ANTI PLASMIN
PLASMIN
(in) — PLASMIN

OD

PLASMIN +

PLASMIN

FIGURE 5. Two concepis (A and B) regarding the mechanism of action of plasmin inhibitors or antiplasmin.

special facilities for monitoring coagulation
changes are available. Studies to define the upper
ranges of safe dosage are currently underway in
our laboratory and others.

What are the best signals that the coagulation
mechanism has become unduly disturbed? Recent
observations indicate that the risk of hemorrhage
correlates best with the appearance of so-called
antithrombin activity in the blood. This anti-
thrombin activity, apparently due to digestion
products of fibrinogen and fibrin, may appear
without marked depression of the fibrinogen level

or other clotting factors although it does prolong
the one-stage prothrombin time. At the present
time, serial measurements of the prothrombin
time and, where available, the antithrombin ac-
tivity and the fibrinogen level will permit satis-
factory detection of hemorrhagicrisk. Inthedosage
ranges mentioned previously, such risk is mini-
mal. Furthermore, we now have an effective in-
hibitor of fibrinolytic activity in the compound
epsilon amino caproic acid (EACA). EACA
promptly inhibits both activator and plasmin
activity in the blood. (It is the vitamin K, or pro-

TABLE 3.
Period February 1957- January 1958- August 1960— April 1961-
December 1957 December 1960 February 1961 February 1962
Dose range* 30-90 5-100 100-300 500-3,000
Patients 12 42 a 44
Temperature
elevation
O°-1° F. 56% 52% 40.0% 68.8%
2° F 15% 19% 28.6% 29.5%
2°-3° F. 13% 22% 14.3% 2.2%
3°-4° F, 18% 2% 10.0% 4.5%
> ar 4% 5% T1% 0.0%
Chills, nausea 25% 33% 8% 2.2%

* In thousands DCGH units

Experience with pyrogenicity of one SK-plasminogen preparation (Actase) in successive trials since
1957. All doses were given intravenously over a two-hour period. Note diminution in pyrogenicity in most

recent material despite increased dosage employed.

GP November 1962

99



Current Status
of Clot Dissolution Therapy

safety check. If this exceeds twice the baseline
value, infusion is slowed; if three times, it is dis-
continued. After infusion, a fibrinogen level is
obtained. If the initial course of therapy is not
successful, the same regimen is repeated the next
day.

In peripheral arterial thrombosis, we prefer
direct installation into the affected vessel. Our
experience indicates that direct injection permits
thrombolysis at dosage levels below those neces-
sary with intravenous therapy. One-third of the
total dose is rapidly injected into the affected
vessel. If there is no response within 30 to 60
minutes, the remainder is given via a polyethy-
lene catheter during the next three hours. Fur-
ther dosage depends on the clinical response and
the coagulation changes induced. If direct injec-
tion cannot be achieved, the same intravenous
regimen as in venous thrombosis is used. Where
prompt surgical intervention is feasible, throm-
bolytic therapy is often used as preparation is
made for surgery. If complete response is not
achieved, surgery is then carried out, followed by
further thrombolytic treatment. Such medical-
surgical cooperation appears to offer advantages
by reducing both pre- and postoperative throm-
botie extension.

Comment

There is no longer any question that various
fibrinolytic agents—activators, activator-plas-
min compounds and plasmin—can dissolve sus-
ceptible intravascular thrombi. However, this
review has indicated that many problems must
be resolved before these agents can be used with
optimum expectation of therapeutic success and
minimum risk. Furthermore, the fibrinolytic
agents now available for clinical use cannot be re-
garded as ideal. Viewed in proper perspective, the
current status of the fibrinolytic agents compares
with that of the anticoagulant drugs in the early
stages of their development. The controversy
which still surrounds the efficacy and proper
dosage of anticoagulant drugs after two decades
of use gives us a glimpse of what lies ahead for
the fibrinolytic agents. Those of us interested in
these agents hope that our efforts may spare
thrombolytic therapy such a prolonged period of
uncertainty and debate. At this point, we feel
such agents do have a place in the treatment of
peripheral venous and arterial occlusion. We
recommend against their general use in other
thrombotie disorders until further experience is
accumulated.

A coupon for ordering a bibliography accompanying this article may be found adjacent to the Index to Advertisers.

102

KENNETH M. MOSER, m0. is assistant professor of medicine at George-
town University School of Medicine, and is chief of the pulmonary section,
Georgetown Clinical Research Institute, Aviation Medical Service, Federal
Aviation Agency. A graduate of Johns Hopkins Medical School, he also is
director of the enzyme research laboratory, D.C. General Hospital. Dr.
Moser is vice president and director of the National Foundation for Research
in Medicine, an associate of the American. College of Chest Physicians and a
member of the New York Academy of Sciences, the AMA Council on Drugs
and the American Thoracic Society.

Volume XXVI, Number 5 GP

GPO 982-97


Current Status
of Clot Dissolution Therapy

Caseinolytic assays (Figure 4) measure potency
in terms of casein digested per unit time. While
they are cumbersome and subject to theoretical
objections, their ability to distinguish activator
from plasmin activity has been helpful. The label-
ing of available agents in “casein activator” and
“casein plasmin” units, as well as in arbitrary
fibrinolytic units, has been a step in the right
direction. The problem of standardization, how-
ever, is still not resolved. When evaluating
thrombolytic agents, physicians should be aware
of these prohlems and be informed as to the true
significance of the words and numbers appearing
on the labels of any such agents.

PLASMIN
Pure plasmin, activated spontaneously or with
small amounts of urokinase, is now available for
study. Current thinking about such compounds
is divided into two camps because of a con-
troversy over the nature and function of its in-
hibitors. All workers agree that normal plasma
contains a considerable amount of “antiplasmin”
activity. As shown in Figure 5, those who sub-
scribe to concept A contend that the combina-
tion of plasmin and antiplasmin in vivo is a firm
one. Therefore, enough plasmin must be given to
bind all circulating antiplasmin before any is free
to attack the blood clot. Accordingly, a detectable
blood level of plasmin must be reached and, in

CASEIN ASSAY

PLASMIN ACTIVATOR
AGENT + AGENT +
CASEIN CASEIN +
PLASMINOGEN
Incubate. Incubate

CASEIN DIGESTION/UNIT TIME

FIGURE 4. The scheme of the caseinolytic assay for measuring
the potency of a drug in terms of casein digested per unit time.

98

doing so, there is risk of destroying other clotting
factors.

Others in this field support concept B (Figure
5); namely, that the plasmin-antiplasmin com-
bination is largely dissociable in the presence of
fibrin. They hold that fibrin, because of its high
affinity for plasmin, competes very successfully
with antiplasmin for plasmin. This means that
antiplasmin serves merely as a carrier of plasmin
in the blood, so that plasmin cannot attack other
clotting factors but retains the ability to attach
to and dissolve fibrin. Furthermore, detectable
“free” plasmin need not be present in the blood.
Investigations are under way in several labora-
tories to discover which of these two concepts is
more correct.

Clinical Considerations

Having reviewed basic theories, concepts and
laboratory observations, it is time to discuss the
problems surrounding clinical application of the
two fibrinolytic agents which are now available.

TOXICITY

Pyrogenicity has been the outstanding toxic
feature of these SK-plasminogen compounds in
the past. However, constant improvement in
the components used in these agents has signifi-
cantly reduced the incidence and severity of febrile
reactions. In our view, pyrogenicity no longer
limits the clinical use of these compounds (Table
3).

Hemorrhage is the second major toxic risk.
This risk, unlike pyrogenicity, depends upon the
dosage. With the dosage schedules we have used
to date, our group has not encountered hemor-
rhage attributable to these agents in more than
500 patients. This experience includes patients
who have received doses of 1,000,000 “fibrino-
lytic units” per hour (“fibrinolysin” A units in
Table 2) for four hours. At the present time, we
cannot recommend doses above this level unless

Volume XXVI, Number 5 GP


Current Status
of Clot Dissolution Therapy

tamine, of fibrinolytic therapy.) Lysine serves a
similar purpose.

Other toxic responses to these compounds have
been infrequent. Perhaps the most striking of
these has been the appearance of severe epigastric
pain several hours after infusion. The etiology of
this rare but dramatic response is unclear. It is
not associated with fever and usually responds
promptly to analgesics. Close clinical and labor-
atory observation has disclosed no residual ab-
normality.

Other toxic responses have included occasional
urticarial rashes and one instance of bona fide
serum sickness. To our knowledge, true anaphy-
laxis has not yet been recorded, although it is
likely that such a response will eventually occur.

FACTORS INFLUENCING DOSAGE

THE AGENT USED
3 METHOD OF ADMINISTRATION
ANTICOAGULANT THERAPY
—THE THROMBUS

THE TISSUE THREATENED

THE PATIENT

FIGURE 6. The criteria for dosage in clot dis:
involve many factors, some of which are unk

100

DOSAGE:

Obviously, proper dosage is a requisite for
achieving optimum therapeutic effect. Unfor-
tunately, no practical and reliable laboratory
methods are yet available for predicting the dos-
age regimen necessary to achieve thrombolysis
in a given patient. The difficulty in defining ideal
dosage criteria reflects the fact that this dosage is
the resultant of a complex equation involving
many unknowns (Figure 6). The composition and
structure of the thrombus is one such unknown.
It has been demonstrated that susceptibility of a
clot to lysis is inversely related to the fibrinogen,
lipid and platelet content of the clot. Therefore,
with identical dosage schedules, thrombolysis
may succeed in lysing a clot with low fibrinogen,
lipid or platelet content and fail to dissolve one
rich in any of these components.

The age of the thrombus is a very critical factor,
and one which is difficult to assess clinically.
Susceptibility to dissolution begins to decrease
shortly after clot formation occurs. It progres-
sively declines until, after three to seven days,
endothelialization or organization renders the
clot totally resistant to fibrinolytic attack.

The dosage requirement is also affected by the
method of administration chosen. Should the agent
be given as close to the thrombus as possible, or
systemically by any peripheral vein? Should it be
given as a single, large injection, or as multiple
small injections or as one prolonged infusion? If
given as one infusion, at what rate and for how
long a period? Our own views on these questions
are discussed at the end of this section, but the
fact is that definitive answers to them do not yet
exist.

Whether or not concomitant anticoagulant
therapy is given also influences the selection of
fibrinolytic dosage. Anticoagulant therapy here
refers only to the use of heparin. We have ad-
vocated concomitant heparin therapy for several
reasons: to prevent rethrombosis after clot disso-

Volume XXVI,Number5 GP


KENNETH M. MOSER, TM.D.

2. ‘rom <(o,
eemtrg — Amutos

November 1962

Current Status
of Clot Dissolution Therapy

Department of Medicine, Georgetown University School of Medicine

and Coagulation Branch, Georgetown Clinical Research Institute

Aviation Medical Service, Federal Aviation Agency
Washington, D.C.

Clot dissolution using fibrinolytic agents

is a promising approach to the treatment

of thromboembolic disease, particularly
peripheral venous and arterial occlusion.

In their present state of development,

these agents are not recommended for general
use in thrombosis or embolism in the cerebral
or coronary circulations. Dosage units

are still not standardized. The commercially
available “fibrinolysins” are, in reality,
combinations of plasmin (or fibrinolysin)
and an activator of plasminogen

(or profibrinolysin). Physicians must

know the limitations of these preparations :
and the factors that alter dosage requirements.

THE THREAT of venous and arterial thromboem-
bolism is a constant companion of virtually every
practicing physician. The sudden formation of
an intravascular clot can injure or destroy any
tissue, any organ. It is not surprising, therefore,
that thromboembolism has been the object of
intensive clinical and laboratory study during
this century. Nor is it surprising that much of this

GP November 1962

study has centered upon efforts to devise effective
means to treat thrombotic disorders. Recently, a
new therapeutic approach to thrombotic disease
—clot dissolution using fibrinolytic agents—has
come upon the scene. During the last several
years, a number of investigators have demon-
strated conclusively that fibrinolytic agents can
dissolve susceptible intravascular thrombi.

The Concept

The concept which underlies fibrinolytic
therapy is disarmingly simple. It is based on the
fact that mammalian blood contains an enzyme
system capable of dissolving fibrin, the protein
which forms the matrix of blood clots. This en-
zyme system is outlined in Figure 1. Plasminogen
(or profibrinolysin) is an inactive precursor sub-

INHIBITORS
ia a
/ ‘
/ ‘
x 1
ACTIVATORS 1
H
H
+
PLASMINOGEN—-—-—-—*—-—-—- —) PLASMIN
(Profibrinolysin) (Fibrinolysin)

FIGURE 1. The intrinsic fibrinolytic system present in the
circulating blood.

95



lution is achieved; to protect patients with non-
thrombotic ocelusive disease, and to prevent in-
terval extension of the thrombus if initial throm-
bolytic dosage is inadequate. Clinical and labor-
atory evidence also indicates that true heparin-
fibrinolytic synergism exists, that is, clot dissolu-
tion can be achieved with smaller dosages of
fibrinolytic agent in the presence of heparin.
Studies are currently under way in our laboratory
to identify the reasons for this synergism. While
we advocate concomitant heparin therapy, a
word of caution is in order. Obviously, heparin
can increase hemorrhagic risk if used with high
fibrinolytic dosage regimens. When doubt exists,
it is wise to sacrifice synergism for safety by
starting heparin after fibrinolytic therapy.

The tissue threatened by the thrombus also in-
fluences dosage decisions. In peripheral venous
thrombosis, thrombolysis within 24 hours will be
attended by little permanent injury. In peripheral
arterial thrombosis, the limb may survive with
minimal defect if the vascular supply is restored
within a few hours. The same cannot be said of
arterial occlusion in the heart, the brain or the eye
where rapid dissolution of critically located
thrombi is essential if structure and function are
to be preserved. Unfortunately, we cannot ex-
pect the relationship between dosage and speed
of thrombus dissolution to be linear. Our ex-
perience suggests that, beyond a certain point,
increase in dose per unit time will not speed dis-
solution but will merely provide additional toxic
risk.

The last variable in the equation for proper
dosage is the patient. Among the many character-
isties of the patient which may influence dosage
are the presence of any disorders which limit or
contraindicate the use of fibrinolytic drugs, the
level of naturally occurring or induced inhibitors
and the status of the patient’s coagulation ap-
paratus.

The apparent complexity of the equation for
dosage selection does not make such selection an

GP November 1962

impossible task. The situation is no less complex
with many widely used therapeutic agents. We
recommend dosage regimens which we know are
safe, can be simply monitored and offer a reason-
able prospect of therapeutic response. Rather
than suggesting general use of the maximum-
tolerated dose to achieve maximum response at
this time, we suggest a practical middle-ground
of well-tolerated dosage with recognition that
often this is not an optimum therapeutic dose.
As larger doses are proved safe and more effec-
tive, they will be suggested. But at each step, we
follow the dictum “do no harm” while attempting
to help.

What, then, is a good current procedure for the
determination of a dosage regimen? These re-
marks apply only to treatment of peripheral
venous and arterial thrombosis, since, in our view,
these are the only disorders in which thrombolytic
therapy should be generally used at the present
time.

We carry out a “predicted dose test,” using the
patient’s plasma and serial dilutions of the agent
to be used. The amount of the agent necessary to
achieve complete lysis of 1 ce. of the patient's
clotted plasma, in 20 minutes, times the caleu-
lated plasma volume is the “predicted dose.”
Just what this test—or any variant—predicts, is
subject to question. We use it as arough guide to
the total composite inhibitory activity in plasma.
If this predicted dose is less than 3,000,000
fibrinolytic units, (units of “fibrinolysin” A in
Table 2) we give the entire amount. If it is above
3,000,000 units, total daily dosage is limited to 50
to 75 per cent of the predicted dose. Heparin
(1 mg. per 1kg.) is given just before fibrinolytic
infusion.

In venous thromboembolism, the fibrinolytic
drug is given via any convenient peripheral vein.
Twenty per cent of the dose is given as a rapid,
priming injection, followed by 20 per cent per
hour for four hours. We follow, as a minimum,
the one-stage prothrombin time hourly as a

101



Current Status
of Clot Dissolution Therapy

stance or proenzyme which can be converted to
an active proteolytic enzyme plasmin (or fibrinol-
ysin) by a number of agents which serve as ac-
tivators. While plasmin can attack many proteins,
it exhibits an adsorptive affinity for fibrin. As in
any system associated with coagulation, the
plasmin system is subject to inhibition.

Available Agents

Inspection of this intrinsic fibrinolytic mecha-
nism indicates several methods which might be
used to promote dissolution of thrombi in vivo.
First, activators of endogenous plasminogen
might be administered. Second, plasminogen it-
self might be isolated from human blood, activat-
ed in vitro and readministered as plasmin. Third,
one might attempt to reduce plasma inhibitor
levels. Finally, several of these approaches might
be combined. Agents of each of these types are
currently under investigation (Table 1). This dis-
cussion will be concerned only with the “leading
contenders.”

TABLE 1,

TABLE 2.

ACTIVATORS,

Urokinase (UK) is obtained from human urine.
It is a direct activator which does not require in-
teraction with any other substance to convert
plasminogen to plasmin. While animal studies
have been promising, only recently. has this ma-
terial been purified enough to permit trials in
man.

Streptokinase (SK), the most widely known ac-
tivator of plasminogen, is isolated from filtrates
of streptococcal cultures. To activate plasmino-
gen, SK must first combine with some component
of human plasma. There is evidence that this
component (proactivator) may be plasminogen
itself. Thus, SK action involves two steps (Fig-
ure 2). First, SK combines with proactivator
(?plasminogen) to form activator complex; then,
this activator converts plasminogen to plasmin.

SK itself has several disadvantages in terms of
general clinical application. Despite extensive ef-
fort, even the most highly purified SK is not
consistently free of pyrogenicity. Furthermore,

Available Thrombolytic Agents

“Fibrinolysin” “Fibrinolysin” — Ratio
A

BA/B
ACTIVATORS
Streptokinase LABELED Porency:
Urokinase “Fibrinolytic units” 1,000,000.0 50,000.0  20.0/1
ACTIVATOR-PLASMIN COMPOUNDS CASEIN —
SK-Plasminogen mixtures . on
UK-Plasminogen mix Fibrinolysin units 81.4 23.8 = 1.8/1
Activator units 7,621.0 6,120.0 1.2/1
PLASMIN
Spontaneously activated plasminogen .
Chloroform-activated plasminogen * Modified from Remmert-Cohen
UK-aetivated plasminogen
OTHER Comparison of labeled “fibrinolytic units” per vial of the two commercially
Nicotinic acid available SK-plasminogen compounds with resulis of casein assays in our
‘Thrombin E laboratory. Note the predominance of activator activity. Also note the lack of

Bacterial pyrogens

96

correspondence between arbitrary “fibrinolytic units” and caseinolylic units.

Volume XXVI,Number5 GP


ACTION OF STREPTOKINASE (SK)
1

[ster 1]

SK+PROACTIVATOR —+ "ACTIVATOR COMPLEX"

(Plasminogen?)

“ACTIVATOR COMPLEX" + PLASMINOGEN—) PLASMIN

SK alone has no fibrinolytic activity

FIGURE 2. The steps by which streptokinase (SK) activates
plasminogen into plasmin.

streptococcal infection is so ubiquitous that in-
hibitors to SK exist in most people. The level of
inhibition varies so widely that the dose of SK
necessary to achieve a given pharmacologic ef-
fect must be determined for each patient indi-
vidually. The unquestioned antigenicity of free
SK also raises questions regarding its effectivity
and safety. For example, administration of SK
results in a rise in plasma inhibitory capacity
sufficient to render retreatment impractical for
six months or more. Finally, the long-term toxic
consequences of infusing and reinfusing an anti-
genic material into man provide concern.

ACTIVATOR-PLASMIN COMPOUNDS

Since urokinase-plasminogen mixtures have so
recently become available, only mixtures of strep-
tokinase with human plasminogen will be dis-
cussed here. These compounds are of the greatest
contemporary interest since both fibrinolytic
materials released for clinical use (Actase® and
Thrombolysin®) fall into this category. As
mentioned previously, the interaction between
human plasminogen and SK produces an activa-
tor complex which can convert profibrinolysin to
fibrinolysin. By manipulating the ratio of SK to
plasminogen, and by changing other features of
the reaction, an end-product which ranges from
virtually pure activator complex to virtually
pure plasmin can be obtained.

Plasmin and activator complex are different
biochemical, physiologic and immunologic en-
tities. Therefore, the potency of any SK-plas-
minogen compound should be defined in terms of
both its true fibrinolysin content and its plas-
minogen-activator activity. Since this has not
been the case in the past, some words of caution
are in order:

Beware the term “fibrinolytic wnit.” Each

GP _ November 1962

pharmaceutical house and many investigative
groups have defined separate fibrinolytic units.
None are equivalent. One unit may represent five,
10 or 20 times more activity than another, as
indicated in Table 2.

Beware the term “fibrinolysin.” The two “fibrin-
olysin’” preparations now available commercially
are, as we have indicated, combinations of plas-
minogen-activator activity and true plasmin.
Their activator potency is many times their fibrin-
olysin content (Table 2). There are preparations
of virtually pure fibrinolysin which may become
available for general use. It is essential that true
fibrinolysin, activator-fibrinolysin compounds
and pure activator compounds be differentiated,
for each material has its own particular toxic and
pharmacologic characteristics.

The diversity of preparations with thrombo-
lytic capabilities has made selection of standard
assays difficult and various assays have been
used. Currently, two major types of assay are in
general use: fibrinolytic and caseinolytic. Fibrin-
olytie assays (Figure 3) measure the potency of
an agent in terms of the speed of clot lysis. Such
assays have suffered from their lack of ability to
distinguish between activator and true plasmin
activity.

FIBRINOLYTIC ASSAY

PLASMIN ACTIVATOR
AGENT + AGENT+
THROMBIN® + THROMBIN* +

FIBRINOGEN* +
PLASMINOGEN

FIBRINOGEN*

Incubate Incubate

jCLOT LYSIS
| eee

*Plasminogen-free

FIGURE 3. The scheme of the fibrinolytic assay for measuring
the potency of a drug in terms of the speed of clot lysis.

97