The coagulation cascade involves a complex series of interactions between coagulation factors that leads to thrombin generation and fibrin clot formation. Originally thought to be initiated by exposure of plasma to damaged tissues, it is now understood that tissue factor initiates coagulation. Tissue factor pathway inhibitor (TFPI) regulates coagulation by inhibiting the tissue factor-factor VIIa complex. The coagulation cascade can be separated into an initiation phase mediated by tissue factor and a propagation phase mediated by factors VIII and IX. Deficiencies in these factors result in hemophilia and impaired propagation of coagulation.

1. Focus Article
The Coagulation Cascade
Guest Contributor: George J Broze, Jr, MD
Introduction
Blood coagulation is part of the hemostatic response to injury and assists in
maintaining the integrity of the vascular system. It involves a complex series of
interactions between protease zymogens, enzymes, and cofactors that leads to the
generation of thrombin and a fibrin clot. Owing to the influence of Schmidt and
Morawitz, it was widely accepted in the first half of the 20th century that the initiating
event in blood coagulation was the exposure of plasma to damaged tissues. The
substance in tissues responsible for this induction of coagulation was initially called
tissue thromboplastin and later tissue factor (factor III). Early studies suggesting an
alternative pathway of coagulation that does not require tissue factor were rationalized
to conform to the prevalent theory of Schmidt and Morawitz. Mounting evidence, in
particular the observation that blood from hemophiliacs apparently clotted normally after
the addition of tissue factor, however, eventually forced a reassessment of the
coagulation mechanism.
In 1964, the "cascade" theory of McFarlane and the "waterfall" theory of Davie and
Ratnoff separated the known coagulation factors into two pathways, the intrinsic and the
extrinsic, which converge at the activation of factor X (FX) with the subsequent

2. Coagulation Cascade
generation of thrombin proceeding through a single, "common" pathway (Figure 1).
The intrinsic pathway, in which exposure of the contact factors (factor XII [FXII], high-
molecular weight kininogen, and prekallikrein) in plasma to a surface leads to the
initiation of coagulation, appeared to be the pathway most important for hemostasis
since it contained factors VIII (FVIII) and IX (FIX), whose deficiencies cause the severe
bleeding of hemophilia. Tissue factor-mediated extrinsic coagulation was relegated to a
lesser role.
Figure 1 Cascade & Waterfall Hypothesis of Coagulation
Surface
HMWK
Prekallikrein
XII XIIa
X
XI XIa
IX IXa VIIa
VIIIa TF
Xa
Va
Prothrombin Thrombin
Fibrinogen Fibrin
The cascade and waterfall hypothesis of blood coagulation. The intrinsic pathway of coagulation
is initiated by exposure of the contact factors (factor XII, prekallikrein, high-molecular weight
kininogen) to an appropriate surface with subsequent activation of factor XI by factor XIIa. The
extrinsic pathway of coagulation is initiated by exposure of factor VIIa to tissue factor. The
requirement for phospholipids and calcium ions in certain of the reactions is not indicated.
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3. Coagulation Cascade
The segregation of the known coagulation factors into the intrinsic and extrinsic
pathways and the availability of assays to test each pathway (partial thromboplastin
time [aPTT] and prothrombin time [PT], respectively) proved invaluable in the diagnosis
of hemorrhagic diseases. Today, these tests still provide the foundation of any
coagulation evaluation. The cascade and waterfall hypotheses, however, failed to
reflect hemostasis accurately. Individuals deficient in one of the contact factors required
for the initiation of intrinsic coagulation are asymptomatic, whereas individuals deficient
in factor VII bleed abnormally. Moreover, if the factor VII(a)/tissue factor (FVIIa/TF)
complex can activate FX, albeit considerably less effectively than FIXa/FVIIIa, why do
hemophiliacs bleed? An important clue to the resolution of this dilemma was provided
in 1977 by Osterud and Rapaport who showed that FVIIa/TF can activate FIX of the
intrinsic pathway as well as FX. Finally, the rediscovery of an endogenous inhibitor of
the FVIIa/TF complex, now called tissue factor pathway inhibitor (TFPI), led to a
reformulation of the coagulation cascade in which tissue factor is responsible for the
initiation of coagulation, but subsequent amplification of the clotting process through the
action of FVIII and FIX is absolutely required for sustained hemostasis 1,2.
Tissue Factor Pathway Inhibitor (TFPI)
In 1947, Thomas and Schneider independently observed that incubation of crude
tissue thromboplastin with serum prevented the lethal disseminated intravascular
coagulation that follows thromboplastin infusion in animals. Later, Hjort showed that the
serum inhibitor recognized the FVIIa/TF complex rather than FVIIa or TF alone. About
the same time, Biggs and her colleagues showed that coagulation was delayed and
incomplete after the addition of low concentrations of TF to hemophilic plasma. This
result contrasted with that of the standard PT assay in which relatively large quantities
of TF are used to initiate coagulation, and in which plasma deficient in FVIII or FIX is
indistinguishable from normal plasma. With the subsequent demonstration of an
"intrinsic" pathway of coagulation, however, interest was diverted away form the
possible connection between Biggs' work and the thromboplastin inhibitor. Finally, a
report by Rapaport and colleagues revitalized interest in the inhibitor in 1985. They
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4. Coagulation Cascade
showed that, in the presence of FX, an agent in the lipoprotein fraction of plasma
produced inhibition of tissue factor-mediated coagulation. Other investigators soon
confirmed this result and found that the properties of this inhibitor were identical to those
of the FVIIa/TF inhibitor described by Hjort in 1957.
TFPI directly inhibits FXa and, in a FXa-dependent fashion, produces feedback
inhibition of the FVIIa/TF catalytic complex. The latter process involves the formation of
a quaternary inhibitory complex that contains FVIIa/TF, FXa, and TFPI. The formation
of this complex is frequently described as a two-step process in which TFPI first binds to
soluble FXa and then the FXa-TFPI complex binds to FVIIa/TF. Instead, kinetic studies
strongly suggest that TFPI interacts with FXa that has not yet been released from the
FVIIa/TF or remains at the phospholipid surface in close proximity to FVIIa/TF3. As a
result, FVIIa/TF inhibition by TFPI is extremely rapid and the concentration of active
FXa and that escapes regulation is linearly dependent on the level of available of TF.
A Revised Coagulation Cascade
1,2
The properties of TFPI led to a revised coagulation cascade (Figure 2).
Coagulation ensues when damage to blood vessels allows the exposure of blood to the
TF produced constitutively by cells beneath the endothelium. The FVII or FVIIa present
in plasma then binds to TF and the FVIIa/TF complex activates limited quantities of FX
and FIX. Some of the FXa generates low levels of thrombin sufficient to activate
platelets and the critical coagulation cofactors FV and FVIII, but TFPI dampens the
clotting process by producing FXa-dependent feedback inhibition of the FVIIa/TF
complex. Persistent and amplified FXa and thrombin generation then proceeds through
the actions of FIXa with its cofactor FVIIIa. Consistent with its regulatory role, inhibition
of TFPI activity has been shown to ameliorate bleeding in an animal model of
hemophilia 4.
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5. Coagulation Cascade
Figure 2 Tissue Factor Pathway of Coagulation
Vessel IX IX
Injury
VII a
TF IX a
TFPI XI a
VIII a
X X
Throm bin XI
Xa
Va TAFI
Throm bin
Prothrom bin
Throm bin TAFI a
Fibrinogen FIBRIN Fibrinolysis
Hemostasis is initiated when factor VII and factor VIIa in plasma gain access to tissue factor at a
site of blood vessel injury. Limited quantities of factor IXa and factor Xa are produced before
feedback inhibition of the factor VIIa/tissue complex mediated by TFPI. The subsequent generation
of factor Xa and thrombin is then amplified through the action of factor VIIIa and factor IXa, the latter
produced initially by factor VIIa/tissue factor and supplemented by factor XIa formed through the
action of thrombin. Thrombin activation of TAFI, which is enhanced by the cofactor thrombomodulin
(TM), inhibits fibrinolysis of the clot. Activated platelets provide a membrane surface critical for
many of the reactions: for example, the activation of factor X by factors IXa/VIIIa; the activation of
prothrombin by factors Xa/Va; and factor XI activation by thrombin.
In this scheme, thrombin generation can be artificially separated into two phases:
initiation and amplification/propagation (Figure 3). In normal blood it is FXa generation
that determines the rate and extent of thrombin production 5. FVIIa/TF is predominantly
responsible for the slow rate of FX activation at the initiation of coagulation. Enhanced
FXa production through the actions of FIXa and FVIIIa leads to a rapid acceleration of
thrombin generation and the amplification phase of coagulation. In hemophilia (FVIII or
FIX deficiency), the duration of the initiation phase is extended and the amplification
phase of coagulation is dramatically reduced when low levels of tissue factor are used
to trigger coagulation. Note that initial fibrin formation (the end-point of clinical
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6. Coagulation Cascade
coagulation assays) occurs at low thrombin concentrations (~10 nM), long before the
bulk of the thrombin has been generated (arrows in Figure 3).
Figure 3 Initiation & Amplification Phases of Coagulation
Thrombin Activity
Time
Depiction of thrombin generation in normal and hemophilic blood following
their exposure to low levels of tissue factor. Arrows denote time of clot
formation
The high rate and level of thrombin produced during the amplification phase of
coagulation, however, is critical for the stability of the clot and hemostasis. This "extra"
thrombin overcomes the effect of coagulation inhibitors, affects the structure of the
developing fibrin network, and reduces the rate of subsequent fibrinolysis (see below).
Depression of the amplification phase of coagulation appears to be responsible for the
delayed hemorrhage that is the clinical hallmark of hemophilia.
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7. Coagulation Cascade
Factor XI and Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)
In the intrinsic pathway of coagulation, activation of FXI by FXII of the contact
system is responsible for the initiation of coagulation. In the revised scheme, it is
FVIIa/TF that triggers coagulation suggesting that FXI functions later in the coagulation
cascade. In 1991, two groups found that thrombin could activate FXI in a FXIIa-
6,7
independent manner . Importantly, Baglia and Walsh subsequently showed that this
thrombin activation of FXI was dramatically enhanced at the surface of activated
8
platelets . FXIa generated at the wound can then produce additional FIXa to
supplement that initially produced by FVIIa/TF and limited by TFPI. The model predicts
that this ancillary FIXa would be most important at sites with limited TF exposure and/or
high fibrinolytic activity. This is consistent with the phenotype of FXI deficiency: overall
a moderate hemorrhagic diathesis, but with a substantial risk of bleeding following
surgical manipulation in the mouth or the urinary tract.
The thrombin generated during coagulation also inhibits fibrinolysis by activating
(TAFI) 9. Clot lysis involves a positive feedback loop. Plasmin cleaves fibrin at sites
following lysine residues. These exposed lysine residues serve to enhance the fibrin
binding of plasminogen, which contains lysine-binding sites. As plasminogen bound to
fibrin is activated to plasmin much more effectively by tPA and uPA than plasminogen in
solution, fibrinolysis is enhanced. Activated TAFI (TAFIa) is a carboxypeptidase enzyme
that proteolytically excises basic (lysine, arginine) residues from the end of
polypeptides. By removing the C-terminal lysine residues from the polypeptides in the
partially digested fibrin, TAFIa interferes with the positive feedback mechanism and
inhibits fibrinolysis. The therapeutic inhibitors of fibrinolysis, ,ε-amino caproic acid
(Amicar®) and tranexamic acid, are mimics of the amino acid lysine and produce a
similar effect by binding to the lysine-binding sites in plasminogen. The premature lysis
of hemostatic clots in hemophiliacs and individuals with factor XI deficiency appears to
be due in part to inefficient TAFI activation 10,11.
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8. Coagulation Cascade
Important New Wrinkles
Figures 1 and 2 only depict the proteins that are involved in the coagulation. It is
important to realize that many of the critical reactions in coagulation occur at the surface
of cells. Although phospholipid vesicles are frequently used to provide the procoagulant
surface for coagulation factor assembly in vitro, they do not completely replicate the
results obtained with cells whose membranes contain a variety of additional
constituents. Activated platelets, which accumulate at the site of a wound, appear to
provide the optimal surface for coagulation reactions and inhibitors of platelet function
affect thrombin generation (e.g. ref. 12). Based on an in vitro cell-based system,
Hoffman, Monroe, Roberts, and their colleagues argue that a major reason for the
bleeding in hemophilia is that FXa formed on the surface of a TF-bearing cell is rapidly
inactivated by proteinase inhibitors as it attempts to transfer to the platelet. In contrast,
FIXa produced by FVIIa/TF is much less susceptible to proteinase inhibitors, reaches
the platelet surface, and there, with its cofactor, FVIIIa, generates FXa in a protected
environment (see ref. 13 for review).
Recent work by Nemerson and his colleagues suggests that TF plays a role not only
in the initiation, but also in the propagation of coagulation (see ref. 14 for review). They
have shown that circulating leukocytes and their shed microvesicles carrying TF bind to
activated platelets at the site of vascular injury and enhance thrombus growth. This
process involves an interaction between P-selectin on the platelet and CD15 (P-selectin
ligand, sialyl Lewis x [sLex]) on the leukocyte-derived membrane (see ref. 15 for review).
Apparently the circulating tissue factor is "encrypted" or at a level below a threshold
needed to initiate coagulation. Interaction of microvesicles/leukocytes with activated
platelets serves to concentrate and, perhaps, de-encrypt the tissue factor at the site of
the developing thrombus. As the delivery of circulating microvesicles/leukocytes is
dependent on flow rate, this process may be most important at sites of arterial injury.
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9. Coagulation Cascade
References
1. Broze GJ Jr, Girard TJ, Novotny WF: Perspectives in biochemistry: Regulation of coagulation by a multivalent Kunitz-type
inhibitor. Biochemistry 1990;29:7539-7546.
2. Broze GJ Jr. The role of tissue factor pathway inhibitor in a revised coagulation cascade. Sem. Hematol. 1992;29:159-169.
3. Baugh RJ, Broze GJ Jr, Krishnaswamy S. Regulation of extrinsic pathway factor Xa formation by tissue factor pathway
inhibitor. J. Biol. Chem. 1998;273:4378-4386.
4. Erhardtsen E, Ezban M, Madsen MT, Diness V, Glazer S, Hedner U, Nordfang O. Blocking of tissue factor pathway inhibitor
(TFPI) shortens the bleeding time in rabbits with antibody induced haemophilia A. Blood Coag Fibrinol 1995;6:388-394.
5. Rand MD, Lock JB, van't Veer C, Gaffney DP, Mann KG. Blood clotting in minimally altered whole blood. Blood
1996;88:3432-3445.
6. Naito K, Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by
thrombin and factor XIa in the presence of negatively charged surfaces. J Biol Chem 1991;266:7353-7358.
7. Gailani D, Broze GJ Jr. Factor XI activation in a revised model of blood coagulation. Science 1991;253:909-912.
8. Baglia FA, Walsh PN. Prothrombin is a cofactor for the binding of factor XI to the platelet surface and for platelet-mediated
factor XI activation by thrombin. Biochemistry 1998;37:2271-2281.
9. Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI. A thrombin-activatable fibrinolysis inhibitor. J Biol
Chem 1995;270:14477-14484.
10. Broze GJ Jr, Higuchi DA. Coagulation-dependent inhibition of fibrinolysis: Role of carboxypeptidase-U and the premature lysis
of clots from hemophilic plasma. Blood 1996;88:3815-3823.
11. Von dem Borne PA. Bajzar L. Meijers JC. Nesheim ME. Bouma BN. Thrombin-mediated activation of factor XI results in a
thrombin-activatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis. J Clin Invest 1997;99:2323-2327.
12. Butenas S, Cawthern KM, van't Veer C, DiLorenzo ME, Lock JB, Mann KG. Antiplatelet agents in tissue factor-induced blood
coagulation. Blood 2001;97:2314-2322.
13. Hoffman M, Monroe DM. A cell-based model of hemostasis. Thromb Haemost 2001;83:958-965.
14. Rauch U, Nemerson Y. Tissue factor, the blood, and the arterial wall. Trends Cardiovasc Med 2000;10:139-143.
15. Vandendries ER, Furie BC, Furie B. Role of P-selectin and PSGL-1 in coagulation and thrombosis. Thromb Haemost
2004;92:459-466.
Address for Correspondence
George J Broze, Jr, MD
Washington University School of Medicine,
Division of Hematology, Box 8125
660 S. Euclid Avenue
St Louis, MO 63110
T: (314) 362-8809, F: (314) 362-8813
e-mail: gbroze@im.wustl.edu
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