 973-9S2, RHOADS, J. E., AND FITZ-HUGH, T., JR.: Idiopathic hypoprothrombinemia an apparently unrecorded condition. Am. J. M. Sc, 202: , STEFANINI, M.: Studies on the role of calcium in the coagulation of blood. Acta med. Scandinav., to be published. 13. STEFANINI, M., AND QUICK, A. J.: Effect of various adsorbants on the components of the prothrombin complex. Federation Proc, 8: 255, WARE, A. G., GUEST, M. M., AND SEEGERS, W. H.: Plasma accelerator factor and purified prothrombin activation. Science, 106: 41-42, 1947.