CASCADA DE LA COAGULACION

1. HISTORICAL SKETCH
The waterfall-cascade and autoprothrombin hypotheses of
blood coagulation: personal reflections from an observer
C. HOUGIE
Department of Pathology, University of California, San Diego, USA
To cite this article: Hougie C. The waterfall-cascade and authoprothombin hypotheses of blood coagulation:personal reflections from an observer.
J Thromb Haemost 2004; 2: 1225–33.
The most cited and probably the two most important papers on
blood coagulation of the 20th century were the independent
reports on the Cascade or Waterfall hypothesis in 1964. The
Cascade paper by R.G. Macfarlane appeared in Nature [1] and
was shortly followed by the Waterfall report by Earl W. Davie
& Oscar Ratnoff in Science [2]. As the papers are landmarks in
the history of blood coagulation they have recently received
much attention. Davie himself has written a brief history for
the centenary issue of the Journal of Biological Chemistry [3]
while Harold Roberts, in a paper [4] on the works and
achievements of Oscar Ratnoff, has traced the evolution of the
Waterfall theory using Ratnoff’s notes. I will focus more on
aspects which were not considered or only briefly dwelt upon in
these two earlier reviews.
The Waterfall and Cascade reports were not only of interest
to workers in hemostasis, but as they appeared in the two most
prestigious journals widely read by scientists in all fields, had
important impacts on research in other related fields of biology.
The basic concept in both papers was that the formation of
fibrinogen occurs in a series of progressive stepwise reactions in
which the clotting factors act in pairs, one factor behaving as an
enzyme and the other like a substrate (Fig. 1). It was postulated
that each protein clotting factor with the exception of
fibrinogen exists in plasma in an inactive precursor form and
is sequentially converted to an active enzymatic form in the
following order: Factor (F) XII, XI, IX, VIII, X, V and
prothrombin and FXIII; the activated forms of the various
factors was denoted by a lower case a. The initial triggering
event was thought to be the activation of FXII which can occur
by several mechanisms. The activated FXII or XIIa activates
FXI to XIa and so on down the line with all the clotting factors
except fibrinogen being converted to a form possessing
enzymatic activity. In both schemes, phospholipid was an
absolute requirement for two steps. The existence of feedback
mechanisms was recognized and the great enhancement of both
FV and VIII activities by thrombin was cited in both papers.
Each of the reports recognized that inhibitors that bring the
process to a halt have an important role. The extrinsic system
was only very briefly alluded to in the text of the Waterfall but
not as all in the Cascade paper.
Macfarlane [1] observed that from one stage to the next in
the Cascade, a greater amount of proenzyme is involved
resulting in amplification. He estimated that in 100 mL of
plasma, there was 15 mg of prothrombin, 1.8 mg of FX and
1.7 mg of FVIII and the final end product of the reaction
converts as much as 250 mg of fibrinogen to fibrin in a few
seconds. Macfarlane concluded from these numbers that the
physiological need to link the initial minute physical stimulus of
surface contact with the final explosive burst of thrombin has
resulted in the evolution of a photochemical amplifier in which
proenzymes are analogous to photomultipliers or transistors
stages. If each enzyme activates 10 times its own weight of
proenzyme during its time of operation, he calculated the
overall gain in response to be a million-fold, a figure close to
modern observations [4]. Macfarlane took the analogy to a
photomultiplier even further. He suggested that the rapid
disappearance of certain coagulant enzymes, and perhaps
FVIII, almost coincidently with the appearance of fibrin were
analogous to a circuit breaker actuated by the last in the series
of operations which stops the whole process and prevents
overrunning, a safeguard of this sort being a biological
necessity if disastrous thrombosis is to be avoided. Apart from
the concept of amplification, the two reports were virtually
identical and the terms Waterfall and Cascade are used
interchangeably. However the term Cascade is usually pre-
ferred in that it connotes not only sequential events but also
amplification.
At the time of the two reports, Macfarlane and Ratnoff were
two of the most distinguished clinical investigators in blood
coagulation while Davie would soon become the foremost
biochemist in the field. Robert Gywn Macfarlane (1907–87), a
delightful, modest and an almost self-effacing figure, was the
director of the Medical Research Council Blood Coagulation
unit in Oxford from 1959 to 1967 where many major advances
were made in the management of hemophilia (Fig. 2). With
A.S. Douglas and Rosemary Biggs, he devised the thrombo-
plastin generation test (TGT test) which provided an explan-
ation for the function of FVIII and led to the discovery of FX.
Correspondence: Professor C. Hougie, Emeritus Professor of Pathology,
University of California, San Diego, CA, USA.
Tel.: +1 858 459 1479, fax: +1 858 459 1988; e-mail: ch@san.rr.com
Journal of Thrombosis and Haemostasis, 2: 1225–1233
Ó 2004 International Society on Thrombosis and Haemostasis

2. Following the first report of Paul Aggeler and his group of a
male patient with a deficiency of plasma thromboplastin
component (PTC), now designated FIX, Macfarlane and his
associates reported a series of patients with this disorder. They
showed the condition, which they named Christmas disease
after the surname of their first patient with the disease, is
inherited as a sex-linked recessive disease, and segregated it
from the more common type of hemophilia due to FVIII
deficiency. Macfarlane had also discovered the important
coagulant properties of the Russell’s viper venom or Stypven
shortly after graduating from medical school, and later with
Peter Esnouf determined its mode of action. Macfarlane was an
early pioneer in the field of fibrinolysis but his discoveries
encompassed virtually the whole field of hematology. He was
elected a Fellow of the Royal Society in 1957, one of the most
coveted honors for a medical scientist in the UK. At the time of
publication of the cascade paper, Macfarlane was at the
pinnacle of his fame and the most distinguished and senior
hematologist in the UK; he compares favorably with William
Hewson (1732–1774) who was the first to perform meaningful
scientific experiments in blood coagulation and is now consid-
ered to be the father of hematology. Hewson was also a Fellow
of the Royal Society. The life and achievements of Macfarlane
has been the subject of a recent biography [5].
Davie & Ratnoff had been colleagues at Case Western
Reserve University in Cleveland, Ohio, but at the time the
Waterfall paper was written Davie had left Cleveland for
Seattle, Washington. Following a hematology fellowship at
Johns Hopkins, Oscar Ratnoff [4] took a staff appointment at
Western Reserve in 1952 where he stayed for the rest of his
career (Fig. 3). In 1955, with J Colopy, he discovered the
Hageman factor (FXII) and had a major role in the elucidation
of the early stages of blood clotting. Endowed with a very
fertile and brilliant mind, he is a delightful companion with an
exquisite sense of humor. Even at an early stage of his career, he
was never afraid to stand up and challenge an erroneous
viewpoint regardless of the eminence of the speaker. Ratnoff
often remarks that one of his major contributions to hemostasis
was to persuade Earl Davie to take an interest in blood
coagulation, and this indeed can be listed as a major and
significant accomplishment.
Earl Davie received his PhD at the University of Washing-
ton, Seattle, in 1954 under Hans Neurath, a leading authority
in protein chemistry. His thesis involved a study of the
transformation of trypsinogen to trypsin, and he made the
important discovery that this occurred by the hitherto unrec-
ognized process of limited proteolysis [6]. He then spent two
productive postdoctoral years working in Fritz Lipmann’s
laboratory at Harvard before being recruited in 1956 into the
Department of Biochemistry at Case Western Reserve Uni-
HAGEMAN F. (XII)
P.T.A. (XI)
CHRISTMAS F. (IX)
ANTIHEMOPHILIC F. (VIII)
STUART F. (X)
PROACCELERIN (V)
PROTHROMBIN (II)
FIBRINOGEN (I) FIBRIN
THROMBIN
ACT. PROACCELERIN
ACT. STUART F.
ACT. ANTIHEMOPHILIC F.
Ca++
Ca++
Ca++
phospholipid
phospholipid
ACT. CHRISTMAS F.
P.T.A.ACT.
ACT. HAGEMAN F.
?
Fig. 1. Waterfall concept. The cascade was virtually identical.
Fig. 2. Gywn Macfarlane. Courtesy of Paul Giangrande.
Fig. 3. Oscar Ratnoff.
1226 C. Hougie
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3. versity by Harland Wood, who was a giant in the field of
intermediary metabolism. It was Wood who introduced him to
Oscar Ratnoff. In 1962, after a fruitful collaboration with
Ratnoff, Davie was persuaded by Hans Neurath to accept a
faculty position at the University of Washington but he
continued his collaboration with Oscar for a few more years.
His laboratory soon began to play a dominant role in the
isolation and characterization of most of the clotting factors
and their eventual cloning. Earl Davie always seems relaxed
and low-keyed but these appearances may be deceptive. I can
recall several fishing trips I took with him in which Earl, lying
flat on his back because of seasickness, with the fishing line
loosely attached to one of his toes somehow always contrived
to catch more and bigger fish than anyone else on the boat
(Fig. 4).
The collaboration of Ratnoff, a clinical investigator and
Davie, an established protein biochemist with a fresh outlook
on blood coagulation was a highly successful one and
culminated in the Waterfall concept. However despite their
distinction and connections, their paper ran into a major
roadblock. At the time, I was a colleague of Earl at the
University of Washington although in a different department,
and I would frequently seek his advice. I happened to be in his
laboratory several minutes after he had learnt that the
Waterfall paper had been rejected by Science. The paper had
received a severe panning from one of the reviewers. Davie
informed me that Ratnoff who knew the editor of Science
would appeal the decision. This was the first time in my career
that I became aware that one could appeal an editorial decision
which I had always thought to be final and irrevocable. I also
happened to be in Davie’s laboratory when Macfarlane’s paper
on the cascade appeared in Nature some weeks later. The
remarkable similarity of the Waterfall with the Cascade was
noted by Davie and added to his displeasure. Fortunately,
shortly thereafter the editor of Science rescinded his decision
and accepted the paper in its original form without any request
for modification, but as a result publication was delayed several
months. On the other hand, the paper of Macfarlane was
published almost immediately and almost certainly without
any peer review, such was his prestige in the UK.
One of the major reasons for the importance of the papers
was that they constituted the first major and direct challenge to
Seegers’ unique view of blood coagulation, a fact that is now
rarely alluded to. While Seegers’ supporters had become a
dwindling number, they were influential and included most of
the early pioneers with the notable exception of Armand
Quick, the developer of the one-stage prothrombin time test,
who was invariably at loggerheads with Seegers. Indeed, at the
time, almost all biochemists outside the field and many within
believed Seegers’ views were generally accepted.
A towering and formidable figure with an aura of great
authority, Seegers had been for many years the acclaimed
leader of the past establishment. There were very few individ-
uals who dared to argue or trifle with him; indeed he was rarely
challenged, at least before 1964. He was revered by his
colleagues who invariably remained loyal to him and his ideas,
even after they had moved away from his orbit; there were few
exceptions. Seegers dominated the blood clotting field in the
USA from 1944 to1956. This is attested to by the perusal of the
five conferences on blood clotting and allied problems held
between 1948 and 1952 and sponsored by the Josiah Macy
Jr. Foundation in New York [7]. All of the leading workers in
the field were invited to attend; they were encouraged to
actively participate, and their comments published with
minimal editing. It can be seen from the published proceedings
that Seegers was a prominent participant. Many of the
investigators made a point of stating that the purified
prothrombin used in their experiments had been supplied by
Seegers. If the prothrombin had not been supplied by Seegers
its purity would be invariably questioned. Seegers was the
author of well over a thousand papers and his former
colleagues, students and visitors to his laboratory numbered
in the hundreds and included many individuals who were
already distinguished in the field and occupied important
positions all over the world ranging from Tokyo, Buenos Aires
and Istanbul. His political influence was formidable since he
was a highly sought after reviewer of papers on blood
coagulation submitted to the major journals in the USA and
was also a member of review panels for many granting
agencies.
Seegers (1910–96) obtained his PhD from the University of
Iowa and in 1937 was recruited by H.P. Smith, the chairman of
the Department of Pathology at that university. The two-stage
prothrombin time test had been devised there by Warner,
Brinkhous and Smith who had performed important studies on
Vitamin K. As the result of their work, Iowa had become the
major center of research in the world on blood coagulation. It
was there that Seegers was indoctrinated into the field of blood
coagulation and developed his interest in prothrombin and
thrombin. When the Iowa group broke up at the outbreak of
the second World War, Seegers was recruited by Parke Davis
to work on thrombin and his purified bovine thrombin was
successfully marketed and is probably still manufactured. In
1946, he moved on to Wayne State University in Michigan
where he became the head and chairman of the Department of
Physiological Chemistry.Fig. 4. Earl W. Davie with trophy king salmon caught in Alaska in 2003.
Historical Sketch 1227
Ó 2004 International Society on Thrombosis and Haemostasis

4. Seegers had for many years maintained that he had purified
bovine prothrombin to homogeneity completely free of any
contaminating proteins [8]. This conclusion, which was based
initially on the sedimentation characteristics using the ultracen-
trifuge remained virtually unchallenged for many years. He
regarded the absolute purity of his bovine prothrombin
preparations an established fact and this was the basic tenet of
all his work until the seventies. By 1962, he claimed that the
additionalcriteriaofelectrophoresis,immunochemistryandend
group analysis substantiated his claim of its homogeneity [8].
According to the concept of Seegers, which he held until the
early seventies, all the activities associated with FVII, FIX and
FX only appear when pure prothrombin is activated. There-
fore, he reasoned they had to be derivatives of prothrombin, as
he believed there was nothing else in the preparation. He
considered prothrombin to be the only vitamin K-dependent
clotting factor and he devised a new and unique nomenclature
for the ÔderivativesÕ which he labeled autoprothrombins.
Although a founding member of the International Committee
for the Nomenclature of Clotting Factors, he was critical of all
their decisions. He charged them with creating hypothetical
factors and assigning them Roman numerals. Although these
numerals were in current use and had been recommended by
the committee, he refused to accept them [9,10]. His refusal was
not willful, for if he had accepted the established nomenclature,
this would have been an admission that the factors actually
existed as discrete molecular entities or proenzymes rather than
being mere derivatives of prothrombin. He was convinced that
the new factors, other than FV, were merely a passing phase
[11]: ÔWhen the peak of ‘‘attention getting’’ in my contemporary
‘‘factorphilic’’ world has been outgrown it will be doubtless
easier for me to sell the relative importance of AcGÕ (AcG being
factor V in modern terminology). Seegers [12] claimed that Ôthe
beautiful concept of prothrombin derivatives is an original and
revolutionary idea which was accurately conceived.Õ He
believed there were no experimental or clinical findings that
could not be explained by it, and placed the onus on those with
the opposite viewpoint to prove it wrong. Statements, similar to
the above that I have quoted verbatim, are replete in Seegers’
writings.
While the reviewer who wrote the adverse critique of the
Waterfall paper was anonymous, it expressed Seegers’ unique
viewpoints. In any event, the attempt to block publication of
the Waterfall paper failed and to add to the chagrin of the
reviewer, the almost identical Cascade paper was published a
few months later. Seegers’ reaction to the two reports was
predictably both swift and vitriolic, and consistent with the
negative review [10]. He wrote a blistering rebuttal of the
hypothesis for a journal of which he was on the editorial
board. This is a very unusual, although not unprecedented step
in science, even more so as the paper was more than twice the
combined length of the two papers he was attacking. Seegers
wasted no time on niceties and was never one to mince words
ÔI regard these presentations as being at the speculative levels
of science created with the wild use of the imagination.Õ He
found it against his sense of order to believe that six proteolytic
enzymes were needed to obtain thrombin. He argued Ôthat
would require six special sites in proteins where each enzyme
does its work; each enzyme would not split the other five sites
if located as single or multiple frequencies in the five other
substrates.Õ He believed this to be biologically impossible.
Seegers took exception to Macfarlane’s remarks that his
prothrombin might contain some hidden contaminants
responsible for the clotting of fibrinogen. But the main thrust
of his argument was that the theory was totally incompatible
with his own work in which he found no evidence for the
existence of the precursor factors designated by the Roman
numerals VII, IX and X. He disputed the existence of most of
the steps in the cascade and wrote Ôfor they could only be
accurate if it were possible to deny important conclusions
stated in papers which are not at all considered. Evidently
refutation of facts in these papers was not possible and a
parochial kind of authority was promoted on the basis of
‘‘consistence’’ with most of the current investigationsÕ [10]. I
interpret this as meaning that most of the steps outlined in the
cascade or waterfall theories were not consistent with the
ÔirrefutableÕ facts presented in papers, presumably those of
Seegers, which were for that reason not cited in either of the
two reports. Seegers maintained that prothrombin contained
all the material required for its own activation and that it was
in effect a molecular system. He believed: ÔWhoever reflects on
this outline of prothrombin activation and is sincere with
himself will have to recognize a conceptual formulation of
beauty. It may well be that the open secret to the riddle of
blood clotting is now there for anyone prepared to see it.Õ
There were two substantive points buried in Seegers’ paper. He
correctly pointed out that there are no enzymatic forms of FV
or FVIII that will directly convert prothrombin to thrombin or
FX to FXa, respectively.
For many years, Seegers was one of the leading proponents
of the two-stage prothrombin time test. He rarely used the one-
stage prothrombin time test which he described as Ôexplosive
chemistryÕ and deprecated all the conventional assay proce-
dures based on one-stage systems. He regarded as invalid all
assay procedures in which congenital deficient plasmas were
used as a substrate [13]. As a result it was difficult for him to
recognize a deficiency of any clotting factor other than FV, let
alone assay it, providing a possible explanation for his failure to
detect impurities in his prothrombin preparations.
Seegers’s paper [10] was far more than a mere rebuttal; it was
an uptodate compendium of his life’s work and philosophy. He
evidently still clung to the lipid inhibitor theory of hemophilia
first proposed by Tocantins, maintaining that this inhibitor had
to be neutralized before platelet cofactor I (FVIII) could act,
although the theory had long been abandoned by its originator.
In the paper, he reiterates his belief that the most important
discovery pertaining to prothrombin was Ôthe plainest, clearest,
unique, outstanding and often confirmed demonstration of
autocatalysis in prothrombin activation seen for the first time
when prothrombin activated in 25% sodium citrate solutionÕ.
SeegersÕs paper was entitled ÔBasic enzymology of blood
coagulationÕ subtly implying that his theory was consistent
1228 C. Hougie
Ó 2004 International Society on Thrombosis and Haemostasis

5. with basic enzymology while the cascade concept was not [10].
The paper was clearly hastily put together and does not
appear to have been subjected to any peer review process. For
example, in Seegers’ representation of the waterfall scheme,
phospholipid appears to be a cofactor of the FIX to FIXa
conversion instead of the subsequent stage. While this is clearly
a typographical error of which there are several, there are at
least two clear, but undoubtedly unintentional misstatements
of the work of others.
Macfarlane’s response [14] to Seegers’ paper appeared in a
later issue of the same journal as Seegers’ paper. It was
conciliatory in tone, and as one would expect coming from the
pen of a modest but great scientist, carefully crafted and written
in beautiful and lucid prose; he addressed each of Seegers’s
objections point by point in carefully measured terms without
hyperbole or dogma. He started off by pointing out that Ôby
definition a hypothesis is speculative but ‘‘wild imagination’’
implies a charge of scientific irresponsibility which when
publicly made by so influential a scientist as Dr Seegers could
hardly go unchallenged.Õ His objective in writing his rebuttal
was to indicate as briefly as possible the sources of evidence on
which the cascade hypothesis was based so that those interested
could judge the issue for themselves. He agreed that the
evidence for certain steps such as the activation of FV to an
active enzymatic form might be non-existent but he had
included it for completeness.
One of Seegers’ objections was that there was a lack of
evidence that FX (presumably autoprothrombin III in Seegers’
terminology) played any role in intrinsic plasma coagulation.
Seegers had cited some of Macfarlane’s own work as support-
ing this contention, claiming that Macfarlane had shown that
FX was not consumed or utilized during intrinsic coagulation.
Actually Macfarlane and his colleagues had found that
only a small amount of FX, rather than no FX was utilized;
adding more FVIII to the system increased the amount of FX
consumed.
Macfarlane next pointed out that if one accepts the existence
of multiple clotting factors, the next question to arise depends
upon whether one thinks as a physiologist or a biochemist: ÔThe
biochemist wants to know what the factors are; the physiologist
wants to know what they do. It is recognized that, ultimately,
physiological questions will be answered in terms of biochem-
istry or biophysics, but at the onset of the investigation it is a
matter of preference which line of approach is adopted. In
practice it is usually the study of some physiological function
which prompts the later detail investigation of its underlying
mechanism in terms of molecular structure. The physiologically
minded worker faced with seven or eight components of the
functional whole is more concerned with the gross ways
in which they interact than the details of their individual
structure.Õ Macfarlane pointed out that an investigator would
resent, and rightly, any suggestion that he should do no
experiments and produce no new theories until each factor is
purified and characterized.
Seegers defined his prothrombin as a molecular system and
relied on physical and biochemical characterization as the test
of homogeneity despite the fact that several functions appear
during its activation in addition to thrombin. Macfarlane
remarked that a sample of 7S gamma globulins may appear to
be homogeneous by physical and chemical criteria while an
immunologist may show it to be a mixture of four different
antibodies.
Macfarlane pointed out that the body of evidence for the
multiple factor theory of clotting was very large and drawn
from real observations and experiments of independent work-
ers in many countries during the preceding half century. He
recognized that Ôsuch experiments create artifacts and can be
open to more than one interpretation. However laboratory
theory is brought into direct touch with physiological reality by
the natural occurence in humans, and some animals of gross
clotting defects which can be best explained and distinguished
by postulating the separate deficiency of one or other of each of
the plasma factors which have been assigned Roman numerals.
Not only can these defects be diagnosed and quantitatively
assessed on this basis but the use of concentrated preparations
corresponding to the arbitrary laboratory definition of the
missing factor correct the defect in vitro, and above all in vivo.
No one who has seen the dramatic clinical effect of the accurate
diagnosis and specific replacement therapy in cases of these
potentially lethal conditions would doubt the existence of these
factors unless he was given equally impressive evidence to the
contrary.Õ Seegers appeared to have had no great interest in the
study of patients with congenital deficiencies of a clotting
factor, although he conceded such studies might be valuable.
Macfarlane claimed that there was a large area of agreement
between his picture of the clotting mechanisms and that of
Seegers, however bleak the prospect may appear from the other
direction. He suggested that if Seegers would be willing to refer
to his Ôpurified prothrombinÕ as the Ôprothrombin complexÕ the
gap between the two viewpoints would be narrowed. He
proposed a possible compromise between the two viewpoints,
but the effort is beyond even the ingenuity of Macfarlane; the
result was clearly contrived and improbable.
Perhaps the highlight of Macfarlane’s paper is its dramatic
conclusion in which he depicts two shadows, one having the
appearance of a wineglass and the other of a wine bottle. If only
one of the two is seen by two observers and they are each told
there is only one source of the two shadows, they might well get
into an argument as to the source of the shadow even to the
extent of accusing the other of wild imagination. In fact the two
shadows were cast from a single object illuminated by two
beams of light at right angles to each other.
The Macfarlane rebuttal was perhaps too defensive and
restrained for the evidence against the Seegers hypothesis was
becoming overwhelming. Seegers’ views defied both common
sense and the principles of basic enzymology for five compo-
nents could hardly act simultaneously, and it was hard to
conceive one proenzyme having so many different active sites
and functions. Even Seegers [15] indirectly acknowledged this
for he often referred to prothrombin as Ôa lively enzymeÕ. He
even made the claim [16] that prothrombin had Ôa versatility as
marvelous as that of any other unit in biology, such as, for
Historical Sketch 1229
Ó 2004 International Society on Thrombosis and Haemostasis

6. example, the cellÕ. There was the compelling finding that
prothrombin could be separated by Seitz filtration from FVII
and FX. Moreover the levels of prothrombin, VII, IX and X
did not decrease at the same rate when dicoumarol was given
and also returned to normal at different rates when the drug
was stopped. Surely if these factors were merely different
functions of a single prothrombin molecule, each would have
decreased and returned to normal at the same rate. Seegers was
aware of these facts but he had no plausible explanation [17].
The Seegers’ theory did not fit the one protein–one gene
hypothesis. Perhaps last but not least, both Soulier and
Duckert, two of the most illustrious names in the field had each
independently found and informed Seegers that a preparation
of prothrombin purified by his method contained demon-
strable levels of FVII, FIX and FX and also FV; one of these
preparations had actually been provided by Seegers himself [8].
His response was that that the prothrombin preparations tested
had not been sufficiently purified.
Rapid improvements of analytical methods in biochemistry
were being made at a rapid rate in the 1960s. Disk gel
electrophoresis [18] was in general use in 1965; SDS polyacryl-
amide gel electrophoresis was introduced shortly thereafter and
by late 1969 was established as a reliable method of estimating
the molecular weight of proteins. Immunochemical techniques
were also well advanced in the early 1960s [19]. Any one of
these procedures would have detected the presence of gross
impurities in Seegers’ prothrombin and led him to abandon his
theory. However it was not until the beginning of the 1970s
when he could hardly have had a single supporter that he
belatedly joined the mainstream. A contrite Seegers [20]
proclaims ÔI started with a beautiful and time honored
materialistic perspective and sincerely did my best to be
objective just as if this could be possible. I believe my original
orientation continues to retard scientific progress, accounts for
heartaches and for a scientist being regarded circumspectly. It
was eventually my good fortune to notice that idealism is
compatible with progress and health and that subjectivity is the
basis of all that is real.Õ It is only too easy in hindsight to be
overly critical of someone who has been shown to be wrong
and especially if he has conceded his mistakes. As Seegers’
views came under increasing fire from all quarters, the more
important it must have seemed to him to vehemently defend
them. He was still on the editorial boards of many of the
journals devoted to blood coagulation and his papers, liberally
sprinkled with philosophical comments, were being accepted
and published, apparently without critical peer review. He
undoubtedly felt it his duty to take every opportunity to present
what he was sure was the correct viewpoint and prevent others
from marching blindly down a blind alley. Accordingly he
availed himself of every opportunity and venue that came his
way to propagate what he believed to be the truth. Most
scientists in his position and feeling as strongly as he did might
well have done the same, though hardly with the same
compulsive zeal.
Seegers was a philosopher as well as a scientist and his papers
are replete with obtuse philosophical quotations and state-
ments. His scientific philosophy can be found in the book
Living Consciously: The Science of Self which he coauthored
with Dr John M. Dorsey, a psychiatrist [21]. When challenged
in 1963 as to the purity of his prothrombin, Seegers [22]
responded that he had written much on this, but in addition he
would make it very plain that in his book with Dorsey they had
stated: ÔI have never proved anything in all my life, and I am
sure no one ever has. This is the way I look at it. It is an illusion
that you can prove anything.Õ It is difficult to have a meaningful
scientific discourse with anyone holding such a viewpoint and
the response quoted above which he gave to a very fair and
reasonable question may explain why he was so rarely
challenged at meetings. A repeated theme seen in his writings
is that Ôall my science is strictly private and my own subjective
creation.Õ Indeed this is a fairly accurate description of his
attitude to science. But in the modern age, dilettantism is
seldom compatible with the objectivity so necessary in hard
science.
Seegers probably knew more of the history of blood
coagulation than any of his contemporaries with the possible
exception of C.A. Owen, a former colleague of his. But this did
not prevent him from repeating some of the common errors of
the past; his failure to distinguish a conclusion was one of these.
Although outspoken in his views, Seegers was honest in his
belief that the prothrombin he had prepared was truly
homogeneous and contained no impurities. However he was
stubborn in his refusal to accept any new evidence that became
available and called for the abandonment of this belief, so in
this sense the homogeneity of his prothrombin had become an
obsession.
It is not at all unusual in modern science for two or more
groups working independently on the same problem to arrive
at more or less identical conclusions at about the same time. So
it was with the Cascade and Waterfall reports. The papers were
actually submitted for publication within a few weeks of each
other in 1964 but as the Waterfall paper was initially rejected its
publication date was delayed and it appeared later than the
Cascade paper. The concept was not entirely new and was
known to both groups at least two years prior to the Science
and Nature reports.
Both Ratnoff and Macfarlane were participants at a meeting
held in Stockholm in 1962 under the auspices of the Interna-
tional Committee on Blood Clotting Factors. Macfarlane [23]
presented some data on the activation of FX. The paper was
very briefly commented on by Ratnoff [24] who spoke of
Macfarlane’s presentation in glowing terms and then asked the
rhetorical question: ÔWhy cannot a substrate be an enzyme?Õ He
concluded by stating that he understood Ôclotting as a process
in which one enzyme acts on a substrate to change it to an
active enzyme which acts on the next substrate and so on down
the line ad nauseam like the fleas.Õ I believe this to be the first
published reference to the Waterfall hypothesis. Macfarlane
agreed entirely with Ratnoff’s comments and stated that the
sequence of substrate to enzyme was exactly what he visualized
in the activation of FX by Russell’s viper venom. A figure of an
almost identical version of the Waterfall reaction sequence
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7. actually appeared in a paper by Ratnoff and Davie [25] in 1962.
It was included in the discussion section of the paper and was
based on previous work performed in the authors’ laboratories
as well as new data presented in the paper. It is clear from this
paper, and the Stockholm proceedings that the concept had by
early 1962 fully crystallized in the mind of Ratnoff. This was
confirmed by Roberts [4] from his examination of Ratnoff’s
laboratory notes. I also have in my possession copies of two
letters by Davie in which the Waterfall scheme is depicted. The
first, to Fritz Lipmann, was written in March of 1962, while
the second, written in June of the same year, was to Roger
Lundblad. The Waterfall scheme was also shown on an
application by Davie in 1962 for a clinical research project; I
was listed as one of the investigators and received a copy. It was
also known by Macfarlane as it appeared in 1962 in a textbook
by Biggs and Macfarlane [25], although the substrate to enzyme
chain reaction was not specifically outlined.
In deriving the hypothesis, each of the two groups took
advantage of the published data of the other and made the
appropriate acknowledgments. Thus the first three papers cited
by Macfarlane referred to work performed in Davie and
Ratnoff’s laboratory while the first citation of Davie and
Ratnoff was to the page in the book by Biggs and Macfarlane
which contained the early diagram of the scheme. Both
Macfarlane and Davie & Ratnoff are always assigned equal
priority, and rightly so. However in a book published in 1993
on the life of Macfarlane, the author, Dr Alistair Robb-Smith
[5], hints that there may have been plagiarism, although he was
careful to point out that this was not Macfarlane’s opinion. He
made this assertion on the sole grounds of the similarity of the
two papers and the fact that the Waterfall report appeared in
print later than the cascade; he believed that this was too much
of a coincidence. This innuendo, as the facts above show, is of
course totally untrue. In fact, Macfarlane and Davie & Ratnoff
in their 1964 reports each cited the earlier 1962 contributions of
the other as if to disclaim any priority; it is therefore clear that
this question had not occurred to either group, just as one
would expect from outstanding and established scientists.
Needless to say, both Drs Ratnoff & Davie, who have always
been models of fairness and propriety, were extremely and
understandably distressed.
The blood coagulation laboratories at Oxford and Cleveland
were amongst the very few major centers investigating the
various sequential reactions along conventional lines, and
there was really only one logical explanation of the available
experimental data. It was therefore almost inevitable that both
laboratories would arrive at the same conclusions. Unlike
Davie and Ratnoff who started at the top of the sequence
and worked their way downwards, Macfarlane started at the
bottom rungs and extrapolated upwards. Macfarlane is quoted
[5] as stating that Ôas people tunneling from opposite sides of a
mountain are supposed to do, we joined up with the work
being carried out on the other side of the problem, mostly on
the other side of the AtlanticÕ (referring to Ratnoff & Davie’s
work). Macfarlane’s perspective also differs from that of Davie
& Ratnoff in that he is more interested in the concept of
amplification and less on the biochemistry of the interactions.
He had long considered the sequential triggering of the various
factors to be like an amplifier but it suddenly occured to him
just prior to writing the paper that it was in fact an amplifier [5].
The idea of amplification in the electrical sense greatly appealed
to Davie & Ratnoff, who considered the term Cascade more
appropriate than Waterfall.
There are several reasons why the impact of the hypothesis
was so immediate and important. Prior to the publication of
the Cascade and Waterfall papers in 1964, Seegers’ views were
generally accepted by most biochemists, in and outside the
field. But following publication of the two reports, Seegers’
views were supplanted by the new theory. The hypothesis acted
as a unifying banner for workers in the field who now spoke in
the same language and used well defined terms that could be
understood by all. For the first time in years scientists in other
fields who formerly shunned blood coagulation were attracted
into the field. The theory became the standard and orthodox
one and appeared in textbooks and reviews. Many theories of
blood coagulation had been propounded in the 60 years
following the Morawitz theory proposal at the turn of the 20th
century but none had succeeded in replacing it as a practical
working model. The Cascade hypothesis presented a composite
of all the bare facts in a nutshell, providing an easy to
understand and general concept more or less unique in biology.
It filled in the vacuum that had been created when the
Morawitz hypothesis became obsolescent in the early forties.
Moreover the Cascade principle could be applied to other
systems including that of complement, and Cascade became an
accepted biological term. It is doubtful whether the theory
would have met with the same general acclaim and immediate
acceptance had it not been for the authority and distinction of
all three authors and the fact that one was an established
protein chemist. That the papers appeared in the two journals
widely read by a broad spectrum of scientists also played an
important role in its wide acceptance.
Davie & Ratnoff in their paper pointed out that the sequence
would require modification when new clotting factors were
discovered and as further studied revealed the complexity of
substances assumed to be pure. Macfarlane, in his rebuttal of
Seegers, also conceded that there were obvious gaps and
difficulties and that the hypothesis might be disproved in
principle or in detail at any time, but he stressed that it provided
opportunities for experimental testing which whatever the
outcome was likely to provide new knowledge These guarded
predictions proved to be correct and shortly after the reports
appeared in print there was new evidence indicating modifica-
tions were needed. Thus complex formation was demonstrated
between FV and FXa and phospholipid in the presence of
calcium [27] while a similar complex was found to occur at an
earlier step involving FVIII and FIXa, phospholipid and
calcium [28]. It is noteworthy that the former work was
performed in the laboratory of Hanahan, a departmental
colleague and former teacher of Davie and the latter work in
Macfarlane’s laboratory. As the Cascade was a hypothesis, new
findings such as these were merely incorporated into a modified
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8. and improved model. This process is still continuing to this day,
40 years later, attesting to the great utility of the concept.
The Waterfall-Cascade concept was based on in vitro studies
of blood from which the formed elements, often including
platelets, had been removed. It remains a good model for the
understanding of most tests used in clinical diagnostic work in
which platelet poor plasma is usually used. But it had severe
shortcomingswhenitwasappliedtoinvivoevents.Theseinclude
the complete absence of a bleeding tendency in severe FXII
deficiency and only mild bleeding in severe FXI deficiency; there
was also the fact that the extrinsic or tissue pathway could not
by-pass the intrinsic pathway. These facts were so obvious and
well-known at the time that there was hardly any need to point
them out. That the two processes of in vitro and in vivo clotting
were different was known more than 200 years ago to William
Hewson [29] who deduced that the endothelial lining did not
merelyplayapassiveroleinphysiologicclotting.Butsuchmajor
flaws in the theory were in a sense a strength for it stimulated
effortstodiscovertiesbetweenthetwopathwaysandexplainthe
clinical anomalies. This was to be an irresistible challenge to
some investigators. My former colleagues in San Diego, Bjarne
O¨ sterud and Sam Rapaport were amongst those who accepted
the challenge. In 1976, they found that the tissue factor-factor
VIIa- calcium-complex not only activated FX but also activated
FIX thereby bypassing both FXII and FXI [30]. On their
hypothesis, tissue factor initiates blood coagulation in vivo. The
explanation why the activated FX did not completely by-pass
the intrinsic pathway was later explained by the presence of a
tissue factor pathway inhibitor. This was later isolated and
characterized independently by both Rapaport and his group
[31] and Broze [32].
In 1975 the discovery [33] of protein C which could not be
detected by laboratory tests in use at the time and the finding
that its activation required thrombin bound to thrombomod-
ulin [34] on an endothelial cell was further evidence of a cellular
component other than platelets and tissue factor-bearing cells
being involved in in vivo coagulation. Harold Roberts and his
group at Chapel Hill, NC [35] have recently devised an
ingenuous system that appears to closely mimic the events that
occur in vivo. Their experimental results establish that cellular
elements play a key and pivotal role in vivo clotting and the
mechanism involved is quite different from that seen in vitro
clotting and depicted by the cascade hypothesis; their findings
correlate remarkably closely with clinical observations. While
the study of in vivo clotting is difficult, this is clearly the wave of
the future but the days of the Waterfall or Cascade are far from
over.
Acknowledgments
I wish to thank Earl Davie for sending me an advanced copy of
his paper on the same topic which appeared in J Biol Chem,
and also for sending me copies of his papers and letters on the
topic. I am also indebted to my former colleague and good
friend Ms. Kathy Donnelly who kindly read the manuscript
and made many helpful suggestions.
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