Heparin Action
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capacities. More recently, our work has provided
convincing physiochemical evidence that antithrombin activity and heparin cofactor activity are
properties of a single molecular species. Furthermore, we have demonstrated by immunoprecipitation of this protein from plasmas that this inhibitor
is responsible for most, if not all, of the thrombin
neutralizing activity and heparin cofactor activity of
normal blood.
We have studied the neutralization of thrombin
by antithrombin, utilizing purfied human protein
preparations, and have demonstrated that the
equivalence point of the reaction occurs at a 1:1
stoichiometric ratio of enzyme to inhibitor. The
addition of heparin to these components does not
appreciably alter the equivalence point, but does
dramatically accelerate the rate of the reaction.
We have also directly monitored the physical
formation of a thrombin-antithrombin complex. As
we expected from our estimates of reaction stoichiometry, the complex has a molecular weight
approximately equal to the sum of the molecular
weights of thrombin and antithrombin. In the
absence of heparin, complex formation occurs at a
relatively slow rate consonant with the gradual
inhibition of enzymatic activity. In the presence of
heparin, complex formation is virtually instantaneous.
The formation of this enzyme-inhibitor complex,
both in the presence and absence of heparin, is
critically dependent upon the active serine center of
thrombin. If this residue is blocked by the addition
of diisopropyfluorophosphate, interaction between
thrombin and antithrombin is totally inhibited. As
we would expect from the known specificity of
thrombin, the reactive site of the inhibitor which
binds the active serine center of the enzyme
contains an arginine residue. Modification of this
group on antithrombin with 213 butanedione or 112
cyclohexanedione virtually eliminates the ability of
this protein to inhibit thrombin in the presence or
absence of heparin.
Furthermore, given the highly acidic nature of
heparin, one would expect that positive groups on
antithrombin such as E-amino lysvl residues form
the binding site for this negatively charged anticoagulant. The chemical alteration of these residues with o-methylisourea prevents the binding of
heparin to this protein and suppresses the accelera-
THE OBJECTIVE of this editorial is to
describe recent progress in our understanding
of the biochemical mechanism of heparin's anticoagulant action and to consider ways in which this new
knowledge may alter our current methods of
monitoring heparin therapy.
During the coagulation of blood, thrombin is
produced from its precursor, prothrombin, by the
concerted action of Factor V and activated Factor
X (Factor Xa) in the presence of a lipid surface
and calcium ions. Thrombin consists of a heavy and
a light polypeptide chain connected by disulfide
crosslinks.' Once this proteolytic enzyme is generated, it specifically cleaves two pairs of unique
arginine-glycine peptide bonds in fibrinogen,2 and
initiates the development of a fibrin clot. The clotting activity of thrombin is critically dependent
upon a group of amino acid residues which constitute its active center region and predominant
among these is a highly reactive serine on the heavy
polypeptide chain.3
Thrombin gradually loses its enzymatic activity
when added to defibrinated plasma or serum. At the
beginning of this century, Contejean,4 Morowitz,5
Howell,6 and others recognized this phenomenon
and postulated that antithrombin, a specific inhibitor of thrombin, must be present in plasma under
normal physiological conditions. By 1916, McLean7
had isolated heparin from the liver as well as the
heart and demonstrated its potent anticoagulant
action. Twenty three years later, Brinkhous et al.8
had shown that the anticoagulant effects of heparin
occurred only in the presence of a plasma
component which they termed heparin-cofactor.
In the 1950's, the work of Lyttleton,9 Waugh and
Fitzgerald,10 and Monkhouse, France, and Seegers,1" indicated that plasma antithrombin activity and plasma heparin cofactor activity are intimately related. These investigators suggested that
heparin acts to accelerate, by 50 to 100 fold, the
rate at which antithrombin neutralizes thrombin. In
1968, this hypothesis was substantiated by Abildgaard12 who obtained small amounts of a purified
human plasma protein which functions in both
From the Department of Medicine, Beth Israel Hospital,
Boston, Massachusetts.
Address for reprints: Robert D. Rosenberg, M.D., Ph.D.,
Department of Medicine, Beth Israel Hospital, 330 Brookline
Avenue, Boston, Massachusetts 02215.
Circulation, Volume XLIX, April 1974
603
604
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tion of inhibitor action by this anticoagulant. However the slow progressive neutralization of thrombin by antithrombin is not appreciably affected.13
Thus antithrombin neutralizes the activity of
thrombin by complex formation via a reactive site
(arginine )-active center (serine) interaction. Hepa.rin binds to the lysyl residue of antithrombin and
accelerates this interaction. The dramatic increase
in the rate of complex formation is most probably
dependent upon a heparin-induced conformational
alteration of the inhibitor which renders the reactive
site arginine more accessible to the active center
serine of thrombin. Indirect evidence for this
heparin-dependent conformational event has been
obtained.
The coagulation cascade is composed of a series
of linked proteolytic reactions. At each stage of this
mechanism, a parent zymogen is converted to a
corresponding serine protease which catalyzes a
subsequent zymogen-serine protease transition.
Thus of the seven proteins directly or indirectly
involved in the conversion of prothrombin to
thrombin, four proteins are ultimately activated to
serine proteases (Factor XIIa, Factor XIa, Factor
IXa, and Factor Xa) and two proteins are cofactors
for these proteolytic events (Factor V and Factor
VIII).14, 15 The characterization of human Factor
VIIa remains to be determined.
Previous investigators have shown that Factor Xa
is neutralized by antithrombin and heparin."'6 17.18
On the basis of this evidence and our knowledge of
the biochemical mechanism of antithrombin action,
we predicted that all serine proteases produced
within the coagulation cascade would be neutralized by this inhibitor and that heparin would
accelerate each of these interactions.
To test this concept, we have examined the
interaction of antithrombin and heparin with
partially purified Factor XIa, a serine protease
which is generated at an early stage of the
coagulation cascade. The physical properties and
substrate specificity of Factor XIa differ markedly
from those of Factor Xa and thrombin. We have
been able to show that Factor XIa is slowly
neutralized by antithrombin in the absence of
heparin and rapidly inhibited by antithrombin in
the presence of this anticoagulant.'9 Furthermore,
Aronson, utilizing antithrombin prepared by us, has
shown that partially purfied Factor IXa is
neutralized by antithrombin and heparin in a
similar fashion (personal communication).
Thus current evidence strongly favors our concept of heparin's multiple actions within the
EDITORIALS
coagulation system and suggests that the remaining
serine proteases of the clotting mechanism might
be inactivated by this acidic mucopolysaccharide
and its protein cofactor. As pure human coagulation factor proteins become available, we hope to
rigorously test this hypothesis for each step of the
coagulation cascade.
This generalized view of the action of heparin
may require a substantial revision of our current
usage of this acidic mucopolysaccharide. Although
the mechanism of heparin's action suggests that in
vivo suppression of serine proteases should determine the adequacy of anticoagulant therapy,
current techniques for monitoring heparin dosage
measure only the extent to which it has "activated"
plasma antithrombin. Present assays quantitate this
phenomenon by determining the degree to which
plasma from a heparinized patient inhibits the in
vitro addition of an arbitrary amount of either
thrombin20 oif Factor Xa21 or else the extent to
which this plasma prevents the in vitro generation
of an arbitrary amount of serine proteases during
kaolin activation (activated partial thromboplastin
time) .22 In either case no attempt is made to judge
the effect of the heparin activated plasma antithrombin in inhibiting the serine proteases of the
coagulation cascade generated within the patient's
circulatory system. This critique of present methodology is pertinent to situations in which either
standard dosages of heparin are employed after
thrombus formation or low dosages are administered
prior to clot development.
Several groups of investigators are establishing
methods for the direct or indirect measurement of
in vivo levels of the serine proteases active in
coagulation. Nossel and his co-workers have developed an assay for the detection of fibrinopeptide
A which is released from fibrinogen by thrombin.Y3
Our laboratory is currently engaged in establishing
techniques for the direct as well as indirect
measurement of in vivo levels of thrombin and
other serine proteases of the coagulation system. As
these and other sophisticated assays become available, we shall shift from our present approach in
which arbitrary amounts of heparin are employed
to grossly inhibit the clotting mechanism to one in
which a minimum dosage is administered to adjust
each stage of the coagulation cascade to a specific
level of activity. This rigorous control of anticoagulant therapy should permit optimally effective usage
of heparin medication with a minimal incidence of
bleeding complications.
ROBERT D. ROSENBERG
Circulation, Volume XLIX, April 1974
EDITORIALS
605
References
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Circulation, Volume XLIX, April 1974
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Heparin Action
ROBERT D. ROSENBERG
Circulation. 1974;49:603-605
doi: 10.1161/01.CIR.49.4.603
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