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J. H. MILSTONE RELATION TO THE THEORY OF HEMOSTASIS AND THROMBOSISTHE CHAIN REACTION OF THE BLOOD CLOTTING MECHANISM IN

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THE CHAIN REACTION OF THE BLOOD CLOTTING MECHANISM IN

RELATION TO THE THEORY OF HEMOSTASIS AND THROMBOSIS

ByJ. H. MILSTONE, M.D.

THE PHYSIOLOGIC REQUIREMENT

I F OCCASION arose to invent a blood clotting mechanism, it might be arranged

for the blood to remain fluid in the vessels, yet promptly congeal when mixed

with the juice of freshly cut tissue. This would probably work well for small

punctures. However, as this mechanism was tested with larger cuts, an unexpected

difficulty might appear. As the blood flowed through the break, a coagulated

film would be deposited on the cut surface. This layer would seal over the wounded

tissue and hinder the admixture of tissue juice with that portion of blood not yet

clotted. Therefore, hemorrhage would continue through a passageway lined by

freshly clotted blood.

To overcome this difficulty a chain reaction might be introduced. Then, as one

layer of blood was clotting, it would incite the neighboring layer to clot. Thereby

the coagulation process could be propagated from one layer to the next, and tissue

juice would be needed only to exert its effect on the initial layer. The mechanism

could then achieve a hemostatic plug which would grow by accretion, and which,

in this respect, would resemble the natural plug depicted by Tocantins.t

Thus, by teleologic conjecture, we have arrived at some function a chain reaction

might serve. Other functions can be imagined. Ordinary chemical reactions, be they

stoichiometric combinations or enzymatic, begin rapidly and thereafter slow

down. But, when a chain reaction is involved, one of the products accelerates the

reaction, with the result that as more product is formed the reaction goes in-

creasingly faster.* Hence, chain reactions may start with a lag period, but later

are apt to proceed explosively. This offers opportunity for control during the lag

period, but ensures rapid action when the lag has been passed or overcome.

THE BIOCHEMICAL MECHANISM

Whatever its prime function, a chain reaction does occur during the coagulation

of blood. An experiment in which the clotting process was transmitted through

a series of plasma samples was described by Gratia in 1911.2 Once the process had

From the Laboratory of Pathology, Yale University School of Medicine, New Haven, Conn.

This work was aided by a grant from the James Hudson Brown Memorial Fund of the Yale Univer-

sity School of Medicine.

* The concept of chain reaction was originally introduced in connection with the photochemical

combination of hydrogen and chlorine. It has been broadly applied to various series of consecutive re-

actions which repeat themselves over and over again. When, in addition, the reaction velocity increases

as described by Glasstone (Glasstone, S.: Textbook of Physical Chemistry, ed. i. New York, D. Van

Nostrand Co., Inc., 1946. P. 1083), then the chain is said to be nonstationary. It is this type which is im-

plied throughout the present discussion. Here, the terms ‘�cJ��i� reaction’S and “autocatalytic effect”

are used in order to include other possible mechanisms besides a simple autocatalytic reaction; and auto-

catalytic reactions are regarded as a special group of nonstationary chain reactions.

I 2.90




J. H. MILSTONE 12.91

been initiated in the first tube, each tube of fluid plasma was caused to clot by

seeding it with a few drops of serum from the preceding tube. In 1935, Fischer,�

apparently unaware of Gratia’s previous discussion, reported similar serial passage

experiments with minor differences in technic and results. Gratia was impressed

by the formal resemblance between the propagation of bacteriophage and thepropagation of the clotting process with repeated new formation of thrombin.

Fischer wrote of blood coagulation as an endlessly transmissible chain reaction.

Although these demonstrations were spectacular, they were not the first in-

dications of the autocatalytic effect. Beginning in the nineteenth century, two

complementary approaches have been made to the analysis of coagulation mecha-

nism: (a) Separation of coagulation factors; (h) separation of individual reactions.

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FIG. 1.-SOME EVENTS OCCURRING WHEN BLooD CLOTS IN A GLASS TUBE. The chain reaction is the

kind of process which could cause the growth of a hemostatic plug and the propagation of a thrombus.

The decreases in thrombokinase activity, thrombin and fibrin illustrate the antithrombokinase,

antithrombin and fibrinolytic effects, respectively. Such reactions may help to limit the growth of a

hemostatic plug, or of a thrombus.

This type of synoptic diagram does not appear to have been attempted before and modifications

will probably become necessary as more data are obtained. The broken line representing platelet changes

is based on observations less pertinent than is the case for the three continous curves. The historical and

experimental background is outlined in the text. (Figure prepared by Mr. Armin Hemberger.)

By 1904, this twin approach had developed far enough to engender the classic two-

stage theory:

i. Prothrombin -s Thrombin. In the presence of thrombokinase and calcium.

i. Fibrinogen -* Fibrin. In the presence of thrombin.

But, beginning before 1904, and continuing into the present, results have been

obtained with separated factors and separated reactions which show that the

simple two-stage concept must be modified. And all along the line there have been

repeated intimations of autocatalysis. Many studies have been made of the rate at

which the coagulation products appear. A frequent finding has been a lag period

followed by a period of accelerated production-characteristics of a chain re-

action. This is illustrated in figure �, which outlines the events that occur when

blood clots in a glass tube. Actually, such a complete experiment has never been




1191 CHAIN REACTION OF BLOOD CLOTTING MECHANISM

done; and the diagram is a tentative, rough summary of data obtained in various

ways.

During the performance of the routine test for clotting time, and when the blood

is normal, the impression is gained that very little physical change takes place

until the end pint is imminent. Then, in a comparatively brief interval, the

viscosity rapidly increases and a solid clot forms. This gross impression has been

confirmed by finer methods; and the production of fibrin is accordingly represented

in the diagram. At the time the solid clot appears, less than half the fibrinogen has

been converted to polymerized fibrin. As this production of fibrin continues, the

clotbecomes firmer. Aftera haif-houror more, theclor retracts. Then, on standing

many hours, the amount of the fibrin diminishes somewhat, as can be determined by

weighing. This last effect is due to the action of one or more fibrinolytic enzymes,

and represents only a partial development of the fibrinolytic potential of the blood.

Normally, the enzyme is kept for the most part in an inactive state. As a result of

disease or artificial manipulation, fibrinolytic activity can be developed to a surpris-

ing degree. � � It is a remarkable fact that blood normally contains enough potential

fibrinolytic activity to liquefy its own clot. For reasons only partially understood,

the fibrinolytic potential is rarely, if ever, developed to the full.

The explanation for the shape of the fibrin curve may be complex in detail,

but the main reason for its late start is that, up until that time, there is not enough

thrombin to clot the fibrinogen. This delay was reported in 1901 by Arthus,6 who

further noted the accelerated production of thrombin just before the clot appeared.

Soon the amount of thrombin dwindles-the antithrombin effect.

Carrying the discussion one step further, the delay in thrombin formation is

due to the fact that sufficient thrombokinase activity must first be developed.

Reasoning from simple experiments on whole blood, Collingwood and MacMahon

concluded in I9I2.� that a large part of the clotting time was spent in developing

thrombokinase from an inactive precursor which they called prothrombokinase.

Recently, by separating the coagulation process into three stages, carried out in

three succesive sets of test tubes, it has been possible to show that thrombokinase

activity develops as shown in figure i.� The latent period and the period of ac-

celeration still suggested a chain reaction, an interpretation corroborated by

seeding experiments. There was further noted a prompt and rapid loss of thrombo-

kinase activity, another result foreshadowed by the work of Collingwood and

MacMahon.

These are not isolated findings. Using different technical approaches, the inter-

pretations of several recent investigators9 are virtually unanimous on the follow-

ing broad conclusions: (a) The coagulation mechanism comprises at least three

distinct reactions. (b) A chain reaction is involved in the production of a factor

that can accelerate the activation of prothrombin.

Contemporary literature, however, shows that these basic findings can be

elaborated with greater diversity than might be supposed. The diversity stems

partly from the fact that terms like ‘thrombokinase” and ‘thromboplastin”

are used in different ways and that several new terms have been introduced. More





important is the uncertainty concerning which and how many factors participate

in the direct activation of prothrombin. Beyond this is the question of which

factors, now thought to activate prothrombin, really do something else, such

as accelerate the development of thrombokinase. How many different chain

reactions occur? To date no convincing evidence has been presented either for or

against the occurrence of a simple autocatalytic reaction (e.g., x accelerates the

production of x). Scegers and his associates” have brought forth impressive,

although not quite conclusive, evidence in favor of a more complicated chain

( e.g., x accelerates the production of y, which in turn accelerates the production

of x).

It is possible to summarize the present situation so as to emphasize the similar-

ities of individual viewpoints rather than their differences:

I . Prothrombokinase Complex -s Thrombokinase Complex. An autocatalytic

or chain reaction results in the acceleration

of this conversion.

2.. Prothrombin -s Thrombin. In the presence of the active thrombokinase corn-

plex.

3. Fibrinogen -� Fibrin. In the presence of thrombin.

This is an oversimplification, attained by neglecting details, and by grouping in

the thrombokinase complex all factors, including calcium, which may be found

to participate directly in the activation of prothrombin.

For the present discussion there are important differences between this formula-

tion and the old two-stage theory. It may be emphasized that the necessary sub-

stances for these three stages of blood coagulation are present in, and obtainable

from, the blood. The prothrombokinase complex is demonstrably different from

the thrombokinase complex; and the conversion from the inactive form to the

active form has been followed experimentally.8 This conversion is of further signifi-

cance in that it consumes a large part of the time required for blood to clot in a

glass tube. Moreover, the autocatalytic effect is concerned with the development

of thrombokinase activity.* As a consequence, a small amount of coagulating blood

can, when mixed with unclotted blood, accelerate the first of the three reactions,

and get the clotting process off to a good start in the fresh portion of blood.

Thus we have rather detailed biochemical evidence of a chain reaction which

can take place entirely within the blood, and which can propagate the clotting

process.

* To avoid undue complexity, this discussion does not deal with the possibility that one or more

reactions precede the three enumerated, and that the principal locus of the autocatalytic effect might be

in one of them, and thence reflected in the development of thrombokinase activity. This would not

change the main argument.

Also left for future elucidation is the question whether the prothrombokinase of the blood is in the

platelets,7 the plasma (Lenggenhager 1936, 1940, cited in reference 8), or both. Any decision on this

question would leave intact the essential contribution of Collingwood and MacMahon, who brought

forth evidence for a prothrombokinase and a three-stage mechanism in 1912.. When plasma is obtained

with only ordinary precautions, prothrombokinase is found in the plasma euglobulins. Whether platelets

contain prothrombokinase is uncertain.’9




12.94 CHAIN REACTION OF BLOOD CLOTTING MECHANISM

THE ROLE OF THE THROMBOCYTES

This advance, although not yet consolidated, is already reaching out to include

the thrombocytes. On the experimental side it appears that the chain reaction

will proceed in the absence of whole platelets. This does not prove that material

derived from platelets is not involved, or that whole platelets are not concerned

when they are present. In his review on platelets, Tocantins’6 states the general

impression that they will cause changes in the plasma, which in turn

will lead neighboring platelets to alter, and so on.”

The platelet lysis and fusion observed in coagulating blood are brought about

not simply by contact with glass in the presence of calcium, but require particularly

the presence of a heat-labile factor found in the serum globulins. The serum factor

was demonstrated by Wright and Minot in 1917.” Brinkhous,’8 on the basis of

different considerations, has recognized what may be the same factor, and has

suggested the name � ‘ thrombocytolysin. , , How this is related to the other factors

is not known; but it gives the means whereby the serum can change the platelets.

In turn, the platelet material directly or indirectly exerts a pronounced accelera-

tion on the production of thrombin.’’

A reaction series, shuttling between platelets and plasma, could result in con-

tinuously renewed metamorphosis of platelets. Thus a white thrombus might be

formed in flowing and eddying blood, where fresh plasma and platelets would be

supplied continuously to a stationary nidus of metamorphosed platelets. Con-

ceivably this might be independent of the chain concerned in the development of

thrombokinase; but there is much to suggest that the two chain effects are inti-

mately related. Exactly how they are related, and whether the platelets help or

hinder the chain reaction is not known.

A further complexity cannot be ignored. The hemostatic plug, as well as the

white thrombus formed in flowing blood, differ appreciably from the test tube

clot, or the red thrombus formed in stagnant blood. The former have a dispropor-

tionately high content of platelets, along with some fibrin (sometimes, apparently,

very little). Although the fibrin is probably a useful constituent, it is not certain

that it is absolutely necessary. Various suggestions that transformed platelets may

be sticky, even in the absence of fibrin, need to be corroborated. In this connection,

it is curious that patients with ‘congenital absence of fibrinogen . . . . are less

incapacited by their abnormality than is usually the case with “21

Phylogenetic comparisons have suggested that alterations of the thrombocytes

represent a primitive component of the hemostatic mechanism, upon which fibrin

formation has been superimposed. Be that as it may, even if two adhesive materials

are used, it does not necessitate two entirely different mechanisms to apply them.

And as yet, the facts do not compel us to postulate a separate chain reaction for

platelet metamorphosis.

THE PROBLEM OF REGULATION

How the chain reaction starts, or whether it is always in progress and needs only

to be brought to an effective intensity or “critical concentration,” is still a mystery.

We are likewise ignorant of the precise mode of control. It is likely that the control





mechanism exerts not merely a static inhibition, but can also dampen the process

when it is already under way. Otherwise, why would not a hemostatic plug or a

fresh thrombus continue to grow until it incorporated all the blood in the cardio-

vascular system?

Several phenomena are known which result in the retardation of the clotting

reactions or disposal of their products. Mere dispersion of coagulant products

by an active circulation may be of great importance. In the test tube the rapid loss

of thrombokinase activity is striking; and here the way is open for its further

investigation.8 If, as Quick’2 and Ware, Murphy and Seegers’4 believe, thrombin

is an important link in the chain reaction, then both the antithrombokinase and

antithrombin effects offer means for breaking the chain. The fibrinolytic enzyme(s)

may accomplish more than is now appreciated. Heparin augments the anti thrombin

effect, and in some ways delays the activation of prothrombin. Although it is still

uncertain whether heparin occurs in significant quantity in normal circulating

blood, it appears to reach a highlyanticoagulant level after anaphylactic shock and

total body irradiation. There have been suggestions that other anticoagulant

secretions are discharged steadily or in response to changes in the blood.

Suggestive data on the turnover of platelets, prothrombin and fibrinogen indi-

cate that these factors are completely renewed every few days. It has been inferred

that they are consumed in the usual blood clotting reactions, slowly proceeding

in the circulation. This implies a degree of dynamic balance to maintain the

fluidity of circulating blood, for the converted factors must be removed fast enough

to prevent gross coagulation. As illustrated in figure i, the antithrombokinase and

antithrornbin effects could take part in this balance. They inactivate coagulant

products which have already been formed. Depending on quantitative relations,

this kind of action could slow up the clotting process when it was already under

way. Such action could help to limit the growth of a hemostatic plug to the

requirements of physiologic necessity. The failure, or overpowering, of such action

might contribute to the excessive propagation of a thrombus.

It is quite possible that the critical juncture of the entire system is the develop-

ment of thrombokinase activity. But detailed study of this has just begun.

RELATION TO THROMBOSIS

Undoubtedly, there are many who hope that few factors remain to be disclosed.

Nevertheless it is clearly necessary to continue a detailed analysis of coagulation

if we are to understand why a thrombus starts, how it propagates and what makes

it stop. These theoretical questions are fundamental to the prognosis, prophylaxis

and therapy of thrombosis. For the present, efforts are being made to estimate the

likelihood of thrombosis from tests which attempt to detect a hypercoagulable

state in the patient’s blood. 22. 23 Modern anticoagulant prophylaxis and therapy

are based on the prevailing view that the state of the coagulation system is a

significant factor in thrombosis, and coagulation tests are extensively used as

guides in the administration of heparin and dicumarol.

For the future this outline has noted only some of the prospects that beckon

to the investigator. Among these is the question of the extent to which the chain




12.96 CHAIN REACTION OF BLOOD CLOTTING MECHANISM

reaction furnishes the biochemical basis for the propagation of a thrombus. � In

biochemical experiments the chain reaction has long been in evidence. In pathologythe formation and propagation of a thrombus has been the subject of classic stud-

24 One might speculate what the outcome will be as these data from different

disciplines are brought together and extended. The chain reaction is so intimately

a part of the coagulation mechanism that it is likely to occur whenever a thrombus

forms, unless the individual’s blood is unusual. Consequently, it would usually

play some part in the extension of the thrombus. The relative importance of its

contribution in the genesis of various types of thrombi remains to be evaluated.

There may be some conditions where a thrombus would propagate even if there

were no chain reaction; in some circumstances propagation might be impossible

without one. Of the latter case, the extension of a mural thrombus far into the

chamber of the left ventricle might be an example.

Here the growing surface is far from the injured myocardium; and the ventricular

blood could hardly be called stagnant. Is the continued deposition of platelets and

fibrin sustained by diffusion of tissue factor through the thrombus? Or does it

depend on repeated formation of fresh coagulant substances at the free surface,

through operation of the chain reaction? Anticipation of this question is to

he found in the statement of Solandt, Nassim and Best21: “It seems reasonable to

suppose that, once agglutinating platelets have covered an injured region, the

nature or degree of the underlying tissue damage will have little or nothing to do

with subsequent growth of the thrombus.” The success of their pioneer experi-

ments in suppressing cardiac mural thrombosis by heparinization emphasized anew

that the state of the clotting system is very important. This may include the chain

reaction, but does not yet single it out as the sine qua non of cardiac mural throm-

bosis.

SUMMARY: A WORKING HYPOTHESIS

Detailed evidence has been accumulating that at least one chain reaction occurs

during the coagulation of blood. Both the metamorphosis of platelets and the

development of thrombokinase appear to be involved. The autocatalytic effect may

serve a function in making possible the growth of a hemostatic plug. It also offers

advantages in the physiologic control of the clotting mechanism. It is likely that

the chain reaction occurs in most instances where a thrombus forms, and plays

some part in its propagation.

The chain reaction is a potentially explosive phenomenon which demands an

adequate countermechanism. With materials derived from blood, reactions have

long since been demonstrated which can reduce thrombokinase activity, inactivate

thrombin and liquefy fibrin. These reactions may help to maintain the fluidity of

the circulating blood by removing the products of smoldering clotting reactions.

* This question has been raised independently in two articles which appeared since the present paper

was submitted for publication. It was briefly mentioned by B. Alexander, A. deVries and R. Goldstein:

Blood �: 739-746, 1949. The idea and a discussion of its implications were presented by J. H. Milstone:

J. Insur. Med. 4: 5-7, July 1949. For a step toward the same idea, see reference i�, page 74.




J. H. MILSTONE 1197

Such effects could help to delimit the growth of a hemostatic plug, or to end the

propagation of a thrombus.

While it is now possible to correlate in this way the data on blood coagulation

with present knowledge of hemostasis and thrombosis, critical gaps in our under-

standing still remain.

REFERENCES

I TOCANTINS, L. M. : The mechanism ofhemostatis. Ann. Surg. 125. 2.92.-3I0, 1947.

2 GRATIA, A. : Discussion on the bacteriophage (bacteriolysin) IV. Brit. M. J. 2.’ 2.96, 192.2..

3 FISCHER, A. : Blutgerinnung als unbegrenzt ubertragbare Kettenreaktion. Biochem. Ztschr. 279.’ io8-

“4, 1935.

-I MILSTONE, H. : A factor in normal human blood which participates in streptococcal fibrinolysis. J.Immunol. 42. 109i16, 1941.

I CHRISTENSEN, L. R. : The activation of plasminogen by chloroform. J. Gen. Physiol. 30� 149-157, 1946.

6 ARTHUS, M. : Etude sur la production de fibrinferment dans le sang extrait des vaisseaux. Compt. rend.

Soc. de biol. 53: 102.47, 1901.

� COLLINGWOOD, B. J., AND MACMAHON, M. T.: The anti-coagulants in blood and serum. J. Physiol.

45: 119’145, 1912..

MILSTONE, J. H.: Three-stage analysis of blood coagulation. J. Gen. Physiol. 3l� 301-32.4, 1948.

LAKI, K.: The autocatalytic formation of thrombin and the clotting defect of hemophilic blood.

Studies Instit. Med. Chem. Univ. Szeged 3: 5-15, 1943.

I� A5TRUP, T.: Biochemistry of blood coagulation. Acta Physiol. Scandinav. 7: Suppl. 2.1, 1944.

11 OWREN, P. A. : The coagulation of blood. Investigations on a new clotting factor. Acta Med. Scandinav.

Suppl: 194, 1947.

12 QUICK, A. J.: Studies on the enigma of the hemostatic dysfunction of hemophilia. Am. J. M. Sc. 214.’

2.72.-2.80, 1947.

i3 MILSTONE, J. H.: Prothrombokinase and the three stages of blood coagulation. Science io6: 546-547,

‘947.‘� WARE, A. G., MURPHY, R. C. AND SEEGERS, W. H.: The function of Ac-globulin in blood clotting.

Science io6: 6i8, 1947.

1� SEEGERS, W. H., AND WARE, A. G.: Protein equilibrium reactions in the blood clotting mechanism.

In Blood Clotting and Allied Problems, Josiah MaCy,Jr. Foundation i. 64-84, 1948.

� TOCANTINS, L. M.: The mammalian blood platelet in health and disease. Medicine I7� 155-2.60, 1938.

17 WRIGHT, J. H., AND MINOT, G. R.: The viscous metamorphosis of the blood platelets. J. Exper. Med.

26. 395409, 1917.

“ BRINKHOUS, K. M.: Clotting defect in hemophilia: Deficiency in a plasma factor required for platelet

utilization. Proc. Soc. Exper. Biol. & Med. 66: 117-12.0, 1947.

‘� MILSTONE, J. H.: Activation of prothrombin by platelets plus globulin. Proc. Soc. Exper. Biol. &

Med. 68: 2.2.5-2.2.8, 1948.

�#{176}WARE, A. G., FAHEY, J. L. AND SEEGERS, W. H.: Platelet extracts, fibrin formation and interaction of

purified prothrombin and thromboplastin. Am. J. Physiol. 15.1: 140-147, 1948.

II MACFARLANE, R. G.: Normal and abnormal blood coagulation: A review. J. Clin. Path. I: 113-143,

1948.

22 SILVERMAN, S. B.: A modification of the Waugh-Ruddick test for increased coagulability of the

blood, and its application to the study of postoperative cases. Blood �: 147-154, 1948.

23 HURN, M., BARKER, N. W., AND MANN, F. D.: Variations in prothrombin and antithrombin in

patients with thrombosing tendencies. Am.J. Clin. Path. i�: 709-711, 1947.

24 ASCHOFF, L.: Thrombosis. In Lectures on Pathology. New York, Paul B. Hoeber, Inc., 192.4. Pp. 2.53-

2.78.

25 SOLANDT, D. Y., NASSIM, R., AND BEST, C. H.: Production and prevention of cardiac mural thrombosis

in dogs. Lancet 2. 592.595, 1939.




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