ISOLATION AND PROPERTIES OF THE THIRD CLOTTING FACTOR IN BLOOD-PLATELETS

23 DIBCL’SSI01

that the barium enema, a most useful investiin its place, is often used unnecessarily. This, I gation rather than exclude think, is because it is used to to confirm the diagnosis of carcinoma of the colon. Any colonic symptom is taken to justify its use, and this policy has caused us to lose sight of the value of a good history and a proper examination. A history and physical examination, followed by digital examination of the rectum and sigmoidoscopy, was sufficient to warrant a laparotomy or to make the diagnosis practically certain in 96% of the cases in this series. Of the 40 barium enemas given in the 110 cases of carcinoma, 29 showed the site and colonic nature of a mass that had already been felt. 2 enemas were given where the lesion had already been seen through the sigmoidoscope, one confirming the growth and the other being reported as normal (carcinoma confirmed by operation). One enema failed in a case of intestinal obstruction ; another confirmed the presence of a pelvic colonic growth that had been felt through the rectal wall ; and another showed the site of the lesion in a case presenting with ascites. There remained 4 cases which may be said to have been diagnosed by means of the enema. One of these was virtually obstructed (case 1), there having been no bowel action for two weeks in spite of treatment. The other 3 patients (cases 2-4) had bloody diarrhoea, and 2 of them (cases 2 and 3) had symptoms suggesting subacute obstruction. If this series gives a true picture of the diagnosis of carcinoma of the colon, it appears highly improbable that patients complaining only of pain, constipation, or diarrhoea, without blood in the stools, and showing no abnormal physical signs or abnormality on rectal or sigmoidoscopical examination, will be found to have a carcinoma of the colon if a barium enema is given. This applies also to what has well been called the " much ballyhooed phenomenon " of alternating diarrhoea and constipation (Bockus 1946), which is usually due to the misuse of aperients. A discussion of the differential diagnosis between carcinoma of the colon and diverticulitis with its complications is not the purpose of this paper; but it must be remembered that this point can sometimes be settled with a barium enema, and sometimes only at operation. It might be argued that at some stage in the history of patients with carcinoma the symptoms of diarrhcea, constipation, or pain only are present. But whether an enema would show anything at this time is dubious. Certainly no example occurred in a large series of patients during these three years. Further, diagnosis is concerned with probabilities, and the conduct of medicine with practicabilities. Most radiologists would agree that a satisfactory barium enema cannot be performed except In many hospitals this on properly prepared patients. can be accomplished only by admitting the patient to the ward. But it is clearly impracticable that every patient with alteration in bowel habit or colonic pain should be admitted to hospital for examination by a It

seems

‘‘

"

competent radiologist. In this series there seemed to be no great difference between the histories of many patients with carcinoma and those of many with nervous and functional disorders of the colon. information obtained from old notes is necessarily limited, one significant fact emerges : 81% of the patients with carcinoma had a history of less than six months’ duration (40% of those with obstruction and 20% of the total had symptoms for less than a month). In clear contrast with this, 60% of the patients with functional disorders had a history dating back for a year or more (only 4O for a month or

Although

presenting

less).

SUMMARY

The notes of 110 consecutive cases of carcinoma of the colon have been examined to discover how the diagnosis was made. -

In 51 cases a palpable abdominal mass was present when the patient was first examined ; in 35 there were signs of acute intestinal obstruction, and in 16 the growth was felt per rectum or seen on sigmoidoscopy. 4 presented in unusual ways, and in 4 only was the diagnosis made with the barium enema in the absence of abnormal physical signs. Out of 227 barium enemas given during the year 1948, 163 showed no abnormality. Most of these had been given to exclude a carcinoma of the colon in patients with With 1 colonic symptoms without abnormal signs. none of these had patients developed possible exception a carcinoma two vears later. The barium enema seems to be most useful in revealing the site and nature of a mass in the abdomen. The of colonic must investigation symptoms invariably include digital examination of the rectum and sigmoidoscopy. These examinations should precede the barium enema and will often render it unnecessary in the diagnosis of carcinoma of the colon.

especially indebted to Dr. R. R. Bomford, who sugthis investigation and helped me in the preparation of this paper ; to Dr. M. H. Jupe, who allowed me access to the records of his department; and to the physicians and surgeons of the London Hospital for permission to cite their cases. I

am

gested

REFERENCE

Bockus, H. L. p. 757.

(1946) Gastro-enterology.

Philadelphia;

vol. II,

ISOLATION AND PROPERTIES OF

THE THIRD CLOTTING FACTOR IN BLOOD-PLATELETS *

S.

VAN

M.D.

CREVELD

Groningen

PROFESSOR OF PÆDIATRICS

M. M. P. PAULSSEN Ph.D. Amsterdam HEAD OF BIOCHEMICAL

With the technical assistance PÆDIATRIC

CLINIC, UNIVERSITY

LABORATORY

of

R. VONK

OF AMSTERDAM

WE have already described our demonstration of third clotting factor in the blood-platelets (van Creveld and Paulssen 1951). This we called factor 3, and to it we ascribed the anti-heparin activity of the bloodplatelets (Conley et al. 1949). Here we report a further study of factor 3. Chargaff et al. (1936) isolated from platelets a phosphatide fraction containing cephalin and a non-phosphatide (lipid) fraction. The phosphatide fraction, according to these workers, should strongly promote coagulation in normal blood. We applied their method of isolation to the water-insoluble fraction of human platelets which we obtained after factors 1 and 2 had been removed from the platelets by the method already described (van Creveld and-Paulssen 1951). In this way a phosphatide fraction and a non-phosphatide (lipid) fraction were obtained. A suspension of the phosphatide fraction proved to possess a strong anti-heparin activity even in high dilutions ; in this it differed from the non-phosphatide lipid also present in the remnants of the platelets. Table i clearly shows the anti-heparin activity of a suspension of the phosphatides (factor 3) isolated from the remnants of human platelets, compared with the activity of the acetone-soluble fraction (containing cholesterol, &c.) and with that of a suspension of the remains of the platelets containing no lipids. In recent years it has often been asked to what extent thromboplastic substances are present in the bloodplatelets. Ware et al. (1948) and McClaughry and Seegers (1950) showed that a watery extract of normal platelets had only a slight thromboplastic effect. These workers defined thromboplastins as substances which, a

* This investigation was made possible by the Netherlands tion for Applied Scientific Research (T.N.O.).

Organisa-

24 TABLE I——ANTI-HEFAHESr ACTIVITY OF FACTOR

i

Factor 3

pension (ml.)

Hepari- i nised

I(j

in dilution

plasma *I (ml.)

*

i

sus-

I

i

1: i

3 I

Limpid- BI chlorIde chlorideIclottingAcetoneClottingsoluble free let time soluble ’I 0-9 % ,I fraction remnants: platelet I (min.) (min.) (ml.) remnants. (ml.) 0.9% I

free i SodÎl!m

!1 : 10:1:10C!) _

IB time

Platelet-deficient normal plasma prepared according to the silicone technique of Jaques et a]. (1946). To this plasma was added 4g. of heparin per ml.

in

the

presence

factor, Seegers’s

of

serum-accelerator (Quick’s labile globulin) and calcium, can

serum-AC

obtained after the clotting of this plasma an abnormally large amount of prothrombin t could still be demonTherefore the A.H.F. cannot, or strated (table IIA). cannot only, act as a thrombocytolysin. Normal values obtained for the clotting-time in such experiments do not prove that the process of coagulation is normal. Normal formation of a fibrin clot does not necessarily mean that the process of coagulation is normal. Coagulation can only be normal if the consumption of prothrombin during coagulation (determined by a two-stage method) is also normal. In agreement with Brinkhous we further found that the addition of normal platelet-deficient plasma (source of A.H.F.) to hæmophilic platelet-deficient plasma greatly shortened the clotting-time of the hæmophilic plasma, TABLE II—INTERACTION OF A.H.F. AND FACTOR

3

convert pure prothrombin into thrombin. As it has been known for many years that the anticoagulant activity of heparin is neutralised by tissue thromboplastins (Howell and Holt 1918), we investigated how far the phosphatide fraction, containing factor 3, exerted a thromboplastic activity according to the definition of McClaughry and Seegers (1950). We used their technique, which is based on the following principle : In a buffered solution of pure prothrombin and serum-a in which sodiumchloride, calcium chloride, and colloid content (acacia) are also present and controlled, no thrombin can be formed. Only after the addition of a thromboplastin can prothrombin be converted into thrombin.

globulin,

The thrombin is measured by determination of the clotting-time of purified fibrinogen after the addition of the incubation mixture. Instead of serum-Ac globulin we used a solution of factor 1, which has an activity analogous to that of After the addition of a suspension serum-AC globulin. of factor 3 to this mixture containing prothrombin, factor 1 (from human platelets), and calcium, the prothrombin was converted into thrombin (see figure). In the absence of factor 3 no thrombin was formed after several hours’ incubation. The experiment proves that in platelets the two factors necessary for the conversion of prothrombin are present : factor 1 as platelet accelerator, and factor 3 as

thromboplastic

agent. Brinkhous

(1947) has shown Thrombin formation during incubation of pure prothrombin with factor I, factor 3, and calcium.

Creveld

1935, Patek and

that theantihæmophilic factor (A.H.F.) present in

normal

plasma

(Bendien and van Taylor 1937) can exert its

in the presence of platelets. Brinkhous found that the delayed clotting-time in haemophilia could be restored to normal with platelet-deficient normal plasma, and that the incomplete clotting of plateletdeficient normal plasma could be restored to normal by the addition of a suspension of platelets. Brinkhous suggested that the antihsemophilic factor deficient in haemophilia is required for the utilisation of the platelets, and suggested that this factor is a thrombocytolysin. As regards this last suggestion, however, we found that the addition of mechanically lysed platelets to hæmophilic plasma deficient in platelets greatly shortened the clotting-time of this plasma, but that in the serum

activity only

did not become normal. We found that the serum of this plasma still contained a large amount of prothrombin. The addition of factor 3 to hsemophilic plateletdeficient plasma also shortened the clotting-time of the hæmophilic plasma. However, in the absence of A.II.F. this clotting-time did not become completely normal, and in the serum of the plasma a large amount of prothrombin could still be demonstrated. The addition of both factor 3 and normal platelet-deficient plasma to haemophilic platelet-deficient plasma caused a normal clotting-time of the latter plasma as well as a normal consumption of prothrombin (table IIA). It seems that factor 1 and factor 2 of the platelets cannot take the place of factor 3 in this respect-e.g., after the addition of factor 1 and factor 2, together with

but the

clotting-time

moreover

(normal platelet-deficient plasma), to hsemophilic platelet-deficient plasma the clotting-time did not become completely normal and the prothrombin content of the serum was still much increased (table IIB). We therefore conclude that A.H.F. requires for its activity the presence of blood-platelets, as already found by Brinkhous. The factor in the platelets which is responsible in this process is factor 3. Factors 1 and 2 of the platelets cannot take the place of factor 3 in this respect. A.H.F.

SUMMARY

The platelet-factor which neutralises heparin is a phosphatide, which we call factor 3. Factor 3 has thromboplastic activity-e.g., together with calcium and an accelerator, it converts prothrombin into thrombin. The antihsemophilic plasma requires for its

substance present in normal activity factor 3 as a co-factor.

t For prothrombin determinations in

serum we

technique of Ware and Seegers (1948).

used the two-stage

25 Factors 1 and 2 of the platelets cannot take the factor 3 in this respect.

place

off

REFERENCES

Creveld, S. (1935) Acta brev. neerl. 5, 135. Brinkhous, K. M. (1947) Proc. Soc. exp. Biol., N.Y. 66, 117. Chargaff, E., Bancroft, F. W., Stanley-Brown, M. (1936) J. biol. Chem. 116, 237. Conley, C. L., Hartmann, R. C., Morse, W. I. (1949) J. clin. Invest. 28, 340. Howell, W. H., Holt, L. E. (1918) Amer. J. Physiol. 47, 328. Jaques, L. B., Fidlar, E., Feldsted, E. T., Macdonald, A. G. (1946) Canad. med. Ass. J. 55, 26. MoClaughry, R. I., Seegers, W. H. (1950) Blood, 5, 303. Patek, A. J. jun., Taylor, F. H. L. (1937) J. clin. Invest. 16, 113. van Creveld, S., Paulssen, M. M. P. (1951) Lancet, ii, 242. Ware, A. G.. Fahey, J. L., Seegers, W. H. (1948) Amer. J. Physiol. 154, 140. Seegers, W. H. (1948) J. biol. Chem. 172, 699. Bendien, W. M.,

van

—

to loss of the stomach reservoir or to overloading of the upper small bowel. More recently Butler and Capper (1951) have shown experimentally that stretching of the stomach remnant by food and the unsupported afferent jejunal loop will cause most of the symptoms associated with the early post-gastrectomy syndromes, and they have demonstrated that this stretching can be controlled operatively in man (Capper and Butler 1951). The new technique should not produce these syndromes if, as seems probable, they are largely anatomical in origin. Further, this technique should largely prevent postgastrectomy hypoglycæmia, said to be caused by excess production of

syndrome due, they believe,

insulin

COLONIC REPLACEMENT OF THE STOMACH EARLY RESULTS OF RADIOLOGICAL INVESTIGATION

B. B. HARRISON M.B., M.Rad. Lpool, D.M.R.D. CONSULTANT

RADIOLOGIST,

ISLE OF MAN HEALTH SERVICES AND

NOBLE’S HOSPITAL, DOUGLAS ; LATE BROADGREEN HOSPITAL,

ASSISTANT RADIOLOGIST, LIVERPOOL

PARTIAL gastrectomy, as at present practised, consists in removal of up to three-quarters of the stomach, with subsequent anastomosis of the remnant to the jejunum several inches beyond the duodenojejunal flexure. Consequently most of the reservoir effect of the normal stomach, whereby the food is macerated and mixed with gastric secretions is completely lost, and with the formation of a surgical fistula between the stomach and the small bowel a considerable length of the most active and important part of the small intestine is bypassed. Serious interference with the normal physiology of the gastro-intestinal tract is therefore inevitable. To overcome these deficiencies of the Polya type of gastrectomy, Moroney (1951) substituted a length of the transverse colon for the resected portion of the stomach (fig. 1). This was intended to increase the capacity of the " gastric " reservoir and to ensure that chyme entered the small bowel in the normal manner. It was felt that this would not only prevent distressing postgastrectomy symptoms but might also be a means of relieving them when already present. Wells and Welbourn (1951) reviewing the post-gastrectomy syndromes, distinguish no fewer than seven types of early postcibal

owing rapid absorption of

to

too

sugar from the upper small bowel; the general malnutrition due to inefficient

absorption by a ’’ physiologically shortened" small bowel ; fat with

inadequate absorption

steatorrhoea, Brain believes to be due to inefficient admixture of pancreatic juice with the food ; and possibly the poor absorption of vitamin B. Whatever which

(1951)

Fig. 2-Radiograph taken immediately after a barium meal, showing how colon retains normal haustration. part of duodenum.

Note bolus in second

subsequent experience may show, early results seem to indicate that the substitution of colon for stomach will, as was hoped, prevent most of the post-gastrectomy syndromes, and alleviate the distressing symptoms which have so often followed, early or late, the Polya type of gastrectomy.

_

I have now followed 30 cases radiologically for six months or more, and the information gained from radiography at one, three, and six months is described here, with particular reference to the physiological aims of the operation. GASTRIC

On

screening

a

RESERVOIR

normal stomach it is

common

to find

that, immediately after ingestion of the first mouthful of barium, it is possible to force a little through the pylorus. Very quickly, however, the pylorus closes and

Fig- I-Tracing

of

radiograph showing relationship of

to stomach remnant

and duodenum.

colonic segment

for some minutes remains so. Not until after peristalsis has begun in the stomach does the pylorus again open to permit passage of the meal into the duodenum. This delay allows admixture of food and gastric juices and disintegration of the solid particles of the meal. When the stomach begins to empty, the meal passes through the pylorus intermittently, allowing the duodenum to accommodate itself to one bolus at a time. After partial gastrectomy, however, barium immediately fills the efferent loop of jejunum, and continues to leave the stomach remnant quickly and continuously. When colonic replacement has been carried out, the appearances approximate much more nearly to the normal. Barium enters the stomach remnant and soon afterwards fills the colonic segment. In some of the earlier cases, in which only 21 2—3 in. of colon was used,