THE COMPLEX MECHANISMS OF THE QUICK PROTHROMBIN TEST AND THE EFFECT OF DICUMAROL*

Transcription

1 THE COMPLEX MECHANISMS OF THE QUICK PROTHROMBIN TEST AND THE EFFECT OF DICUMAROL* FRANK D. MANN, M.D., AND MARGARET M. HURN, M.S. From the Section on Clinical Pathology, Mayo Clinic, Rochester, Minnesota Investigations in a number of laboratories 16 25, ' 26 have necessitated revision of the concept that the one-stage Quick prothrombin time measures a single substance, prothrombin, while at the same time the concept of prothrombin as a definite substance has been greatly strengthened by the work of Seegers and his associates. Nevertheless, the Quick prothrombin time has been directly related to the physiologic state of the coagulation system of the blood by a great accumulation of clinical experience; it must remain the main reliance of the physician unless and until it is superseded by procedures backed by an equal weight of clinical data. Although at present we have no intention of attempting such a replacement, we have tried to study some of the individual factors which influence the Quick prothrombin time in the hope of obtaining a better understanding of the over-all result. The procedures which have thus far been most useful to us in this endeavor are (1) the well-known two-stage prothrombin determination, which measures the total amount of thrombin the plasma can yield, and (2) the co-thromboplastin assay. The latter test, with which this paper is largely concerned, is based upon a preliminary reaction between thromboplastin and very dilute plasma or serum which influences the rate of subsequent conversion of prothrombin to thrombin. METHODS One-stage 8 and two-stage 10 prothrombin determinations and co-thromboplastin assays 13 were performed as previously described. One tenth of 1 ml. of human serum was added to the 2 ml. of conversion mixture in the two-stage determination. The specimens of serum used (at least four hours subsequent to coagulation) were assayed for prothrombin by the two-stage method, and those containing more than 1 unit of prothrombin per milliliter were rejected. For the co-thromboplastin assay, normal control plasma and thromboplastin were used which had approximately the characteristics previously reported. 13 Some preparations of thromboplastin, although suitable for the two-stage determination, could not be used for co-thromboplastin assay, probably for reasons which will be discussed later. RESULTS Specimens of plasma from patients receiving routine Dicumarol therapy were studied (Table 1). It may be generalized that while a marked Dicumarol effect * Presented at the Twenty-Eighth Annual Meeting of the American Society of Clinical Pathologists, in Chicago, October 13, Received for publication, November 16,

2 226 MANN AND HURN was rapidly developing, the one-stage test gave far lower values for "prothrombin" than did the two-stage. The levels of prothrombin found by the two-stage method were remarkably high in some patients who had abnormal prothrombin times. (In a previous study, 9 the difference between the one-stage and twostage methods tended to be less, probably because the necessity of adding serum in the two-stage procedure was not realized at that time.) In specimens with such an extreme difference between values for prothrombin as demonstrated by the two-stage and one-stage procedures, the co-thromboplastin assay always gave very low values. Thus, to measure the co-thromboplastin effect in these abnormal plasmas it was necessary to use a dilution no greater than 1 to 25, in contrast with 1 to 200 (on the average) for the normal control. In prolonged Dicumarol therapy TABLE 1 EFFECT OF DICUMAROL ON BLOOD COAGULATION FACTORS TREATMENT WITH DICUMAROL QUICK PROTHROMBIN ASSAY Seconds "Per cent Normal" TWO-STAGE PROTHROM BIN ASSAY Units Per Cent Average Normal CO-THROMBO PLASTIN ASSAY, PER CENT OE CONTROL Two days Two clavs Two clays Three clays Nine clays 12 days 125 clays Two clays One clay later, after Vit. K Two days One day later, after Vit. K IS S t 10 actual low values for prothrombin were obtained with the two-stage procedure, but the value for co-thromboplastin usually remained lower. During recovery from Dicumarol, co-thromboplastin rose rapidly. (No conclusions are drawn at present as to the role of vitamin K.) While decreased co-thromboplastin certainly appears to be an important, and perhaps the principal, factor in the initial or increasing stages of the Dicumarol effect, it is equally apparent that it is not a complete explanation of the findings. In fact, we have deferred a more extensive investigation of the problem pending new methods because it became immediately evident that large differences occurred in the Quick prothrombin time when the differences produced by the cothromboplastin method and the two-stage prothrombin procedure were small or were in the opposite direction. In hypoprothrombinemia caused by damage to the liver or deficiency of vitamin K (Table 2) a decreased value as obtained by the one-stage method usually was associated with a marked although not necessarily equal decrease in the

3 PROTHROMBIN TEST 227 value secured from the two-stage prothrombin procedure, which was not greatly affected by the use of serum in the conversion system. (As previously reported, in hepatic disease it is not unusual for the value resulting from the two-stage prothrombin determination to be actually lower than the prothrombin time would suggest.) Co-thromboplastin is decreased, but not ordinarily to the disproportionate extent noted in patients receiving Dicumarol. It should be pointed out that since the changes in both prothrombin and cothromboplastin rapidly reverse themselves on recovery, the foregoing relationships depend to a considerable degree on the tempo of the processes. Thus, the differences between Dicumarol and other types of hypoprothrombinemia may not always be as sharp as in the examples given. Experimentally in the rat, in which marked hypoprothrombinemia due to deficiency of vitamin K can be TABLE 2 EFFECT OF DEFICIENCY OF VITAMIN K AND DAMAGE TO LIVER ON BLOOD COAGULATION FACTORS QUICK PROTHROM BIN ASSAY TWO-STAGE PROTHROMBIN ASSAY CO-THROMBO- DIAGNOSIS Seconds "PerCent Normal" Units Per Cent Average Normal PER CENT OP CONTROL Carcinomatous obstruction of bile duct Same, after Vitamin K Cirrhosis, terminal hepatic coma Cirrhosis, terminal hepatic coma Infectious hepatitis, terminal hepatic coma SO IS rapidly and consistently produced by external drainage of the intestinal lymph, 18 co-thromboplastin was always higher than the value produced by the two-stage prothrombin determination, in striking contrast to the Dicumarol effect produced in the same species of animal at about the same rate. 17 It seemed likely that the co-thromboplastin effect might explain the differences previously noted 8 in the action, on certain specimens of Dicumarol plasma, exerted by certain thromboplastin preparations which were practically identical in their behavior toward normal plasma. In Table 3 is shown a striking example of such inconsistencies, resembling those characteristically observed with venom from Russell's viper. 30 As might be expected, specimens of Dicumarol plasma which show prolonged one-stage prothrombin times with standard rabbit-brain thromboplastin and much shorter times with these other thromboplastins have relatively normal amounts of prothrombin and low co-thromboplastin. Brief exposure to very dilute serum (Table 4) causes standard thromboplastin to take on the property of these unsuitable thromboplastins; namely, extreme insensitivity to the Dicumarol effect.

4 228 MANN AND HURN COMMENT The report of Hum, Barker and Mann 9 of defective conversion of prothrombin to thrombin in the blood of patients receiving Dicumarol therapy was paralleled by similar observations of Owen and Bollman 22 in the dog. Almost coin- TABLE3 STANDARDIZED THROMBOPLASTIN COMPARED TO A COMMERCIAL* THROMBOPLASTIN WITH USE OF NORMAL AND DICUMAROL PLASMA QUICK PROTHROMBIN TIME, SECONDS Normal 1 Normal 2 Dicumarol 1 Dicumarol 2 Dicumarol 3 Dicumarol 5. Dicumarol 6 Dicumarol 7 Dicumarol S Standardized Thromboplastin S 72 Commercial Thromboplastin 17 IS * A preparation which is not in common use, made from horse tissue. TABLE 4 EFFECT OF EXPOSURE TO VERY DILUTE SERUM O.V STANDARD THROMBOPLASTIN USED IN THE QUICK TEST REACTION MIXTURE CLOTTING TIME 0.1 ml. thromboplastin ml. normal saline ml. Dicumarol plasma mixed, then 0.1 ml M calcium chloride blown in immediately 0.1 ml. Dicumarol plasma ml. normal serum 1:100 mixed, then 0.2 ml. of a mixture of equal parts thromboplastin and M calcium chloride blown in immediately 0.1 ml. thromboplastin ml. normal serum 1: ml M calcium chloride mixed, allowed to stand 3 minutes at room temperature, then 0.1 ml. Dicumarol plasma blown in seconds The Dicumarol plasma used had a Quick prothrombin time of 50 seconds, two-stage prothrombin level of 260 units per milliliter and co-thromboplastin of 8 per cent of control. The serum used contained less than 1 unit of prothrombin per milliliter. cident with the foregoing work, it became generally recognized on the basis of a number of largely independent contributions (reviewed by Seegers and Ware 26 ) that a factor in plasma other than prothrombin plays an important role in the conversion of prothrombin to thrombin. Most of these investigators have written in terms of a single factor, but Quick, 24 ' 25 Mann and Hum, 15 and Flynn and Standley 6 have presented independent and different evidence that there are more

5 PROTHROMBIN TEST 229 than one. Naturally, these different investigators have largely used different terms, and it is often difficult to determine whether one given factor is the same as another. There have even been repeated changes in terminology from the same laboratory. u 25 Despite this rather confusing situation, it remains important to learn what may be responsible for a given defect in the conversion of prothrombin to thrombin, especially that existing in Dicumarol plasma. It should be remembered that it was actually in Dicumarol plasma that a prothrombin conversion factor was first directly demonstrated. (This factor, which the discoverer, Quick, first called "prothrombin A", and later the "labile factor", has been rather generally regarded as synonymous with the factor V of Owren and Ac-globulin of Ware, Guest and Seegers.) Accordingly, it is hard to see how Dicumarol plasma can be so strikingly deficient in this factor as to explain its marked conversion defect. Fahey, Olwin and Ware 8 found a relatively small decrease in Ac-globulin after Dicumarol, but Olwin did not consider this to be adequate explanation for the differences between one-stage and two-stage prothrombin values. Olwin found values obtained with the one-stage prothrombin procedure usually higher than those obtained with the two-stage method during Dicumarol therapy, results just opposite to those of the other workers herein cited. It may be pointed out that if the conversion defect of Dicumarol plasma is not deficiency of Ac-globulin, then the modification of the two-stage method by the supplying of Ac-globulin does not necessarily assure quantitative conversion of prothrombin to thrombin in such plasma. Human serum of the age used in our determinations is reported to contain very little Ac-globulin activity, but it has marked co-thromboplastin activity and is effective in restoring the conversion defect of Dicumarol plasma when it is used in the amount stated. Mawson 19 recently studied Dicumarol plasma by the two-stage and several onestage technics and concluded that the differences observed are not explained by a deficiency of the "labile factor", or "Ac-globulin". In addition to the work already quoted and that reviewed by Seegers and Ware, the studies of Alexander and associates 2 ' 4 and of Jacox 12 should be compared with the foregoing. Not merely is it difficult to determine what coagulation factor is meant by a term in the literature, but it is basically no easy matter to study specifically a given factoi' by the actual use of laboratory procedures. It is not regarded as proved that co-thromboplastin activity is due to a specific factor, co-thromboplastin, but this point of view will be used as a basis for discussion. Co-thromboplastin, as the name indicates, reacts with thromboplastin in the absence of moiji or all of the prothrombin which later is to be converted into thrombin. Co-thromboplastin is resistant to heat and is extremely resistant to aging and to the procedures for inactivation of the third and fourth components of complement, which we have found also to inactivate prothrombin conversion factors. 15 Like prothrombin, it is readily adsorbed by such materials as aluminum hydroxide, and it is affected in the same way as prothrombin by deficiency of vitamin K and damage to the liver. It is normal in hemophilia. This suggests that co-thromboplastin is closely related to prothrombin chemically and physiologically. However, co-thromboplastin is high in serum which is virtually free of

6 230 MANN AND HURN prothrombin, and it is very low in specimens of Dicumarol plasma which contain abundant prothrombin. Although deficiency of co-thromboplastin appears to be characteristic of Dicumarol plasma, it does not quantitatively explain the findings obtained with one-stage and two-stage prothrombin determinations. Additional methods will be needed to explain fully the complex effect of Dicumarol. Changes in fibrinogen and in antithrombin have been found by some investigators 9 u ' and not by others (using somewhat different material and methods); we feel that both factors should remain under consideration. Flynn and Standley 7 have studied the time course of the reaction in the one-stage method by two-stage determinations at short intervals. They found that the thrombin formed in plasma is destroyed far more rapidly than is the case with the commercial thrombin preparations available for antithrombin studies. Hence, physiologically significant antithrombin determinations probably remain to be done. The fact that brief exposure to very dilute serum or plasma (up to 1:2,400 dilution in the co-thromboplastin assay system with certain exceptional thromboplastins) markedly increases the activity of thromboplastin means that it is difficult to rule out the occurrence of such effects during the preparation of thromboplastin from tissue. Thus, specimens of thromboplastin may have been exposed to a partial or maximal co-thromboplastin effect during preparation, resulting in varying degrees of insensitivity to the co-thromboplastin deficiency of Dicumarol plasma, and also unsuitability for the co-thromboplastin assay. It may even be surmised (although we have no evidence on this point) that the very possibility of preparing thromboplastins sensitive to co-thromboplastin effect may depend upon the presence in tissue of inhibitors of the type prepared by Tocantins and Carroll. 29 In view of the stability of co-thromboplastin, one may further speculate that it may have something to do with the peculiar increases in thromboplastic activity noted by Aggeler and associates in stored dried brain. It has been observed in three different laboratories that thromboplastin prepared from the brains of animals receiving Dicumarol was deficient in activity particularly when tested on Dicumarol plasma S The co-thromboplastin effect would appear to provide an entirely reasonable basis for this otherwise unexplained phenomenon. Finally, the foregoing observations provide a rationale for the system of checking thromboplastin on Dicumarol plasma developed by Hum, Barker and Magath. 8 The one-stage prothrombin test, once regarded as very simple, has been shown to be extremely complex; a given prolonged prothrombin time has different meanings in different conditions. Different technics of the one-stage test are variously affected by the several factors concerned. Hence, the basis for control of this useful but empirical procedure should be adequate sensitivity and reproducibility with the precise type of abnormal plasma in which one is interested, and especially the plasma of patients treated with Dicumarol. SUMMARY The increase in the Quick prothrombin time after the use of Dicumarol is explained partly but not entirely by a decrease in co-thromboplastin activity,

7 PROTHROMBIN TEST 231 which is often a far larger change than occurs in prothrombin. Due to co-thromboplastin, exposure to very dilute serum or plasma increases the activity of thromboplastin, especially with respect to Dicumarol plasma. Variations in results obtained with the one-stage prothrombin test may be due to varying degrees of exposure of the thromboplastin to blood during its pi'eparation from tissue. REFERENCES 1. ACGELER, P.M., LEAKE, TILLIEB., AND TALBOT, J. C: Spontaneous increase in potency of thromboplastin from acetone-extracted brain tissue. Science, 108: , 194S. 2. ALEXANDER, B., DE VRIES, A., AND GOLDSTEIN, R.: A factor in serum which accelerates the conversion of prothrombin to thrombin. II. Its evolution with special reference to the influence of conditions which affect blood coagulation. Blood, 4: , BOSE, A. N.: On mode of action of Dicoumarin. Ann. Biochem. and Exper. Med., 5: , DE VRIES, A., ALEXANDER, B., AND GOLDSTEIN, R.: A factor in serum which accelerates the conversion of prothrombin to thrombin. I. Its determination and some physiologic and biochemical properties. Blood, 4: S, FAIIEY, J. L., OLWIN, J. H., AND WARE, A. G.: Effect of Dicumarol on Ac-globulin and prothrombin activity. Proc. Soc. Exper. Biol, and Med., 69: , 194S. f>. FLYNN,,J. E., AND STANDLEY, E. T.: Effect of lecithin in the formation of thrombin. Federation Proc, 8: 5, FLYNN, J. E., AND STANDLEY, E. T.: Unpublished data. 8. HURN, MARGARET, BARKER, N. W., AND MAGATII, T. B.: The determination of prothrombin time following the administration of Dicumarol, 3,3'-methylenebis (4-hydroxveoumarin), with special reference to thromboplastin. J. Lab. and Clin. Med., 30: , HURN, MARGARET, BARKER, N. W., AND MANN, F. D.: Variations in prothrombin and antithrombin following the administration of Dicumarol. Am. J. Clin. Path., 17: , HURN, MARGARET, AND MANN, F. D.: The determination of prothrombin (two-stage method) and antithrombin using commercially available reagents. Am. J. Clin. Path., 17: , IRISH, U. ID., AND JAQUES, L. B.: The effect of Dicumarol upon plasma fibrinogen. Am. J. Physiol., 3: , JACOX, R. F.: Studies on the activation of a serum "prothrombin-converting factor." J.Clin. Investigation, 28: , MANN, F. D.: Co-throniboplastin assay. Am. J. Clin. Path., 19: S61-S64, MANN, F. D., BUTT, H. R,, AND HURN, MARGARET: Prothrombin in liver disease: a clinical evaluation of the two-stage method. Gastroenterology, 11: , 194S. 15. MANN, F. ID., AND HURN, MARGARET: Relation of complement to blood coagulation. Proc. Soc. Exper. Biol, and Med., 67: S3-S5, 194S. 16. MANN, F. D., HURN, MARGARET, AND MATHIESON, D. R.: Platelets as foci in the coagulation of blood. Am. J. Physiol., 158: S4-SS, MANN, F. ID., MANN, J. D., AND BOLLMAN, J. L.: Unpublished data. IS. MANN, J. D., MANN, F. D., AND BOLLMAN, J. L.: Hvpoprothrombinemia due to loss of intestinal lymph. Am. J. Physiol., 158: 311-3, MAWSON, C. A.: The use of Russell viper venom and lecithin as thromboplastin in the estimation of prothrombin. J. Lab. and Clin. Med., 34: 45S-472, MUNRO, F. L., AND LUPTON, A. M.: The influence of Dicumarol treatment of rabbits, rats and dogs on the thromboplastic activity of their brain suspensions. Arch. Biochem., 15: 245^250, 1947., 21. OLWIN, J. H.: The one-stage and two-stage prothrombin methods in the control of Dicumarol therapy, with remarks on Ac-globulin. J. Lab. and Clin. Med., 34: S06-813, OWEN, C. A., AND BOLLMAN,.J. L.: Prothrombin conversion factor of Dicumarol plasma. Proc. Soc. Exper. Biol, and Med., 67: , 194S. 23. OWEN, C. A., AND BOLLMAN, J. L.: Serum and plasma antithrombin. Proc. Soc. Exper. Biol, and Med., 67: , QUICK, A. J.: Congenital hypoprothrombinaemia and pscudohvpoprothrombinacmia. Lancet, 2: , QUICK, A. J., AND STEEANINI, M.: The concentration of component A in blood, its assay and relation to the labile factor. J. Lab. and Clin. Mod., 34: 973-9S2, 1949.

8 232 MANN AND HURN 26. SEEOERS, W. H., AND WARE, A. G.: Recent advances in our knowledge of prothrombin. Am. J. Clin. Path., 19: 41-47, SHAPIRO, S., AND MOORE, D. H.: Electrophoretic patterns after Dicumarol medication. Proc. Soc. Exper. Biol, and Med., 69: , STEPANINI, M., AND BLANCHAER, M. C: Action of 3,3' methylenebis (4-hydroxycoumarin) (Dicumarol) on thromboplastic activitv of rabbit brain. Proc. Soc. Exper. Biol, and Med., 64: 47-50, TOCANTINS, L. M., AND CARROLL, R. T.: Activity curves of crude and purified inhibitors and accelerators of blood coagulation. Proc. Soc. Exper. Biol, and Med., 69: , 194S. 30. WILSON, S. J.: Differences during Dicumarol therapy in the Quick and Russell viper venom methods for prothrombin determination. Proc. Soc. Exper. Biol, and Med., 66: , 1947.