revision cascada de la coagulacion

FFooccuuss AArrttiiccllee 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

Coagulation Cascade

Broze (2002, updated 2005)CLOT-ED, Inc., Copyright 2005. All rights reserved.

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

ThrombinProthrombin

FibrinFibrinFibrinogen

Va

Xa

VIIaTF

X

VIIIaIXaIX

XIaXI

XIIaXII

Surface

HMWKPrekallikrein

ThrombinProthrombin

FibrinFibrinFibrinogen

Va

Xa

VIIaTF

X

VIIIaIXaIX

XIaXI

XIIaXII

Surface

HMWKPrekallikrein

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.

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

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

The properties of TFPI led to a revised coagulation cascade 1,2 (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.

Coagulation Cascade


Figure 2 Tissue Factor Pathway of Coagulation

Thrombin

Thrombin

Thrombin

Fibrinogen FibrinolysisFIBRINFIBRIN

TAFI

TAFI aa

VesselInjury

Prothrombin

TFPI

IX

X

VIIaTF

XaVa

IXaVIIIa

X

IX

XIa

XIThrombin

Thrombin

Thrombin

Fibrinogen FibrinolysisFIBRINFIBRIN

TAFI

TAFI aa

VesselInjury

Prothrombin

TFPI

IX

X

VIIaTF

XaVa

IXaVIIIa

X

IX

XIa

XI

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

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.

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

Time

Thro

mbi

nA

ctiv

ity

Time

Thro

mbi

nA

ctiv

ity

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.

Depiction of thrombin generation in normal and hemophilic blood following their exposure to low levels of tissue factor. Arrows denote time of clot formation

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-

independent manner 6,7. Importantly, Baglia and Walsh subsequently showed that this

thrombin activation of FXI was dramatically enhanced at the surface of activated

platelets 8. 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.

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.

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: [email protected]

revision cascada de la coagulacion

Education