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Blood Coagulation and Fibrinolysis.

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ANGEWANDTE CHEMIE
Internat
V O L U M E 10 N U M B E R 2
F E B R U A R Y 1971
PAGES 85-146
Blood Coagulation and Fibrinolysis
Norbert Heimburger and Heiner Trobisch[*]
As a result of advances in protein chemistry it is now widely accepted that blood coagulation proceeds via a series of reaction steps o f the nature o f enzyme-substrate reactions
catalyzed by phospholipids. This also applies to the compensatory process of fibrinolysis.
The factors taking part in both mechanisms as well as the inhibitors regulating the equilibrium have been shown to be proteins, some of them with a high carbohydrate content.
Modern diagnostic methods and therapy are based on this knowledge.
1. Introduction
One of the most important physiological properties of
blood is its ability to coagulate, and even to become completely solid and liquid in turn. In principle, both phenomena are manifestations of enzymatic reactions of a
plasma protein, fibrinogen. The conversion of the soluble
fibrinogen, present at a concentration of 0.2-0.4% in
human plasma, into an insoluble, easily polymerizable
derivative, fibrin, characterizes the process of blood
clotting, while dissolution of the crosslinked fibrin marks
the process of fibrinolysis. Coagulation and fibrinolysis
succeed each other under physiological conditions. Such
interplay guarantees that fibrin is produced when required for hemostasis and is degraded when it has fulfilled its biological function after the wound has healed.
Hence, fibrinolysis can be considered as a further stage
in the coagulation of blood.
Coagulation and fibrinolysis are under enzymic control.
Thus thrombin catalyzes the change of fibrinogen into
fibrin and plasmin the hydrolysis of fibrin. Both enzymes,
which are endopeptidases, possess no strict substrate
specificity. The body is protected by a safety measure ensuring that under physiological conditions these enzymes
circulate in the blood not in their activated form but as
proenzymes. The activation of the proenzymes is also
under enzymatic control. Thus some enzyme activators must first be generated from their precursors present
in the blood. The factors required are often separated
from the bloodstream by a cell or tissue barrier, which
['I
Dr. N. Heimburger and Dr. H. Trobisch
Behringwerke AG
D-355 MarburgiLahn (Germany)
Angew. Chem. internat. Edit. / Vol. 1 0 (1971) / No.
2
may also hold the activators back. Such an arrangement
ensures that they are formed and released only when tissues are damaged or the surface of cells and especially
of blood vessels is altered. This sequence of events
guarantees that thrombin and plasmin are generated only
locally and only in response to a real physiological need.
Prolhrombin
Activators
in tissues
Activators
/
in blood
Thrombin
Fibrinogen
Peptides,arninoacids
I
Fibrinopeptides#
Peptidases
1
Blood clotting
Fibrin
1
Fibrinolysis
Plasmin
Activators in blood
t-<
Plasminogen
Activators in tissues
Scheme 1. Blood coagulation and fibrinolysis.
The reactions participating in blood clotting and fibrinolysis are very similar, although they exert opposite
effects (Scheme 1).In each process the biological activity
is associated with a proteolytic enzyme, thrombin or plasmin, which exists as inactive proenzyme in the blood and
is activated by activators localized both in blood and in
85
tissues. In a healthy organism clotting and fibrinolysis
are in dynamic equilibrium. If the enzyme reactions overshoot in one or the other direction, the results may be
fatal. Hemorrhage, thrombosis, and vascular disease can
be the biological consequences, and arteriosclerosis may
also ensue from a disturbance of this dynamic
equilibrium [l].
governs the sequence of reaction steps, which lie either
on an extrinsicLzl or an intrin~ic[~1
pathway. The intrinsic
system is the more complicated; it involves several reaction steps, and since it incorporates several safety
measures it tends to be relatively sluggish. In contrast,
the extrinsic system reacts spontaneously. It can maintain
the blood volume constant in an emergency, thus fulfilling what is probably its most important function. Platelets and vessels mutually assist the blood clotting system:
a traumatic stimulus produces arterial contraction; an
annular layer of muscle in the arterial wall regulates vascular tone; vaso-active peptides and biogenic amines affect muscular tension. These pharmacological substances, either vasoconstrictors or vasodilators, are
produced by a kind of side reaction during coagulation
and fibrinolysis.
Disturbances of the hemostatic equilibrium can nowadays be diagnosed and treated appropriately thanks to
the knowledge gained from research on blood clotting.
This means that the factors that determine the state of
the coagulation and fibrinolysis system are known and
can be measured.
2. Blood Clotting
A damaged vessel whose surface is foreign to the blood
As many as twelve factors present in the plasma and ad-
activates the intrinsic clotting mechanism. This applies,
in particular, to collagen, which forms the supporting
matrix of blood vessels, for blood platelets adhere to the
collagen fibers, form aggregates on the unphysiological
surface, and fuse into a viscous, white plug that effects
a preliminary, mechanical closure of the ~ o u n d [ ~ .The
~1.
thrombocytes release their ADP which acts directly on
the platelet membranes and catalyzes aggregationL6].
ditional components derived from thrombocytes and
cells are involved in the process of coagulation, which
extends over several reaction steps. The plasma factors
are predominantly proteins, some of which have enzymatic properties and which circulate in the form of inactive precursors in the blood; they can be characterized
by techniques customary in protein chemistry, but only
Intrinsic system
rE$
;rke
Initial stage
F XII
Starter reaction
Formation of
activator of factor X
Activation ot tactor X
Formation of
activator of factor
Extrinsic system
--+
F M
F Wa-PL-F
4
--+
Tissue damage,
rekase of factor
XI
Pt-CaZ'-
F Ma-Ca'*-PL-F WI
1
L
I
FX
-
-
-
-
i
-
F
IU
-1
Vna+-F-
W
d
F Xa-La"-PL-FV
II
FXIII
Activation of
prothrombin
FII-FIIa
Formation of fibrin
fibrin crosslinking
Scheme 2. Clotting mechanism (schematic; see also Table 1). PL
I
=
Fibrin
monomer
1
CaZ+
,Fibrin
polymer
phospholipid
a few have so far been isolated in pure and biologically
active states. The cellular factors active in the clotting
process are phospholipids, a class of substances widely
distributed as components of cell membranes. These lipids have been shown to catalyze important steps of enzyme activation during the process of clotting. This fact
could explain why cellular disintegration inevitably leads
to hypercoagulability.
The process of blood clotting can be initiated along two
different pathways, both of which lead to the activation
of factor X and hence to the formation of the prothrombin a c t i ~ a t o r [ ~(Scheme
,~]
2). The site of the stimulus
86
-
F Kma
During this viscous metamorphosis intracellular coagulant substances are released among which platelet factor 3 , a phospholipid, and antiheparin, platelet factor
4, are the most importantl61.
111
T. Astrup, Lancet 271, 565 (1956).
[2] W. Sfaub and F. Duckerr, Thrombos. Diathes. Haemorrh. 5, 402
(1961).
[3] G. H. MiiNer, Thrombos. Diathes. Haemorrh. 14,417 (1965).
[4] S. Karpatkin and R. M. Langer, J. Clin. Invest. 47,2158 (1968).
[S] M. Corn, Nature 212, 508 (1966).
161 H. Holmsen, H. J. Day, and H. Srormoken, Scand. 1. Haematol.,
Suppl. No. 8 (1969).
Angew. Chem. internat. Edit. / Vol. 10 (1971) / N o . 2
Table 1. Factors participating in the clotting process. They have been numbered with Roman numerals in the order of their discovery by a decision of the
International Committee on Nomenclature. F IV is Ca**, F VI is not a n independent factor but is probably identical with F V. Activated factors indicated
by “a”.
Factor
Synonyms
Source of
preparation
Electrophoretic mobility/
Molecular weight
Function and special properties
Plasma
concentration
Site of
formation
I
Fibrinogen
Human plasma
8,-Globulin 360000
(peak)
200-400 mg/
100 ml
Liver
I1
Prothrombin
Human plasma
a,-Globulin 52000
fli-Glycoprotein, split by F IIa at
two specific arginyl sequences t o
give fibrin monomers
Proesterase activated bv F Xa to an
endopeptidase which is relatively
fibrinogen-specific [32]
10.15 mg/
100 ml
Liver. deuen.
dent on vitamin
K [35,36]
Ila
I11
Thrombin
Thromboplastin,
thrombokinase
Tissue factor, released on injury
together with phospholipids
129, 301
Caz+
Very sensitive protein, stabilized
by Ca2+.Catalyzes i n conjunction
with phospholipids and Ca’+ t h e
activation of prothrombin by F Xa
[42,43, 541
Proenzyme, activation by contact
with cell fragments, cofactor of F
I11 t o form activator of F X 1311
Normally not
present in
plasma
Cells
Unknown
Liver?
[Inknown
Liver, dependent on vitamin
K [311
No proof of enzyme character,
stabilized by Ca2+.substrate of
F IXa [24]
Unknown
Spleen, RES [a]
Unknown
Liver, dependent
on vitamin K
10 mg/100 ml
Liver,
dependent
on vitamin K
Unknown
RES? [a]
1 mg/lOO ml
RES? [a]
IV
V
VII
Accelerin,
accelerator
globulin, labile
factor
Bovine plasma
1541
Proconvertin
Bovine plasma
1311
Convertin
Antihemophilic
globulin A,
antihemophilic
factor
Christmas factor,
antihemophilic
globulin B
VIIa
VIIl
IX
34000 I551
U p t o 3 x 106
Human and animal
organs. preferably
lung and brain.
Human, bovine,
porcine, and
rabbit plasma
Human and bovine
plasma
8-Globulin Unknown
8-Globulin 63000
1311
48000 [31]
p,-Globulin Unknown
[231
p,-Globulin Unknown
Unknown
IXa
Stuart-Prower
factor
X
Xa
XI
Plasma thrombo- Not yet
plastin antecedent isolated
XI1
XIIa
XI11
Hageman factor,
contact factor
[13,141
Bovine plasma
1161
Fibrin stabilizing
factor, fibrinase
Human plasma
a,-Globulin 50000
34000 [32]
8,-GlobuIin Unknown
P,-Globulin
[I61
Substrate of F XIIa, both form a
complex with phospholipids
catalyzing activation of F IX
Proesterase, activation on foreign
surfaces t o give arginine esterase
(F XIIa) I161
.~
co. 240000I14-16]
Transglutaminase, activated by
$,-Globulin 290000
thrombin: links fibrin monomers
through peptide bonds into a firm
network
1151
1-2 mgI100 ml
Liver
Plasma transglutaminase
XIIIa
[a] RES
Bovine plasma
1321
Present in a complex with F VIII,
C a l f , and phospholipids t o form
activator of F X [21]
Proesterase, converted by endogenously or exogenously formed
activators to arginine esterase
19,321
=
reticuloendothelial system
Table 1 lists the factors involved in the process of coagulation, showing their most important properties. Several
of the factors are enzymes. Thrombin (F IIa) and the
activated factors X and XI1 (Xa and XIIa) are esterases,
and factor XI11 is a transamidase in its activated form
(XIIIa)[81.The biochemical nature of most of the factors
has not yet been clarified. There are indications that factors 11, VII, IX, and X are related to one another. According to Seegerd9],they do not exist as individual
proteins but are dissociation and association products
of prothrombin (F 11). Since clotting factors are particularly labile this question has not yet been finally
answered; even the most modern techniques do not
prevent denaturation and inactivation. The function of
coagulant proteins naturally requires that they should
be highly sensitive (see Section 2.1.1).
[7] F. Jobin and M. P.Esnouf, Biochem. J. 102, 666 (1967).
[S] W. H. Seegers, Annu. Rev. Physiol. 31, 269 (1969).
[9] W. H. Seegers: Blood Clotting Enzymology. Academic Press, New
York 1967, p. 129.
[lo] 0. D. Ratnoffin E. 5.Brown and C. V. Moore: Progress in Hematology. Grune and Stratton, New York 1966, p. 204.
Angew. Chem. internat. Edit. / Vol. 10 (1971) / N o . 2
2.1. The Intrinsic Pathway
2.1.1. Initial Stage
In the starter reaction contact of blood with nonphysiological surfaces, such as skin, cellular debris, collagen fibers, and arteriosclerotic layers initiates the clotting process. At the same time, the Hageman factor (F XII), a
proesterase, is changed into an active arginine esterase110-17].Other proteins acting as protective colloids pre[111 H. L. Nossel, Proc. Soc. Exp. Biol. Med. 12.2, 16 (1966).
[12] S. Niewiarowski, E. Bankowski, and 1. Rogowjcka, Thrombos.
Diathes. Haemorrh. 14, 387 (1965).
[13] 0. D. Ratnoff, J. Lab. Clin. Med. 44, 915 (1954).
[I41 0. D. Ratnoft E. W. Davie, and D. L. Mallet, J. Clin. Invest. 40,
803 (1961).
[15] C. Haanen and I. G. G. Schoenrnakers, Thrombos. Diathes.
Haemorrh. 9,557 (1963).
[ 161 I. G. G. Schoenrnakers, R . Matu, C. Hannen, and F. Zilliken, Biochim. Biophys. Acta 93, 433 (1964).
[17] H. Temrne, R. Jahrreis, E. Habermann, and F. Zilliken, HoppeSeylers Z. Physiol. Chem. 350, 519 (1969).
87
vent an accidental activation of the Hageman factor in
units. Neither theory has yet been proved. The most
the blood. The technique used for enrichment and isouseful procedure for preparing therapeutically active
lation makes use of the high affinity of the proenzyme
concentrates is also the simplest, i e . cryoprecipitation,
for foreign s ~ r f a c e s [ ~however,
~1;
the activated Hageman
where use is made of the differential solubilities of
factor (F XIIa) obtained in this way is not stable indefplasma proteins on freezing and thawing.
initely[181. That is probably why the physicochemical
Activation of factor X is the first step in the cascadeproperties of the Hageman factor are not yet fully known.
like sequence of reactions common to the intrinsic and
A deficiency of factor XI1 does not apparently constitute
extrinsic mechanism of clotting and yet catalyzed by aca hemorrhagic disease, as may be seen from the clinical
tivators of different composition (Scheme 2). Enzymes
history of the first patient, by name of Hageman, with
catalyze the transformation of factor X to yield an ara hereditary deficiency of factor X11[221.However, if facginine esterase (F Xa) with a high affinity for calcium
tor XI1 is activated inside the body disseminated inions[28],which is evidently an important prerequisite for
travascular coagulation will occur, with fatal results.
the formation of the prothrombin activator from factor
Since the Hageman factor also catalyzes the conversion
Xa, calcium ions, phospholipids, and plasma factor V.
of kallikreinogen into kallikrein, the kallikrein-kinin sysThe intrinsic and extrinsic mechanisms converge at the
tem (and indirectly also fibrinolysis) is activated at the
point where the prothrombin activator is formed; details
same time as the intrinsic clotting m e c h a n i ~ m [ ’ ~ ~ ~ ~ I
will therefore be discussed in the appropriate Section
(Scheme 4).
in order to facilitate an understanding of the subsequent
Factor XI1 once activated (F XIIa) forms an enzymeactivation of prothrombin.
substrate complex with factor XI in the presence of
phospholipids; the starter reaction consists essentially
2.2. The Extrinsic System
in the formation of this complex, which then catalyzes
the activation of factor IX, the most important com2.2.1. Initial Stage
ponent of the intrinsic system activating factor X[211.
The chemistry of factor XI is almost unknown: it has
hitherto defied all attempts at isolation. However, its existence is demonstrated by a rare hereditary hemorrhagic
disease caused by its deficiency in or absence from the
blood.
When factor IX in its activated form (IXa) combines with
another plasma protein, factor VIII, and with phospholipids in the presence of calcium ions, the complex thus
formed constitutes the activator of factor X. No conclusive proof for this enzymatic step has yet been obtained because factor IX has not hitherto been isolated or
characterized in any way.
Factor IX appears to possess neither protease nor esterase properties, because its action is not affected by diisopropyl fluorophosphonate to which esterases having
serine in the active site are sensitive. Calcium ions catalyze the activation of factor IX, heparin blocks it. The
synthesis of factor IX which takes place in the liver is
dependent on vitamin K. A deficiency of factor IX which
is inherited is responsible for hemophilia B, the second
most frequent hemorrhagic disease.
Factor VIII, antihemophilic globulin A (AHG-A), is one
of the best known clotting factors. Its inherited absence
or deficiency is the cause of the most frequent familial hemorrhagic disease, i e . , hemophilia A. The term “antihemophilic globulin A” derives from the fact that its administration represents the only effective treatment of
hemophilia A. This globulin, which is probably synthesized in the spleen, belongs to the group of &globulins
of the plasma and has a relatively high molecular
weight[z3.241.
It has not been possible to prepare AHG-A in a pure
state, because it is very labile. CheIating agents inactivate
it irreversibly by removing calcium ions which may
stabilize AHG-A; alternatively, factor VIII may be a
complex made up of several individual proteins or sub-
88
When a wound is inflicted, blood will rush out of the
injured capillary vessels into the surrounding tissue.
Simultaneously substances differing in chemical nature
and capable of promoting blood coagulation will be
washed out of the damaged cells. Such substances are
often still bound to cellular components; they are present
in particularly large amounts in microsomes. This fraction of cell particles is called tissue thromboplastin or
factor III[29.301;
its release may be regarded as the starter
reaction.
The interaction between tissue factor I11 and a plasma
cofactor (F VII, proconvertin), which is carried by the
blood to the wound, is the next step in the formation
of the activator of factor X. The activation of factor VII
to give FVIIa, which occurs on contact with factor
111, is accompanied by a reduction of the molecular
1181 I. G. G. Schoenmakers, R . M. Kurstjens, C. Haanen, and F. Zilliken, Thrombos. Diathes. Haemorrh. 9, 546 (1963).
1191 S. G. Jafridis and J. H. Ferguson, Thrornbos. Diathes. Haemorrh.
6, 411 (1961).
[20] R. W. Colman, Biochem. Biophys. Res. Comm. 35, 273 (1969).
[21] 0 D. Rarnoff and E. W . Davie, Biochemistry 1, 677 (1962).
[22] 0. D. Ratnoff and A. G. Sfeinberg, J. Lab. Clin. Med. 59. 980
(1962).
I231 S. van Crefeld e t al., Thrombos. Diathes. Haemorrh. 17, 188
(1967).
[24] E. J. Hershgold, L. Silverman, A . M. Davison, and M. E. Janszen,
Selecta 9, No. 35, p. 2384 (1967).
[25] H. C. Hemker, A. C. W. Swart, and R. G. Macfarlane, Nature 215,
248 (1967).
1261 F. Jobin and M. P. E. Esnouf, Biochem. J. 102, 666 (1967).
[27] M. P. E. Esnouf: Biochem. J. 115, P1 (1969).
[28] M. P. Esnouf: Proc. 10th Congr. Intern. SOC. Blood Transf.
Stockholm 1964. Karger, Basel 1965, p. 1337.
[29] H. C. Hemkerand C.Haanen, Tijdschr. Geneeskunde 113, No. 5,
p. 203 (1969).
[30] A . D. Bangham, Nature 174, 791 (1954).
[31] H. Prydz, Scand. J. Clin. Lab. Invest. 17, Suppl. 84, 78 (1965).
Angew. Chem. internat. Edit. / Voi. 10 (1971) / N o . 2
weight[31],as investigations on isolated factors obtained
from bovine plasma and serum have shown[31].The difference in the molecular weights between the inactive
and activated forms of F VII may indicate that activation
is enzymatic, but no experimental evidence has yet been
obtained 341.
f333
An enzyme, factor Xa, arising on activation of factor
X catalyzes the hydrolysis of N-substituted arginine esIt is liberated from the proesterase enzymatically; the reaction is catalyzed by trypsin or Russel viper
venom in vifro. The activators formed either endogenously or exogenously catalyze this activation in a comparable manner in viv0[~1.
Factor X was prepared in high purity for the first time
from bovine plasma[32].The isolation is carried out by
adsorption onto BaSO, or Ca,(PO,),, fractional elution
from these adsorbents, chromatography of the eluates
on cellulose ion exchangers, and final separation on a
molecular sieve. A uniform preparation is obtained under
these conditions only if diisopropyl fluorophosphonateis
this inhibitor prevents an enzyadded to the buffer~[~*1;
matic degradation and inactivation during the isolation.
The molecular weight is decreased and the electrophoretic mobility altered as a result of activation[32].Factor
Xa is the enzymatic and also the most important component of the prothrombin activator complex.
The prothrombin activator catalyzes the transformation
of prothrombin (FII) to thrombin (F IIa). Several components of the prothrombin activator take part in the
reaction: factor Xa and factor V, which is a plasma globulin of uncertain nature, phospholipids, and calcium ions.
The phospholipids provide the reaction surface on which
the enzymatic conversion of prothrombin by factor Xa
takes place. Since factor V determines the reaction rate,
it is referred to as accelerator globulin. It is assumed that
factors V and Xa are held inside the lipid micelles in
such a way that both come into close contact with the
prothrombin m ~ l e c u l e [ ~ ~thus
- ~ ~satisfying
],
an essential
condition for activation, i. e. a partial hydrolysis.
2.2.2. The Activation of Prothrombin
Although prothrombin (F 11) is one of the trace proteins
in human plasma, its presence in plasma can be detected
by modern electrophoretic techniques as well as by biological tests.
[ 3 2 ] C M. Jackson and D. J. Hanahan, Biochemistry 7, 4492, 4506
(1968).
(331 Y. Nemerson, Biochemistry 5, 601 (1966).
1341 W. J. WiUiams and D G. Norris, J . Biol. Chem. 241, 1847 (1966).
[35] H. C. Hemker, J. Van der Meer, R. Hodge, and E. A . Loeliger in
J. Van der Meerr Pharmacological Aspects of Vitamin K1. SchattauerVerlag, Stuttgart 1968.
[36] E. F. Mammen, Internist 1, 2 (1969).
[37] P. 0. Ganrot and J. E. Nilkhn, Scand. J. Clin. Lab. Invest. 21,238
(1968).
[38] J. E. Nilebn and P. 0. Ganror, Scand. I.Clin. Lab. Invest. 22, 17
(1968).
I391 D. Heene in H. Scbreer: Biochemie und Aktivierung des Prothrombins. Schattauer-Verlag, Stuttgart 1968, p. 3.
[40] G . F. Lancbantin and J. A . Friedmann, Vox Sanguinis 9, 228
(1964).
[41] W. H. Seegers et al., Texas Rep. Biol. Med. 23, 675 (1965).
Angew.
Chem. internat. Edit. / Vol. 10 (1971) / N o . 2
When human plasma is subjected to electrophoresis in agarose
gel containing fibrinogen, a turbidity arises in the a2-globulin
region if tissue thromboplastin (F 111) and calcium ions are
allowed to diffuse from a slot parallel to the direction of migration. The turbidity occurs in the electrophoretic position of
prothrombin where it encounters the tissue thromboplastin diffusing in, so that thrombin could be formed. The turbidity in
the plate is due to the formation of fibrin. This biological test
can be combined with an immunological assay using a specific
antiserum against prothrombin but not a multivalent antihuman
serum. The concentration of prothrombin in plasma can be determined quantitatively in this manner. It is 10-15 mg/100 ml,
and thus of the same order of magnitude as that of human
p~asminogenl~~!
The synthesis of prothrombin (F II), which depends on
vitamin K, occurs in the liver together with that of factors
VII,IX, andX[35,361.Theprothrombin complex thus comprises four protein factors. Serious diseases of the liver
upset the synthesis of these proteins. This also applies to
vitamin K deficiencies caused by partial or complete
elimination of the intestinal flora, e.g. in diarrhea, after
treatment with non- absorbable antibiotics, or in cases of
obstructive jaundice when fats are not readily reabsorbed and vitamin K is not taken up at all.
The dependence of the synthesis of factors comprising
the prothrombin complex on vitamin K is pharmacologically important because the dicoumarols and indandiones, being antagonists of vitamin K, can be employed successfully in the prophylaxis of thrombosis and
the treatment of cardiac infar~tion[~’,~~1.
Recent findings
on the effects of these long-acting anticoagulants contradict the original idea of a total inhibition of synthesis.
These drugs act not so much by affecting the rate of synthesis of the prothrombin complex as by inducing the
formation of a biologically unusable prothrombin molecule which will react with the prothrombin activator but
not give rise to any t h r ~ m b i n [ ~ ~ - ~ * ] .
In principle, the same techniques are applied to the isolation of prothrombin as to the preparation of factor X.
Since the two proteins have such similar properties, they
can be separated only with difficulty even when using
the most varied methods of fractionation. It has not been
possible to obtain a uniform preparation satisfying all
purity criteria of proteins, because autocatalytic inactivation cannot be prevented entirely139-41].
Prothrombin, which has a molecular weight of 52 000,
breaks up during activation into at least three components, one of which is identical with thrombin (mol.
wt. = 34000)[25-271.
The prothrombin activator, a complex of phospholipids
with factors Xa and V and with calcium ions, catalyzes
the transformation of prothrombin to thrombin; the reaction appears to be a specific proteolytic hydrolysis,
which is also catalyzed by trypsin and which proceeds
autocatalytically in a 25 96 solution of sodium
Factor Xa on its own catalyzes the reaction only inadequately, although it cleaves arginine
The
reaction proceeds very sluggishly in a purified system
containing prothrombin, but once factor V, phospholipids, and calcium ions are added, the rate of the reaction
increases 1000-fold without simultaneous enhancement
of ester hydrolysis. This result shows that factor V ac89
celerates the activation of prothrombin specifically, but
only in a medium containing phospholipids. For this reason it is assumed that the activation of prothrombin takes
- ~ ~ a] .
place on the surface of the lipid m i ~ e l l e s [ ~ ~Such
concept would also explain the effect of prolonged-action
anticoagulants on the organism: competitive blocking of
the complex of factors Xa and V and of lipid (prothrombin activator) by pathologically modified prothrombin
greatly reduces the rate of
Thrombin (F IIa) is a highly specific protease whose most
important function is to catalyze the coagulation of
fibrinogen. However, thrombin loses this property progressively from the moment of its formation while retaining its ability to hydrolyze esters for a relatively long
An acetyl derivative of thrombin has been described which, though not catalyzing coagulation, cleaves
synthetic esters as well as thrombin ( t h r ~ m b i n - E ) [ ~ ~ ] ,
Prothrombin and factor X have much in common: the
site of formation, the methods of isolation, and properties
such as their enzymatic nature. These facts may justify
the notion that factor X is a subunit of prothrombin from
which it is generated together with thrombin or as a byproduct, according to the prevailing conditions[g].
2.2.3. Formation and Crosslinking of Fibrin
The transformation of fibrinogen (F 1)'into fibrin monomers and their subsequent crosslinking mark the last
stage of the clotting process. Both reactions are catalyzed
by enzymes, ie. thrombin (F IIa) and factor XIIIa, a
transglutaminase.
First of all thrombin removes two relatively acidic peptides, referred to as fibrinopeptides A and B, from fibrinogen, thus generating fibrin monomers which in turn
polymerize into long strands with the diameter of one
fibrinogen molecule. Fibrin filaments are formed by lateral aggregation of double strands of fibrin polymers[46].
A freshly formed fibrin network is not held together by
covalent bonds but is stabilized by hydrogen bonds and
hydrophobic forces. Hence it is soluble in denaturing
agents. Covalent crosslinking is effected by the plasma
transglutaminase (F XIIIa) which thrombin sets free
from factor XI11 in the presence of calcium ions (see
Scheme 2). The €-amino groups of lysine and the yglutaminyl groups are then linked via amide bonds,
mainly between the fibrin monomers of a double
(Fig. 1).
[42] P. G. Barton and D. J. Hanahan, Nature 214, 923 (1967).
[43] P. A. Owen: Coagulation of Blood, Investigations on aNew Clotting Factor. J. Chr. Gundersen, Oslo 1947.
[44] M. P. Esnouf and F. Jobin, Biochem. J. 102, 660 (1967).
(451 R.H. Landaburu and W. H. Seegers, Canadian J. Biochem. Physiol.
37, 1361 (1959).
[46] R. Gollwitzer, E. Karges, H. Hormann, and K. Kuhn, Biochim.
Biophys. Acta 207, 445 (1970).
[47] R.&el, A. G. Loewy, K. Dunathau, and H. J. Wolfinger, J. Biol.
Chem. 236, 2625 (1961).
[48] A. G. Loewy, S. Maiacic, and H. J. Darnell, Arch. Biochem.
Biophys. 113, 435 (1966).
[49] B.BIomback in W. H. Seegers: Blood Clotting Enzymology. Academic Press, New York 1967, p. 628.
90
-CO-NH-CH-CO-NHI
( y M 4
-CO-NH-CH-CO-NHI
p 2 ) 4
-CO-NH-CH-CO-NH-
1Fig. 1. Reaction mechanism of plasma transglutaminase (factor XIIIa).
This covalent crosslinking completes the clotting process. Fibroblasts enter the clot, where factor XIIIa promotes their
and synthesize collagen which
increasingly replaces fibrin; the latter is now no longer
required and will be degraded. The healing of a wound
has entered its final stage[52,53!
3. Fibrinolysis
The fibrinolytic pathway is simpler and more comprehensible than the clotting pathway: the plasminogen
activators are available directly and do not have to be
first formed from a series of plasma factors with participation of cellular components. All the factors involved
in fibrinolysis are proteins which can be characterized
by customary methods of protein chemistry.
3.1. Human Plasminogen (Proactivator Plasminogent*') and Plasmin
Human plasminogen differs in one important respect
from that of many animals: the action of streptokinase,
a metabolite of P-hemolytic streptococci, converts it into
plasmin. This interaction yields an intermediate complex
which has the same biological effect as intrinsic activators
(see Sectidn 3.2). Hence we also apply the term proactivator plasminogen (PP) to human p l a s m i n ~ g e n [ ~ ~ ] .
Plasmin is the predominant protease circulating as an
inactive precursor in blood. The concentration of the
proenzyme is 15-20 mg/100 ml human
Since
human plasma contains 7-8g of protein/100 ml isolation
of the proenzyme therefore represents a 350-fold enrich[*] We prefer this functional term because human plasminogen combines
with streptokinase, a metabolite of 6-hemolytic streptococci, to form activator as well as plasmin (see Section 3.2); in this respect it differsfrom
the plasminogen of most mammals [56].
[SO] E. Beck, F. Duckert, A. Vogel, and M. Ernst, Z. Zellforsch. 57,
327 (1962).
[51] K. Laki and L. Lorand, Science 208,280 (1948).
[52] F. Beck, F. Duckerr, and M. Ernst, Thrombos. Diathes. Haemorrh.
6, 485 (1961).
[53] I? Duckert, Thrombos. Diathes. Haemorrh. Suppl. 13,115 (1963).
[S4] D. Papahadjopoulos, C. Houge, and D. Hanahan, Biochemistry
3, 2 (1964).
[ 5 5 ] S. Magnuson, Arkiv Kemi 24, 367 (1965).
1561 N. Heimburgerand H. G. Schwick, Thrombos. Diathes. Haemorrh.
7, 444 (1962).
1571 H. G. Schwick and N. Heimburger: Internat. Symposium uber therapeut. u. experiment. Fibrinolyse, Ulm 1967. Schattauer-Verlag. Stuttgart 1969.
Angew. Chem. internat. Edit. / Vol. 10 (1971) / N o . 2
ment. Starting material for the preparation from human
plasma is Cohn fraction I11 which is obtained by stepwise
precipitation with alcohol; it may be extracted either with
mineral acids[58]or with buffers containing lysine and
E-aminocaproic acidls91.
These amino acids are then also used for elution from
ion exchange columns[60].
physical properties and the appearance of the two ad
ditional end groups appear to be largely independent
of the mode of activation. This has been demonstrated
for autocatalytically activated plasmin as well as for the
following activators: pig-heart activator, urokinase,
streptokinase, and trypsin.
These basic amino acids, which enhance the solubility
of human plasminogen but simultaneously inhibit activation, probably form salt linkages with the plasminogen
molecule[56].Use of buffers containing these amino acids
in support media for electrophoresis has proved useful
since many plasminogen preparations are poorly soluble
under physiological conditions.
LA 800.21
The sedimentation coefficient of highly purified human plasminogen measured in the gravitational field of the ultracentrifuge is S20,w=4.1.A molecular weight approaching 90000 may
be calculated on the basis of a diffusion coefficient of D2,,w
= 3.96 and a partial specific volume of 0.72. In an electric field
human plasminogen migrates uniformly, with a mobility corresponding to that of the &globulins of human plasma. Human
plasminogen is detected and estimated quantitatively in plasma
by immuno-electrophoresis and by use of a univalent antiserum.
Human plasminogen migrates in several bands under the conditions of poiyacrylamide gel electrophoresis, in which proteins
are separated according to their charge and molecular shape
and size (Fig. 5 ) . The reason for this can be sought in the slight
differences existing in the charge or in the molecular size and
shape, due possibly to structural changes in the molecule induced by p H shifts. It can be shown by reaction with the activators and with homologous antisera that all the bands have the
same specificity. When an unstained pherogram of human
piasminogen is covered by a thin agar film, an immune precipitate is formed if the antiserum is allowed to migrate against
the components that have diffused from the polyacrylamide
gel into the agar. This precipitate extends over the whole region
in which the bands are observed. Human plasminogen bound
to serum migrates to the same electrophoretic position when
this technique is applied.
Human plasminogen is composed almost entirely of amino acids[61];it contains only 1.4% of carbohydrate. The
polypeptide chain is held together by 22 disulfide
bridges; its N-terminal amino acid is lysine and the Cterminal one is asparaginef631.
There are two additional terminal groups in plasmin, the
activated molecule: N-terminal valine and C-terminal
arginine (Fig. 2). Interestingly, the activation of human
plasminogen is characterized by cleavage of an argininevaline bond but the two resultingpeptide chains separate
only after one of the 22 disulfide bridges has been reduced. Obviously, large peptides are not removed on
activation. However, the shape of the molecule is
changed: its sedimentation coefficient increases and the
coefficient of friction decreases. The changes in these
[58] D. L. Kline, J. Biol. Chem. 204, 949 (1953).
[59] N Alkjaersig, P. Fletcher, and S. Sherry, J. Biol. Chem. 234, 832
(1959).
[60] P. Wallen and K. Bergsrriim, Acta Chern. Scand. 13,1464 (1959).
[61] K. C. Robbins, L. Summaria, D. Elwyn, and G. H. Barlow, J. Biol.
Chem. 240, 541 (1965).
[62] H. G. Schwick in: Verhandl. d. Dtsch. Arbeitsgemeinsch. f.
Blutgerinnungsforsch., 9. Tagung, Freiburg 1965. Schattauer-Verlag,
Stuttgart 1967.
[63] K. C. Robbins, L. Summaria, B. Hsieh, and I. Shah, J. Biol. Chem.
242, 2333 (1967).
Angew. Chem. internat. Edit. / Vol. 10 (1971) / N o . 2
Fig. 2. Activation of plasminogen (top) by urokinase to form plasmin
(bottom), schematic. Only three of the 22 disulfide bridges present are
shown [63].
Since plasmin is an endopeptidase, its activity may be
determined by measuring the rate of hydrolysis of fibrin,
casein, or synthetic esters of lysine or arginine. However,
plasmin catalyzes the hydrolysis not only of fibrin but
also of other plasma proteins including certain clotting
factors such as fibrinogen (Scheme 4). This reaction is
of particular physiological importance because it can account for a pathological increase in fibrinolysis.
3.2. Activation of the Fibrinolytic System
The fibrinolytic system can be activated by three different mechanisms.
1) Direct conversion of the proenzyme by intrinsic activators and also by certain proteases.
2) An activator produced as an intermediate in a twostep reaction has the same biological properties as the
intrinsic activators. Streptokinase is one of the relatively
rare proteins having this action.
3 ) Some compounds of widely differing structures can
have an indirect effect on the
3.2.1. One-Step Activation
Natural activators are widely distributed throughout the
body. As enzymes can be detected by their ability to hydrolyze synthetic esters of lysine and arginine but not
high-molecular proteins such as
Apart from
this latter feature, their substrate specificity resembles
that of trypsin which, too, is capable of activating human
plasminogen in catalytic amounts. This fact is consistent
with the finding that activation of the proenzyme is
characterized by the hydrolysis of an arginine-valine
(Fig. 2).
Different amounts of activator are present in various
tissues1661:Uterus, adrenals, lungs, and prostate are rich,
[64] L. Summaria, 8.Hsieh, and K. C. Robbins: J. Biol. Chem. 242,
4279 (1967).
[65] P. Kok and T. Asfrup, Biochemistry 8,79 (1969).
[66] 0. K. Albrechtsen, Acta Endocrinol. 23, 207 (1956).
91
and the testicles and spleen poor in the activator; the
liver contains none at all. A high content of activator
ensures that the blood maintains its fluidity. This explains
why menstrual blood remains liquid o r is reliquified.
It should be mentioned again that human plasminogen changes
ta plasmin autocatalytically and that the activation of plasminogen can also be catalyzed by t r y p ~ i n [ ~However,
~].
these reactions are of no biological interest.
Numerous problems are encountered in the isolation of
3.2.2. Two-step Activation
pure tissue activators, because the active principle is
often bound to structural elements. It can be extracted
In addition to the intrinsic activators, which are esterases
and convert plasminogen directly into plasmin, certain
with concentrated thiocyanate solution but will represubstances of biological origin are capable of changing
cipitate in physiological saline. So far, two activators
proactivators into activators which in turn catalyze the
have been isolated from animal tissues, one from the
heart muscle[67],the other from pig o v a r i e ~ [ ~the
~ lat~ ~ ~ ] ; formation of plasmin in a further reaction.
ter has a molecular weight of 58000 and its properties
Although the animal organism makes little use of this
as a protein and as an enzyme resemble those of urokimechanism, which should therefore be regarded as an
nase obtained from human urine. Neither of the tissue
exceptional case of plasminogen activation, it is of great
activators is very soluble under physiological conditions.
interest therapeutically. A kinase having these properties
has hitherto only been isolated from ascites cells[74].The
Thrombocytes and erythrocytes are some of the blood
corpuscles in which activators have been demonstrated;
mode of action of streptokinase, one of the many methe active principle from erythrocytes, a pure erythrotabolites of f3-hemolytic streptococci has been more exkinase, has already been obtained16’1. Certain body
tensively investigated than that of this lysokinase. For
fluids and secretions are relatively rich in activators, e. g.
several years streptokinase has been manufactured o n
urine (urokinase), amniotic fluid, and human milk, but
an industrial scale by modern fermentation techniques
activators are also found in lacrimatory fluid, saliva, and
for use in fibrinolytic therapy.
semen. In fact, the body makes them plentifully available
Streptokinase is a protein and has been characterized by the
wherever body fluids and secretions flow through capilusual methods. It is homogeneous in an ultracentrifuge; it milaries liable to b e endangered by occlusion.
grates uniformly like an a2-globulin of human serum during free
One of the soluble activators is urokinase, which has been prepared in analytical purity from human urine. A volume of 2300
liters gave 29 mg of crystalline substance, the yield being
24%c7O1. Urokinase, which is formed in kidney cells, has a molecular weight of 53000~”]. Like all intrinsic activators, it is
an esterase that does not attack fibrin. A modified immunoelectrophoresis technique using an agar film containing fibrin
as the supporting medium can demonstrate this very ~learly[’~1.
The opalescent gel is a sensitive indicator for protease activity:
clear areas appear on the agar plate wherever enzymes are located or are being generated following electrophoresis of enzyme or activator samples. Urokinase and other activators do
not attack the fibrin film themselves. However, when human
serum is permitted to diffuse at right angles to the migration of
the activators in the fibrin film, an arc-like area of lysis is formed
upon confluence as the result of proenzyme activation to plasmin. A result such as this illustrates the organization of the
fibrinolytic system: neither activator nor human serum alone has
fibrinolytic activity; the activators must be liberated before
plasmin can be formed (Fig. 3).
a i Urokinase
Human serum
bi Streptokinase
cI Streptokinase
Human serum
Anti - streptokinase serum
Human serum
Fig. 3. Detection of plasminogen activators by electrophoresis on fibrinagar plates and activation of the plasminogen system in human serum
(a + b) with simultaneous immunological demonstration (c).
(671 F. Bachmann, N. Retcher, N. Alkjaersig, and S. Sherry, Biochemistry 3, 1578 (1964).
1681 7. Asfrup and P. Kok, Thrombos. Diathes. Haemorrh. 13, 587
(1965).
[69] A. J. JO~RSOR,10. Kongr. Int. Ges. f . Haemat., Stockholm 1964.
I701 A. Lesuk. L. Terminiello. and J. H . Traver, Science 147, 3660
(1965).
92
electrophoresis, immunoelectrophoresis, and polyacrylamide
gel electrophoresis. The molecule (molecular weight = 47000)
consists of a single peptide chain in which the amino acids
are arranged without any intramolecular stabilization afforded
by disulfide bridges, because cysteine is
3.2.3. The Mode of Action of Streptokinase
According to the available evidence, streptokinase is not
an enzyme: it is neither an esterase like the intrinsic activators nor a protease. Also, it is not affected by treatment with diisopropyl f l u o r o p h ~ s p h o n a t e [ ~Hence
~ ] . its
mode of action must differ from that of urokinase or
other intrinsic activators. The fact that animal plasminogen, e.g. bovine plasminogen, is not activated by streptokinase agrees with the finding that it is largely specific
for human p l a ~ m i n o g e n [ ~ * , ~ ~ ] .
A definite amino-acid sequence and conformation of the
plasminogen molecule therefore appear necessary for
interaction with streptokinase. The activator is the initial
product of the reaction between human plasminogen and
streptokinase; plasmin is generated only during a second
reaction. The activator, which has the physical characteristics of a complex between human plasminogen and
[71] R. H. Painter in: Intern. Sympos. Anticoagulants and Fibrinolysis.
Lea and Febiger, Philadelphia 1961, p. 351.
[72] N. Heimburger and G. Scbwck, Thrombos. Diathes. Haemorrh.
7, 432 (1962).
[731 J. H. Lewis and J. H. Ferguson, Amer. J. Physiol. 170,636 (1952).
[74] H. C. Kwaan. Thrombos. Diathes. Haemorrh. Suppl. 1 ad 6, 75
(1961 ) .
[75] N . Hejrnburger, Behringwerk-Mitt. 41, 84 (1962).
[76] H. G. Schwjck, Behringwerk-Mitt. 44, 103 (1964).
[77] F. Buck and E. C. De Renzo, Biochem. Biophys. Acta 89,348
(1964).
(781 S. Muffertz and M. Lassen, Proc. Soc. Exp. Biol. Med. 82, 264
(1953).
[79] S. Miillerfz, Biochem. J. 61, 424 (1955)
Angew. Chem. internat. Edit. / Vol. 10 (1971) / N O . 2
-
tokinase concentrations the capacity of the activator increases to the same extent that the plasmin yield
decreases. Quantitative stoichiometric evaluation of
these results shows that the optimum for plasmin
formation is at a molar ratio of plasminogen:
streptokinase, catalyzes the conversion of human and
animal plasminogen in a manner comparable with natural a c t i ~ a t o r s [ ~ ~(Scheme
.~’l
3). For this reason we have
proposed the functional term “proactivator-plasminogen” to describe human plasminogen.
Plasmin
T-
Streptor
Human
Tissue activator,
urokinase or
lSKl
P P - SK complex
lactivatorl
plasmi nogen
erythrokinase
I
lPPl
Plasmin
Scheme 3. Activation of animal and human plasminogen with endogenous activators and with streptokinase.
Measurement of the rate of hydrolysis of a fibrin clot, prepared
by adding thrombin to a solution of fibrinogen, is the basis for
assays of activator and plasmin. Clots of bovine fibrin are employed for the estimation of activator. Bovine plasminogen is
adsorbed in relatively large quantities by fibrin prepared by the
usual methods; activators but not streptokinase convert it into
plasmin, as emphasized above. Plasmin, a proteolytic enzyme,
is assayed by its hydrolysis of casein or of clots prepared from
plasminogen-free fibrinogen. The dilution at which the clot
is lyzed in the test solution within 15 min affords a measure
of the activity of the activator or plasmin; its reciprocal value
is used in the calculation.
The relative amounts of activator and plasmin formed
by the catalytic action of streptokinase on human plasminogen can easily be appreciated[B0].Figure 4 is a plot
of the results of a series of experiments showing how
the activator and plasmin activities are affected by reaction of varying amounts of streptokinase (SK) with
a constant amount of human plasminogen (PP). It can
be seen that a relatively low proportion of streptokinase
is sufficient for catalyzing a maximum formation of plasmin. It is important to realize that with increasing strepMolar ratio SK PP +
01 1
12 rl
21
11
I
i
11200
I
m
5000 10000
50000
100000
Streptokinase ilUI-----
Fig. 4. The capacity of a constant quantity of human plasminogen for
plasmin and activator formation as a function of the units of streptokinase
used for activation. Determination on clots from pure bovine fibrinogen
(a, plasmin) and plasminogen-containing bovine fibrinogen (b, activator). U = units.
I801 N. Hemburger in: Sympos. d. Dtsch. Gesellsch. f. Angiologie,
Miinchen. Schattauer-Verlag, Stuttgart 1967, p. 7.
Angew. Chem. internat. Edit. / Vol. 10 (1971) / N o . 2
streptokinase = 1:0.1, but the activator yield reaches
its maximum only when the components are present in
equimolar amounts. It might be added that the activator
is only weakly proteolytic, in contrast to plasmin. The
values obtained under the same conditions by measuring
the hydrolysis of casein agree with the above figureslaO1.
On the basis of these findings we have developed a working hypothesis according to which plasmin loses its proteolytic activity but retains its ability to hydrolyze esters
once it is bound to streptokinase to form the activatorI57,g11.
To prove this hypothesis it is essential that plasmin can
be shown to combine with streptokinase in an activator
complex. The technique of fibrin-agar electrophoresis
enables us to demonstrate this clearly. Streptokinase is
first subjected to electrophoresis on a fibrin-agar plate
and plasmin that has been prepared by reacting streptokinase and human plasmincgen at a molar ratio of
0.2: 1 is then allowed to diffuse into the gel from a slit
parallel to the direction of electrophoretic separation.
An inhibition of the fibrinolytic activity of plasmin may
be observed within the zone of migration of the streptokinase. If a solution of bovine serum, which does not
react with streptokinase, is dropped onto this inhibition
zone a clear halo indicating hydrolysis will appear. This
result is not surprising, since the molar ratio of streptokinase to plasminogen reaches a value of 1where plasmin (molar ratio of the components = 0.2: 1) meets
streptokinase. Here the conditions for activator formation are optimal, so that only residual proteolytic
activity can be detected with casein or plasminogen-free
bovine fibrinogen as the substrate (Fig. 4). Such activity
cannot be ascribed to the activator with certainty because
it is relatively labile and decomposes with the release
of plasmin.
Plasmin and activator, which arise by the interaction between human plasminogen and streptokinase, can be
[81] N. Heimburger, 16th Annual Sympos. on Blood. Detroit, Wayne
State Univ., January 1968; Thrombos. Diathes. Haemorrh. 19, 598
(1968).
93
characterized by the usual methods of protein chemistry.
Polyacrylamide gel electrophoresis is an efficient tool
to achieve a clear separation (Fig. 5).
m
a
b
c
d
e
'
g
Fig. 5. Polyacrylamide gel electrophoresis. a, human plasminogen
(proactivator plasminogen). b, human plasminogen activated by urokinase. c, human plasminogen activated by streptokinase; molar ratio
1 : 0.2. d, human plasminogen activated by streptokinase, molar ratio
1 : 1 e, streptokinase. f, human serum. g, human plasminogen activated
by streptokinase, molar ratio I : 1, in 8 M urea.
It has been mentioned above that human plasminogen
migrates in several bands on polyacrylamide gel electrophoresis although it behaves as a homogeneous protein
on paper and immunoelectrophoresis and in the ultracentrifuge. in contrast, plasmin appears almost homogeneous when it has been prepared by activating human
plasminogen with urokinase. The electrophoretic pattern
of a reaction mixture of streptokinase and human plasminogen (molar ratio = 0.2: 1) contains a component
with the same mobility as well as a faster one. Since it
is known that urokinase only catalyzes the formation of
plasmin, the slower moving band can obviously be attributed to plasmin and the faster one to the activator,
particularly since an activation mixture consisting of
equimolar quantities of the components gives rise to only
this one component, which migrates almost exactly between human plasminogen and streptokinase. Plasminogen as well as streptokinase can be demonstrated in this
band by immunological methods. This means that the
activator behaves like a complex of the two reactants[56].
The molecular weight calculated from sedimentation
analysis corresponds to the sum of the molecular weights
of plasminogen (or plasmin; cf. Fig. 2) and streptokinase[821.
The activator is a relatively IabiIe intermediate product;
for that reason traces of plasmin always appear, whatever
its composition. Moreover, the complex remains stable
for several days at O'C but disintegrates within a few
hours at 37°C[831.The plasmin formed catalyzes the degradation of the activator by hydrolyzing streptokinase.
The complex is bound together only by physical forces;
it disintegrates under conditions where hydrogen bonds
are broken, e. g. in concentrated solutions of urea. During this dissociation a relatively large amount of plasmin
is formed within a short time, which leads to total hydrolysis of streptokinase to form numerous by-products.
All the available evidence supports our concept of the
mode of action of streptokinase: the intermediate product of the activation of human plasminogen is a complex
[82] E. C. De Renzo, Thrombos. Diathes. Haemorrh., Suppl. 1 ad 6,
134 (1961).
183) N. Heim burEer, unpublished.
94
held together by hydrogen bonds and hydrophobic
forces. As far as its specificity is concerned, it may be
compared with the intrinsic activators (Scheme 3); nevertheless, it is not stable but is catalytically decomposed
by plasmin in a temperature-dependent reaction.Plasmin
and streptokinase combine in the cold to form the activator. Human plasminogen that has previously been inactivated with diisopropyl fluorophosphonate will still
bind streptokinase but is no longer biologically active.
This indicates that the pIasmin and the activator have the
same active site, ie. a reactive serine
It follows that the activator is actually a modified plasmin
which has lost its broad proteolytic action by association
with streptokinase, but at the same time has acquired the
specificity of intrinsic activators. How this complex is
formed from the proenzyme is not yet clear, because
streptokinase appears to possess n o enzyme activity.
3.2.4. Indirect Activation
Fibrinolysis can be stimulated by many chemically defined substances, with often varying pharmacological efficacy. Such an e€fect is exhibited by heparin and its derivatives, a wide range of salicylic and nicotinic acid
derivatives, hormones including their anabolic forms,
and certain antidiabetic
All these have a mode
of action about which little is known. They are regarded
as indirect fibrinolytic agents, because some of them are
not active in vitro. Heparin and its derivatives are used
extensively in the prophylaxis and therapy of thromboembolic diseases; the nature of the fibrinolytic action
of heparin has not yet been discovered. Derivatives of
nicotinic acid act by mobilizing endogenous factors, and
those of salicylic acid either activate plasminogen or
block antiplasmins. The salicylic acid derivatives have
widely varying structures and display activities measurable in vitr0[57,8s-s71.
It may be assumed that metabolic
stimulation and a shift in the metabolic equilibrium constitute the basis of the action of the hormones including
their anabolites, and also of antidiabetic drug^[^^-^^].
4. Inhibitors of Blood Clotting and Fibrinolysis
Both blood clotting and fibrinolysis are under the control
of inhibitors that are capable of blocking activation at
various stages.
According to a no longer contemporary classification,
human serum contains six antithrombins1116~,
all of them
proteins (Table 2 ) . Only two of them are thrombin in1841 L. Summaria, B. Hsieh, W. R . Groskopf, and K. C. Robbins, J.
Biol. Chem. 242, 5046 (1967).
[85] K. N. von Kaulia: Chemistry ofThrombolysis: Human Fibrinolytic
Enzymes. C. Thomas Publ., Springfield (USA) 1963.
[86] K. N. von Kaulla, Arzneimittelforsch. 15, 246 (1965).
(871 K. N. von Kaulla, Federation Proc. 25,57 (1966).
1881 G. R. Fearnly, Brit. Med. Bull. 20, 185 (1964).
[89] G. R. Fearnly, R. Chakrabarti, and C. T. Vincent, Lancet 11, No.
7151 622 (1960).
1901 R . Chakrabarti, G . R . Fearnly, and E. D. Hocking, Brit. Med. J.
No. 5382 534 (1964).
[91] R. Holemans, Amer. J . Physiol. 208, 511 (1965).
Angew. Chem. internar. Edit. / Vol. 10 (1971) / N o . 2
Antithrombin 1111951and a , - m a ~ r o g l o b u l i n [are
~ ~ ~the
most potent inhibitors found in blood; they are thrombin
inhibitors in the strict sense. They were isolated only a
few years ago and found to be glycoproteins (Table 3);
in Table 2 they are still listed under a general name (antithrombin I11 or progressive antithrombin), because
they couId not previously be estimated separately. Together they are capable of inhibiting amounts of thromIt may
bin four to five times that present in the blo~d[~’l.
be recalled that these substances belong to the type of
progressive inhibitors which do not neutralize thrombin
immediately but progressively at a rate of inactivation
that is constant during the first few minutes. Heparin
accelerates the neutralization of thrombin by antithrombin very considerably by catalysis. In Table 2 this effect
is listed as antithrombin I1 or heparin cofactor; originally
it had been ascribed to another protein before we were
able to show that antithrombin I1 and 111 are identical
proteinsIg51.
hibitors in the strictest sense. The functional relationships can be recognized from the nomenclature of the
antithrom bins.
Table 2. Antithrornbins of human serum.
Antithrombin
Origin or action
I
Fibrin (adsorption effect)
Heparin cofactor
Progressive antithrombin [a]
Reaction product of prothrombin activation
Pathological increase of immunoglobulins:
antibody
Degradation products of fibrinogen
11
111
lV
V
VI
[a] See also Table 3.
Fibrin has been called antithrombin I because it is capable of binding relatively large amounts of thrornbid9’1.
Degradation products arising from the action of plasmin
on fibrin and fibrinogen are classified as antithrombin
V1[93].Their presence constitutes a diagnostic aid because they are found in blood in cases of pathologically
raised fibrinolytic activity. These hydrolysis products act
by inhibiting t h e polymerization of fibrin. It is of particular physiological interest that plasmin is able to split
off an antithrombin substance from fibrin, for clotting
The following proteinase inhibitors present in human serum, listed together with their physical and chemical
properties in Table 3 , have antiplasmin action: a,-antitrypsin, antithrombin 111, the Ci-inactivator, and a2macroglobulin. All these received their names from the
most important or the first biological property discovered
(Table 4).
Table 4. Enzymic specificity of inhibitors.
Inhibitors
Enzymes inhibited
chymotrypsin
trypsin
u,-Antitrypsin
+
a,-Antichymotrypsin
Inter-a-trypsin inhibitor +
Aotithrombin I l l
+
Cl- iniictivator
weak
a,-Macroglobulin
+
plasmin
thrombin
kallikrein
Ci-esterase
-
+
+
+
-
-
-
+
weak
-
-
-
-
-
-
weak
+
+
+
+
+
-
+
-
+
+
-
+
-
a2-Globulins account for ca. 10%of the antiplasmin activity of human serum. a,-Antitrypsin is the most important antiplasmin - it inactivates plasmin irreversibly like
a progressive inhibitor. Human serum is capable of exTable 3. Concentration and properties of inhibitors in human serum.
1
I
Conc. in humai
Carbohydrate
content
mgllOO ml
(%I
Inhibitors
a,-Antitrypsin
a,-Antichymotrypsin
Inter-a-trypsin inhibitor
A_ntithrombin I l l
C 1-inactivator
q-Macroglobulin
290.0
48.7
50.0
29.0
23.5
260.0
+ 45.0
+
6.5
+
2.9
3.0
+
12.2
24.6
8.4
13.4
34.1
7.1
+ 70.0
701.2
I
I
and fibrinolysis are linked through this reaction in compensatory fashion. Antithrombin IV is set free during
prothrombin activation[94]in proportions related to the
quantity of thrombin generated. This neutralization of
thrombin is based on a feedback mechanism. Antithrombin V[’”I is a pathological factor probably identical with
an antibody against thrombin.
Angew. Chem. internat. Edit. / Vol. 10 (1971) / N o . 2
[92] W. H. Seegers, M. Nieft, and E.C. Loomis, Science 101, 520
(1945).
1931 S. Niewiarowski, 2. LataIIo, and J. Stachurska, Rev. Haemat. 13,
320 (1958).
I941 W H. Seegers, J. E Johnson, and C. Fell7 h e r . J. Physiol. 176,
97 (1954).
1951 N. Heimburgei, 1st Inrernat. Symp.stn Tissue Factors in the Homeostasisof the Coagulation-FibrinolysisSystem. Florence 1967, p. 353.
1961 M. Sreinbuch, C. Blafrix, and F. Josso, Nature 216, 500 (1967).
95
hibiting 30 times as much antiplasmin activity as
potential plasmin activity.
is known to hydrolyze fibrin and fibrinogen while at the
same time setting free antithrombin VI which blocks the
polymerization of fibrin. Finally, plasmin is capable of
catalyzing the liberation of kinins from kininogen, an a2globulin of human pla~ma['~'1.Here too the reaction
product is biologically highly active, since it is a vasodilator, increases capillary permeability, and promotes the
migration of leukocytesI'08]. These events characteristic
of inflammations demonstrate how a narrowly circumscribed biological phenomenon can express itself through
many complicated chemical reactions. It should be mentioned that clotting factors I, V, and VIII are initially
inactivated when fibrinolysis overshoots, but antiplasmins prevent the attack by plasmin on other plasma
proteins or biological factors.
Two of the plasmin inhibitors, a,-macroglobulin and antithrombin 111, also possess thrumbin specificity, an observation which is of particular physiological interest.
Hence these two proteins are able to control the
equilibrium between clotting and fibrinolysis: the
quantity expended during an increased release of
thrombin is compensated by its not being available as
an antiplasmin. The biological result is an increase in fibrinolysis.
The lack of specificity of proteinase inhibitors present in
the blood is a phenomenon exploited by the body for
the regulation of other proteolytically controlled reactions. These proteinases have many amino-acid sequences in common, including those at the active site,
thus making them broadly specific[99].This fact applies
to trypsin, thrombin, chymotrypsin, and elastase; a phylogenetic relationship is obviously expressed. Therefore,
it appears only logical that the reactions regulated proteolytically in the blood are also linked through these
enzymes.
Inactivation and also generation (antithrombin VI) of
clotting factors should be looked upon as biological measures designed to prevent reclotting after fibrinolysis. Finally, it should be stressed once more that the broad
specificity of proteinases in blood is apparent in all these
reactions.
Various drugs may intervene in the processes just described. Prothrombin synthesis may be influenced by
vitamin K antagonists of the dicoumarol type, and complexing agents can prevent clotting because the calcium
ion is the essential catalyst of the most important stages
of the clotting mechanism.
It can be seen in Scheme 4 that the Hageman factor
(F XII) activates not only the coagulating but also the
fibrinolytic mechanism~lOO~lO*~
and the kallikrein-kinin
system['02-106~.
Neither thrombin nor plasmin are strictly
specific enzymes: thrombin converts fibrin into fibrinogen and hydrolyzes fibrin as thrombin E[45,1181.
Plasmin
Clotting pathway
Katlikrein-kinin system
Fibrinolytic pathway
Surface contact
Plasma - kallikreinogen
Fibrinogen
4
I
L
.................
t
E
Fibrin
Kininogen
6
Kinin
a
I
Fibrin
_ _ _Thrombin
_ _ _ _ _ _ _ _ _I_ _ _ _ _ _ _ _ _ _ _ - - - - - -
7
' Fibrinopeptides
I
J
Scheme 4 . Interaction of physiologically important enzyme systems in blood.
[97] H. G. Schwickand N. Heimburger, 12. Tagungd. Dtsch. Arbeitsgemeinschaft f. Blutgerinnungsforsch.,Deidesheim 1968.
[98] H. G. Schwjck, N. Heimburger, and H. Haupt, 2. inn. Med. 21, 1
(1966).
[99] D. E. Koshland, Science 142, 1533 (1963).
[loo] V. Eisen, Brit. Med. Bull. 20, 205 (1964).
[loll R.W. Colman,Biochem Biophys. Res. Commun. 35,273 (1969).
[I021 H. Kraur, E. K . Frey, and E. Werle, Hoppe-Seylers Z . Physiol.
Chem. 189, 99 (1930).
96
[lo31 E. Webster and J. Innedield, Enzymol. Biol. Clin. 5, 129 (1965).
[lo41 G. P. Lewis, J. Physiol. 140,285 (1958).
[lo51 R. W. Coleman, L. Matffer, and S. Sherry, J. Clin. Invest. 48,
11 (1969).
[lo61 R. W. Cofernan, L. Mattler, and S. Sherry, J. Clin. Invest. 48,
23 (1969).
[lo71 G . P. Lewis, J. Physiol. 140, 258 (1958).
[lo81 E. K. Frey, H. Kraut, and E. Werle:Das Kallikrein-Kinin-System
u. seine Inhibitoren. Enke-Verlag, Stuttgart 1968.
Angew. Chem. internat. Edit. / Vo1. 10 (1971) / N o . 2
5. Biochemid Aspects of Thrombolysis Therapy
Heparin is administered in the conventional treatment
can be treated by hydrolyzing the clot [1151 (Fig. 6). Recent heart infarctions promise to become another important indication.
of thrombosis. It is known to accelerate catalytically the
neutralization of thrombin by antithrombin 111, and thus
prevent reclotting following fibrinolysis. A lipoprotein
lipase with the functional name of “clearing factor” is
released following an injection of heparin[109-113].The
nature of the effect of heparin on the fibrinolytic mechanism is still unknown.
The fact that thrombosis is a vascular occlusion consisting
predominantly of fibrin and therefore accessible to proteolysis suggests the following therapeutic possibilities:
local application of enzymes to hydrolyze the clot; infusion therapy with enzymes; infusion therapy with
fibrinolysis activators.
For local application the cIot should be compact and accessible, so that the activator or. enzyme solution can
reach it through the catheter, thus by-passing inhibitors
in the plasma. The reason why activators by themselves
are so effective is the relatively large quantity of plasminogen that is adsorbed on the fibrin of the clot. Intrinsic substances such as plasmin are most suitable as
enzymatic hydrolyzing agents, because immunologically
foreign enzymes may stimulate the production of antibodies.
Treatment of thrombosis by injecting proteases at a distant site has little chance of success. The level of proteinase inhibitors in blood is too high, and it would not
be sound biological sense to take a gamble. A generalized
proteolysis would be the result, because proteinases are
not sufficiently specific.
Infusion therapy with activators has been well tested clinically; in fact, the mechanism is found in the body. At
first the obvious attempt was made to use endogenous
urokinase for treatment, but experiments have shown
that as much as 400 liters of urine yield only a single
dose for treatment. Thus preparation of urokinase requires the manipulation of large volumes of urine which,
owing to the limited stability of urokinase, must be processed fairly quickly and by applying complicated methods. These procedures make urokinase very costly and
it is not yet available in amounts sufficient for therapy.
In the meantime, the use of streptokinase has been developed. Now, ten years later, the necessary experimental and clinical experience is available for using
streptokinase in treatment[114]:the indications and contraindications are known; with the exception of a few
special cases, the dosage has been largely standardized
and with it supervision of treatment.
Acute arterial and venous occlusions have proved to be
sure and early indications. There are now pointers that
so-called chronic arterial occlusions, several months old,
Angew. Chem. internat. Edit. / Vol. 10 (1971) / N o . 2
a
b
Fig. 6. Chronic obliteration of the common iliac artery and the femoral
artery (left) a) before and b) after treatment with streptokinase (X-ray
photograph by Dr. M. Martin, Aggertalklinik, Engelskirchen, Germany).
The dose of streptokinase is chosen so that the activator
is the predominant product. Application is by infusion
through continuous drip lasting several days. The advantage of this method of administration is that the hemostatic equilibrium remains largely undisturbed. A short
period of plasminemia occurs at the beginning of the
treatment, until the plasminogen and streptokinase are
present in equimolar proportions. That is why a prolongation of the plasma thrombin time may be observed
shortly after the onset of thrombolysis, which has been
caused by the release by plasmin of antithrombin VI
from fibrinogen. When the thrombin time becomes normal again after 4-6 h, the fibrinolysis is known to
proceed smoothly.
Research on fibrinolytic agents tends toward finding oral
prophylactics against thrombosis in the hope of bringing
these endeavors to a successful conclusion.
Received: May 8, 1970 [A 800 I€]
German Version: Angcw. Chemie. 83.89 (1971)
Translated by Express Translation Service, London
[lo91 C. 8. Anfinsen, E. Boyle, and R.K. Brown, Science 115, 583
(1952).
[IlO] E. D. Korn, Science 120, 399 (1954).
[111] E. D. Korn, J. Biol. Chem. 215, l(1955).
[I121 E. D.Korn, J. Biol. Chem. 226, 827 (1957).
[I131 E. D. Korn and 7. W. Quigfey, J . Biol. Chem. 226, 833 (1957).
[114] Behringwerk-Mitteilungen, No. 41 (1962), No. 44 (1964), No.
48 (1967).
Lll5J W. Schoop, M. Martin, and E. Zeifler, Dtsch. Med. Wschr. 93,
2321 (1968).
I1161 C. N. Fell, N. A. Ivanovic, S. A. Johnson, and W. H. Seegers,
Proc. SOC.Exp. Biol. (N.Y.) 85, 199 (1954).
[117] A. Loelingerand J. F. Hers,Thrombos. Diathes Haemorrh. 1,499
(1957).
(1181 R.H. Landaburcland W. H.Seegers,Proc.Soc. Exp. Biol. (N.Y.)
94, 708 (1957).
97
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