Armand J. Quick

Determination of Prothrombin

The author 1 developed a quantitative method for the determination of prothrombin based on the principle that the clotting time of blood or plasma is a quantitative measure of the prothrombin concentration provided an excess of thrombin and a constant concentration of calcium are present. For convenience and accuracy, the blood is oxa-lated and the test done on the plasma. It was demonstrated, however, that the test can be applied to whole blood. 2 In this determination 0.1 cc of thromboplastin emulsion was added to 1 cc of blood obtained by venipuncture. Theoretically there should be essentially no difference between the clotting time of recalcified oxalated plasma and unoxalated plasma or blood provided an excess of thromboplastin is present. This can be demonstrated experimentally as shown by the results of Table I. Recently Smith and his associates 3 have adopted the authors method of determining the clotting time of 1 cc of blood containing 0.1 cc of thromboplastin as a “Bedside Test” for the determination of pro-thrombin. They employ the formula: This formula is based on the assumption that the clotting time is a linear function of the concentration of prothrombin. The writers quantitative studies of prothrombin in man, 4 in the rabbit, in the chicken 5 and other animals have shown that the relationship between the clotting time and the concentration of prothrombin is not linear. If the values are plotted, a hyperbolic curve is obtained which can be satisfactorily expressed by the equation:

Intravenous modification of the hippuric acid test for liver function

A technique for carrying out the intravenous modification of the hippuric acid test for liver function is described. The excretion of 1 gm. of hippuric acid in one hour after 1.77 gm. of sodium benzoate has been injected is recommended as the standard for normal.

Prothrombin Concentration in Newborn

The concentration of prothrombin in the blood of babies 3 to 7 days old has been found to be essentially the same as in adult blood. 1 Curiously, however, a profound fluctuation has been observed to occur during the first 48 hours of life, as shown in Table I. It should be noted that the prothrombin level of babies 6 hours old is relatively high and not strikingly different from cord blood. At the end of 24 hours, however, it may drop to an exceedingly low level as shown by babies 5 and 6. After 48 hours the prothrombin concentration usually has begun to return to normal. In one baby (No. 10) the return was definitely delayed since 5 1/2 days elapsed before the concentration had reached 75% of normal. These results offer a solution to the perplexing problem of the hemorrhagic disease of the newborn. Apparently no reserve of prothrombin is built up in the fetus, and in the first 24 hours of life this clotting factor may be reduced to a distinctly hemorrhagic level. Were it not for the prompt restoration of the prothrombin, many babies would bleed. Any delay in this recovery will naturally give rise to a bleeding diathesis, and this is very probably the cause of the hemorrhagic disease of the newborn. Potentially all newborns are in jeopardy of hemorrhage, and this undoubtedly accounts for the insistence of the mosaic law that circumcision be not performed before the eighth day. The fact that the hemorrhagic disease of the newborn responds promptly to vitamin K therapy, as Waddell, et al., 2 have reported, indicates definitely that a deficiency of this factor is the basic cause. The question remains: why the abrupt recovery? The small amount of food taken during the first 48 hours obviously can furnish little vitamin K.

Activation of plasma thromboplastinogen and evidence of an inhibitor.

Summary The first step in coagulation is the conversion of thromboplastinogen to active thromplastin by a platelet factor. The platelets from hemophilic blood react equally as well as those of normal blood. Evidence of a factor which inhibits the activation of thromboplastinogen has been found in the blood of a patient who has an acquired hemophilia-like condition.

Synthesis of Hippuric Acid in Man Following Intravenous Injection of Sodium Benzoate

With the increasing employment of the synthesis of hippuric acid as a means of determining liver function, several disadvantages of this test have been recognized. Vomiting occasionally occurs after the ingestion of sodium benzoate; the collection of urine for 4 hours is at times inconvenient; and in rare instances the condition of the patient may not permit the oral administration of the drug. To meet these difficulties, the intravenous injection of sodium benzoate was investigated. 1.77 gm. of sodium benzoate (equivalent to 1.5 gm. benzoic acid) dissolved in 20 cc. of distilled water were given intravenously. The subject voided before the test began and a complete urine specimen was collected exactly one hour after the completion of the injection. Approximately 5 minutes were required for the injection. The hippuric acid was determined by the senior authors simple clinical method. 1 The results obtained on normal adult subjects are given in Table I. From these results one can conclude that after the injection of 1.5 gm. of benzoic acid (as the sodium salt), 0.7 to 0.95 gm. are excreted as hippuric acid during the first hour by a healthy adult subject. If exogenous glycine is supplied, the synthesis of hippuric acid is greatly increased, which indicates that the amount of hippuric acid excreted is a measure of the bodys capacity to synthesize glycine.

THE ASSAY AND PROPERTIES OF LABILE FACTOR (FACTOR V)

Human oxalated plasma stored at 4° C. until the prothrombin time is increased beyond 60 sec. is a reliable medium for assaying labile factor (factor V) because its response to added labile factor corresponds quantitatively to that of plasma from patients with congenital deficiency of this factor. Such an agreement is not obtained with plasma stored at 37°C. The stability of labile factor is closely associated with ionized calcium. The addition of thrombin to fresh oxalated plasma causes an apparent hyperactivity of labile factor, but this is completely removed by adsorption with Ca3(PO)2. Oxalated plasma when adsorbed with Ca3(PO4)2 before treatment with thrombin does not develop this adventitious activity, nor does it occur in stored plasma treated with thrombin. The seemingly high labile factor activity in serum can be explained by the activation of this factor which is independent of labile factor but acts synergistically with it. The true labile factor concentration can be determined only after the accelerator is removed by adsorption with Ca3(PO4)2. A close agreement between the consumption of prothrombin and the loss of labile factor during clotting is observed.

ON THE COMPARATIVE PROTHROMBIC ACTIVITY OF HUMAN AND DOG BLOOD

Soon after the introduction of the oneand twostage methods for determining prothrombin, it was noted that while the results obtained by the two procedures generally agreed fairly well, marked discrepancies occurred in certain specific instances. Particularly puzzling has been the observation that the prothrombic activity of human blood as compared with dog blood, when measured by the two tests, is markedly different. Warner, Brinkhous and Smith (1) reported that the prothrombin concentration in dog blood is 350 units per cc. and in human blood 295 units. More recently Murphy and Seegers (2) found 190 to 205 units in dog and 290 to 315 in human blood. Mann and his associates (3, 4) reported that the average for dog blood is 183 units per cc. and for human blood 319 units. These findings indicate that the prothrombin concentration as measured by the two-stage method is approximately the same in the two species. When determined by the one-stage method, the prothrombic activity of dog blood, in marked contrast, is many-fold higher than in human blood (5). The reason for this divergency has not been satisfactorily explained, but obviously as long as this problem remains unsolved, a marked gap in the knowledge concerning prothrombin remains. In the present investigation human and dog bloods were studied by means of the prothrombin consumption test and by the adsorption and elution technique for determining prothrombin with the aim of obtaining new data that might be helpful in arriving at a better understanding of what constitutes prothrombic activity.

Heparin and the Agglutination of Platelets in Vitro.

Heparin inhibits both the coagulation of the blood and the agglutination of platelets. A much higher concentration, however, is required for the latter. Solandt and Best 1 have shown that over 300 mg per kg of body weight must be injected intravenously to prevent the intravascular clumping of platelets. The object of the present study was to determine the minimum amount of heparin that is required for the prevention of platelet agglutination in vitro. Blood was drawn into a dry sterile syringe, and immediately distributed in 1 cc amounts to a series of small test tubes containing 0.1 cc of heparin solution (Roche Organon Liquaemin∗) of varying concentrations as recorded in the table. After thorough mixing, the blood was drawn to the 0.5 mark in a red blood cell pipette and the pipette filled with 0.85% sodium chloride solution. The platelet count was made in the usual manner. A concentration of more than 0.1 mg (or 10 Toronto units) per cc of human blood is required to prevent clumping of platelets. No decrease in the platelet count by heparin as reported by Copley and Robb 2 was observed. Why so much more heparin is required for the prevention of platelet agglutination than for inhibiting coagulation is not clear. Nevertheless, it is well recognized that a definite relationship exists between these two processes. An additional observation in support of this may be cited. The platelets of blood obtained from animals whose prothrombin has been drastically reduced by feeding dicumarol show no tendency to agglutinate in the absence of an anticoagulant. A typical experiment was as follows: Dicumarol was given to a rabbit daily until the prothrombin time was 7 minutes.

Prothrombin and the One-stage Prothrombin Time

We would like to thank Professor D. W. Smithers, of the Radiotherapy Department, and Professor W. V. Mayneord, of the Physics Department, and members of their staffs, for making this work possible. We are also grateful to numerous colleagues in this and other hospitals for referring cases to us, and in particular to Dr. P. E. T. Hancock. The earlier cases in the series were treated by Dr. R. J. Walton, to whom we acknowledge our debt.

Effect of dicumarol on concentration of the labile factor.

Conclusions The labile factor of the prothrombin complex is relatively constant in normal rabbit and dog blood. It is not reduced by dicumarol even after the prothrombin activity falls to a very low level.