COLMAN

Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice http://gateway.ut.ovid.com/gw1/ovidweb.cgi

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Editors: Colman, Robert W.; Clowes, Alexander W.;

Goldhaber, Samuel Z.; Marder, Victor J.; George, James N.

Title: Hemostasis and Thrombosis: Basic Principles and

Clinical Practice, 5th Edition

Copyright 2006 Lippincott Williams & Wilkins

> Table of Contents > Part I - Basic Principles of Hemostasis and

Thrombosis > Section B - Fibrinolysis and its Regulation > Chapter 20 -

Thrombin-Activatable Fibrinolysis Inhibitor AKA Procarboxypeptidase U

Chapter 20

Thrombin-Activatable FibrinolysisInhibitor AKA Procarboxypeptidase

U

Michael E. Nesheim

Judith Leurs

Dirk F. Hendriks

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma

glycoprotein of 401 amino acids that circulates at a concentration

of approximately 5.0 g per mL (1,2). It is the precursor of zinc

iondependent, carboxypeptidase Blike enzyme, designated

TAFIa, that suppresses fibrinolysis. It is activated by proteolysis at

the Arg92Ala93 bond. The amino-terminal portion is an activation

fragment, and the carboxy-terminal portion is the enzyme TAFIa.

Thrombin and plasmin are capable of activating TAFI, but they are

relatively inefficient in doing so. The physiologic activator is

thought to be the thrombinthrombomodulin complex (3). The

gene for TAFI is located on chromosome 13 (4). It contains 11

exons and spans approximately 48 kb of genomic DNA and is

expressed in the liver (5).

As is the case with many complex biologic molecules, TAFI made

its existence known in several laboratories through independent

and unrelated investigations, each of which resulted in a new name


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for the previously unknown entity. As a consequence, the protein

has acquired several monikers, each of which, as learned in

retrospect, refers to the same entity. Among its names are

procarboxypeptidase U (6,7), plasma procarboxypeptidase B (1,8),

procarboxypeptidase R (9), and TAFI (2). The name

procarboxypeptidase U was assigned because the protein is the

precursor of an unstable carboxypeptidase Blike enzyme found in

serum. It was named plasma procarboxypeptidase B because it is

the precursor of carboxypeptidase Blike enzyme homologous to

the carboxypeptidase B precursor found in the pancreas. It was

called procarboxypeptidase R because it has a preference for

removal of arginine, as apposed to lysine, from the carboxy

terminus of proteins and peptides. This distinguishes the enzyme

from the other carboxypeptidase Blike enzyme of plasma, known

as carboxypeptidase N (6). It was named TAFI because it was

discovered in a search for an entity that gives rise to an inhibitor

of fibrinolysis in response to prothrombin activation.

This chapter provides information regarding the presumed balance

between the deposition and removal of fibrin and the role of the

TAFI pathway in it; the activation of TAFI; the mechanisms by

which TAFIa suppresses fibrinolysis; TAFI and the factor

XIdependent pathway of coagulation; assays for TAFI and TAFIa;

the physiologic and pathophysiologic roles of the TAFI pathway;

and an update on the epidemiology of TAFI. Several recent reviews

on TAFI are available

(10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26).

THE BALANCE BETWEEN FIBRINDEPOSITION AND REMOVALThe vasculature system has within it two powerful, well-regulated

systems that operate to both stop blood flow at the site of an

injury and to maintain blood fluidity elsewhere. These systems are

known, respectively, as the coagulation and fibrinolytic cascades.

These systems involve plasma proteins, formed elements of blood,

particularly platelets, and cells lining the blood vessel wall. They

are latent, and therefore their potential is not obvious without

overt stimulation. When triggered, however, they can be very

potent. This point has been demonstrated, for example, in


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chimpanzees that were injected over a 30-second period with trace

quantities of a combination of blood coagulation factor Xa and

procoagulant phospholipid vesicles (27). Within 1 minute, all of the

plasma fibrinogen had been converted to fibrin, and the platelet

count had dropped to zero. This startling and dramatic coagulation

response, which might be expected to be fatal, had no long-term

effects on the animals because it was immediately followed by an

equally potent fibrinolytic response. Within a minute or two, the

circulating level of tissue-type plasminogen activator (tPA) had

risen approximately 800-fold, and all of the fibrin that had been

deposited within the vascular system was solubilized to fibrin

degradation products. These experiments demonstrated, in the

systemic circulation, events that presumably can happen locally

when required to prevent local blood loss or remove

inappropriately deposited fibrin.

A further appreciation of the potential of the coagulation system

can be gained by considering that thrombin added to plasma at 1

NIH U per mL will provide a clot in approximately 15 seconds. The

plasma, however, has in it sufficient prothrombin to generate

approximately 150 NIH U of thrombin per mL if fully activated.

Therefore, were the coagulation system to be fully activated

instantaneously, the blood would be fully gelled within 1 or 2

seconds, and the rate-limiting step would be the polymerization of

fibrin. Likewise, plasmin sustained at a level of approximately 2 nM

will lyse a fibrin clot within approximately 30 minutes. Plasma has

plasminogen in it at a level of approximately 2,000 nM. Therefore,

if the plasminogen were instantly turned to plasmin, a clot could

be expected to be fully solubilized in approximately 2 seconds.

These considerations suggest that the coagulation and fibrinolytic

systems are potentially extremely powerful. They also tend to

rationalize the many levels of control that exist in these systems

so that they can perform their respective functions without doing

untoward damage to the host.

The balance between fibrin deposition and removal is depicted in

Figure 20-1. In response to vascular injury, the coagulation

cascade is upregulated to convert prothrombin to thrombin, which

then converts fibrinogen to fibrin, thereby producing the familiar

blood clot. In response to fibrin, the fibrinolytic cascade can be


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upregulated to convert plasminogen to plasmin, which then digests

fibrin into soluble fibrin

degradation products. When these processes are properly

balanced, the physiologic roles of these systems are realized.

When they are unbalanced, however, bleeding or thrombosis can

occur. The systems are regulated at many levels, most of which

are not depicted in the figure. One very important mode for the

regulation of coagulation, however, is indicated. It involves the

protein C pathway, whereby the thrombinthrombomodulin

complex converts the zymogen protein C to the enzyme, activated

protein C (APC), which, through a negative feedback loop,

downregulates thrombin formation (28). Studies with TAFI have

shown that a similar feedback loop exists on fibrinolytic side of the

balance (2,3). In this case, the thrombinthrombomodulin complex

activates the zymogen TAFI to the enzyme, TAFIa, which

suppresses the fibrinolytic cascade. Because TAFI is activated by

the thrombinthrombomodulin complex, the TAFI pathway provides

an explicit molecular connection between the coagulation and

fibrinolytic cascades, such that activation of the former can

suppress the activation of the latter.


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FIGURE 20-1. The balance between fibrin deposition and

removal. Prothrombin (II) is activated to thrombin (IIa) by the

coagulation cascade, and fibrin deposition occurs.

Subsequently, plasminogen (Plg) is activated to plasmin (PLn)

by the fibrinolytic cascade, and fibrin is removed. The

thrombinthrombomodulin complex (IIa-TM) activates protein C

(PC) and thrombin-activatable fibrinolysis inhibitor (TAFI) to

the enzymes-activated protein C (APC) and TAFIa, which

respectively downregulate the coagulation and fibrinolytic

cascades. The TAFI pathway provides a regulatory link between

the two cascades such that activation of coagulation

suppresses fibrinolysis. FGN, fibrinogen; FDP; fibrin

degradation products.

The importance of the protein C pathway in the regulation of the

coagulation cascade has been demonstrated by the existence of

severe thrombosis in the congenital absence of protein C (28,29)

and the elevated risk of thrombosis in the condition known as

activation protein C resistance, associated with factor VLeiden (30).


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Whether defects in TAFI exist and to what extent they might be

associated with hemostatic abnormalities is not known as yet.

THE ACTIVATION OFTHROMBIN-ACTIVATABLE FIBRINOLYSISINHIBITORTAFI is activated by proteolytic cleavage at the Arg92Ala93 bond

(1,2). The enzyme TAFIa is composed of amino acids 93 through

401 (1). Enzymes known to catalyze this cleavage are thrombin,

plasmin, and trypsin (1,2). The reactions catalyzed by thrombin

and plasmin are very inefficient compared to many other reactions

catalyzed by these enzymes. The efficiency of the reaction

catalyzed by thrombin, however, is stimulated by a factor of 1,250

by thrombomodulin (3). This magnitude of increase accomplished

by thrombomodulin is similar to that obtained in protein C

activation (31). Heparin stimulates the activation of TAFI by

plasmin, but not to the same extent as thrombomodulin stimulates

the thrombin-catalyzed reaction (32). Because thrombomodulin so

potently stimulates TAFI activation by thrombin, the

thrombinthrombomodulin complex is thought to be the physiologic

activator.

The activation of TAFI is calcium iondependent (33). It shows a

monophasic dependence on the calcium ion concentration with a

half maximal effect at a concentration of approximately 0.25 mM.

This is in sharp contrast to the calcium ion concentration

dependence of protein C activation, which is biphasic with a peak

at a calcium ion concentration of approximately 0.25 mM (33,34).

The kinetics of TAFI activation are consistent with what has been

referred to as an enzyme-central, parallel assembly model (3,35).

According to this model, as shown in Figure 20-2, the enzyme

thrombin can bind to either TAFI or thrombomodulin to form the

corresponding binary complexes. These can interact further to bind

the third component (either TAFI or thrombomodulin) to form the

ternary thrombinthrombomodulinTAFI complex, from which the

enzyme TAFIa is generated. Three parameters are associated with

this model: the dissociation constant for the

thrombinthrombomodulin interaction, the Km for thrombin TAFI

interaction, and the kcat or turnover number, of the ternary


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complex. The three respective parameters were evaluated to be 10

nM, 1 M, and 1 per second, respectively (3). The Km number is

high relative to the plasma concentration of TAFI, as is the case

with protein C activation (31). This implies that the rate of TAFI

activation in vivo would be proportional to

the plasma concentration, which has in fact been demonstrated

(36,37). In addition, the relatively low plasma levels of both

protein C and TAFI, compared to their Km values, indicates that

they would not compete appreciably with each other for the

thrombinthrombomodulin complex, and activation of both would

occur simultaneously with little if any interference from each other.

The activation of TAFI has been demonstrated not only with soluble

thrombomodulin but also with the thrombomodulin found in

endothelial cells (38,39). Consistent with a relative lack of

interference with each other, competition for protein C activation

by TAFI on endothelial cells, although present, was only modest

even at concentrations of TAFI several times its plasma

concentration (39). The same was observed regarding the

inhibition of TAFI activation by protein C (38).

FIGURE 20-2. A model of the mechanism of activation of

thrombin-activatable fibrinolysis inhibitor (TAFI) by thrombin

plus thrombomodulin. This is an enzyme-central, parallel

assembly model whereby thrombin (IIa) interacts with either


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TAFI or thrombomodulin (TM) to form the corresponding binary

complexes. These then interact further to form the ternary

thrombinthrombomodulinTAFI complex from which TAFIa is

generated. Thrombin alone will catalyze activation of TAFI, but

thrombomodulin increases the efficiency of the process by a

factor of 1,250.

Thrombomodulin has five recognized domains (40). In order, from

the amino-terminus, they are a lectinlike domain, six tandem

epidermal growth factor (EGF)-like domains, a chondroitin sulfate

rich domain, a transmembrane domain, and an intracellular

domain. The minimal structure required for protein C activation

comprises the fourth, fifth, and sixth EGF-like domains, plus the

small peptide that connects epidermal growth factor domains three

and four (41). This same structure is necessary, but not sufficient,

for TAFI activation (33,42). In addition, the thirteen residues

comprising the third disulfide loop of the third epidermal growth

factor domain are required. Therefore, although the elements of

thrombomodulin structure required for efficient activation of TAFI

and protein C are similar, they are not identical. Two amino acid

residues are particularly intriguing. One is Met388, which is found

in the small peptide that connects the fourth and fifth epidermal

growth factor domains. This residue is essential for protein C

activation, but not for TAFI activation (42). In addition, it can be

oxidized in the presence of neutrophils (43). When this occurs,

activity with respect to protein C activation is lost, but activity in

TAFI activation is retained (33). This, in turn, suggests that in an

inflammatory milieu, a strong shift in the balance between fibrin

deposition and removal could occur in favor of thrombosis, because

the anticoagulant pathway through protein C would be severely

attenuated, but the antifibrinolytic pathway through TAFI would

not. The other residue that stands out is Phe376, which is in the

fourth epidermal growth factor domain. When this residue is

replaced with alanine, activity in TAFI activation is retained, but

activity in protein C activation is lost; therefore, Phe376 appears

very important for protein C, but not for TAFI activation (33).

Because of the differences in elements of structure of


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thrombomodulin required for the two reactions, the potential exists

to create recombinant forms of thrombomodulin that are specific

for either protein C or TAFI activation.

The elements of thrombin structure required for the two reactions

are overlapping, but not identical. Alanine-scanning mutagenesis of

thrombin showed that the activation of both TAFI and protein C

depends on residues of thrombin that map to exosite I, where

thrombomodulin is known to bind (44). Other residues, however,

were identified that are selectively needed for one or the other of

the two reactions. Therefore, a region below the active site was

found to be necessary specifically for protein C activation, whereas

another region above the active site was found to be necessary

specifically for TAFI activation. In addition, mutagenesis studies in

which key tryptophan residues of thrombin were exchanged for

phenylalanine showed that Trp215 of the active site of thrombin is

very important in protein C, but not TAFI, activation, whereas the

opposite is true for Trp60d (45).

The elements of structure of TAFI that confer thrombomodulin

dependence upon its activation have not been identified to date.

They, however, apparently do not map to amino acids comprising

the P6 to P3 positions around the activation cleavage site.

Mutagenesis studies of this region showed that even when several

of these residues were replaced with those found around the

thrombin cleavage site in the chain of fibrinogen, full

thrombomodulin dependence of TAFI activation was retained, and

no catalytic efficiency was lost (46). Remarkably, when the

residues of TAFI around the cleavage site were replaced with those

around the cleavage site of protein C, the mutant TAFI did not

express well, and its activation by thrombinthrombomodulin was

very inefficient.

MECHANISMS BY WHICH ACTIVETHROMBIN-ACTIVATABLE FIBRINOLYSISINHIBITOR SUPPRESSES FIBRINOLYSISTAFIa is a carboxypeptidase Blike enzyme; that is, it catalyzes

removal of basic (arginine and lysine) residues from the carboxy

termini of selected peptides or proteins. This property confers

upon TAFIa its ability to suppress fibrinolysis. When thrombin


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catalyzes the removal of fibrinopeptides A and B from the

amino-termini of the A and B of fibrinogen E-domains, the

cleaved fibrinogen monomers associate noncovalently with the

D-domains of neighboring molecules and the fibrin polymer is

formed, thereby providing the major proteinaceous component of

the familiar blood clot (47). The fibrin polymer is further stabilized

by covalent isopeptide bonds formed between the D-domains of

adjacent fibrin protomers in the clot. These bonds are formed

through the action of factor XIIIa.

In response to fibrin, the vasculature is capable of releasing tPA, a

serine protease enzyme (48). This enzyme then catalyzes

activation of glu plasminogen through a single proteolytic cleavage

to form glu plasmin, also a serine protease. The activation of glu

plasminogen is stimulated by a factor of approximately 500 by

fibrin (49,50), an effect that presumably keeps plasminogen

activation localized to the site of a clot. This stimulation occurs

through a mechanism in which fibrin acts as a template to bind

both glu plasminogen and tPA (50). Once formed, plasmin begins

to digest the clot by catalyzing cleavages after selected arginine

and lysine residues in the , and chain in regions connecting

the D- and E-domains of the fibrin protomers (47,51). These

cleavages expose carboxy-terminal lysine residues in the fibrin

mesh that provide additional binding sites, especially for glu

plasminogen. As a consequence, the cofactor activity of fibrin in

glu plasminogen activation increases by a factor of approximately

3, and glu plasminogen activation is accelerated

(52,53). In addition, the partially cleaved fibrin serves as a

cofactor for a reaction in which a peptide comprising the first 77

amino acids of glu plasminogen and glu plasmin are liberated by a

cleavage catalyzed by plasmin. These reactions produce species

known as lys plasminogen and lys plasmin, respectively. Lys

plasminogen is a much better substrate for tPA than glu

plasminogen (approximately 20-fold) (49,50,54). Therefore, (lys)

plasmin formation is further accelerated. These two phenomena

(upregulation of the cofactor activity of fibrin and generation of lys

plasminogen) comprise potent positive feedback steps in the

process of plasminogen activation. TAFIa interferes with this


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positive feedback by removing the newly exposed carboxy-terminal

arginine and lysine residues as they appear in fibrin (53). It

therefore eliminates the positive feedback steps in plasminogen

activation and slows down the process of fibrinolysis.

TAFIa also functions by modulating the inhibition of plasmin by

antiplasmin. Both glu and lys plasmin are very rapidly inhibited by

antiplasmin, such that free plasmin in plasma has a half-life of

approximately 0.1 second (55). Fibrin, however, attenuates this

effect somewhat, such that the rate constant for inhibition of

plasmin by antiplasmin is reduced approximately threefold by

intact fibrin. As the fibrin is modified by plasmin, the magnitude of

this effect increases an additional 10- to 15-fold, such that the

rate constant for inhibition of plasmin in the presence of

plasmin-modified fibrin is only approximately 2% to 3% of the

value found in the absence of fibrin (56,57,58). As a consequence,

plasmin is highly protected from the inhibitor in the presence of

fibrin, especially when it has newly exposed carboxy-terminal

lysine and arginine residues. The net effect of this is to

substantially raise the steady-state level of plasmin during the

fibrinolytic process, thereby promoting the rate at which the clot is

dissolved (57). TAFIa, by removing the carboxy-terminal lysine and

arginine residues, eliminates most of this protective effect.

Therefore, in the presence of TAFIa, the steady state level of

plasmin is considerably lower than it is in the absence of TAFIa,

and the process of fibrinolysis is prolonged (57).

Another more subtle effect likely occurs directly in the digestion of

fibrin by plasmin. Fibrin is solubilized when the , , and chains

of adjacent protomers within the polymer are cleaved. For adjacent

D- and E-domains on neighboring protomers to be separated, six

cleavages must occur at the same location. Exhaustive digestion,

which includes all such connections, is not necessary for complete

solubilization, because fibrin degradation products can be released

as a family of molecules of various molecular weights, the larger

ones having many D, E connections not severed completely

(59,60). Therefore, fibrin can be completely solubilized with only a

fraction of all D, E connections cleaved through. The cleavages

made by plasmin do not occur randomly, presumably because the

new carboxy-terminal lysine and or arginine residues that result


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from cleavage at one of the , , or tends to bind plasmin and

localize it so that the other chains are cleaved at the same D, E

site (60). As a consequence, relatively few cleavages are needed

to completely solubilize the fibrin. In the presence of TAFIa,

however, this retention of plasmin does not occur to the same

extent, and more cleavages are required to completely dissolve the

fibrin because they occur randomly throughout the fibrin network.

This also can prolong the process of fibrinolysis.

Most of the enzymes of coagulation and fibrinolysis can be

downregulated by protease inhibitors, especially by antithrombin,

antiplasmin, and plasminogen activator inhibitor-1 (PAI-1). TAFIa

is exceptional in this regard in that, to date, no physiologic

inhibitor of it has been reported. Instead, TAFIa spontaneously

loses activity, an effect that presumably represents the means by

which it is physiologically downregulated (6,61). The rate of decay

is highly dependent on temperature, such that at body temperature

the TAFIa half-life is approximately 10 minutes, but at room

temperature it is approximately 2 hours, and at ice temperature it

is indefinitely stable (61). Because TAFIa is unstable, it suppresses

fibrinolysis only transiently, an effect that in part determines the

potency of TAFIa as an antifibrinolytic agent (62). The loss of

activity occurs in the absence of proteolysis, but once TAFIa loses

activity, it is susceptible to proteolysis by thrombin or plasmin at

Arg302 (61,63). Mutagenesis studies have identified a region of

TAFIa comprising residues 302 to 330 to which the tendency to

decay can be attributed (61,63). A naturally occurring

polymorphism is found within this region at residue 325, which

comprises either a threonine or an isoleucine residue (62,64). The

latter variant of TAFIa has a half-life that is double that of the

former; in addition, it is approximately 60% more potent as an

antifibrinolytic agent (62). Approximately 10% of the population is

homozygous for the more stable variant and 40% for the less

stable one (64). Whether pathologic tendencies correlate with one

or the other is currently under investigation.

When the time to lyze a clot is measured in vitro at various input

concentrations of TAFIa, it increases at low concentrations and

eventually appears to reach a plateau (2,3,61,62). Typically, the

time to lyze in the plateau is three to four times that observed in


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the absence of TAFIa. The concentration needed to obtain a

half-maximal prolongation is typically approximately 1 nM, a

concentration which is only approximately 1% of the plasma

concentration of the zymogen TAFI. Therefore, although TAFIa

does not appear to completely eliminate fibrinolysis, even at

relatively high levels, it is very potent in that only a small fraction

of available TAFI needs to be activated to have an appreciable

effect. The magnitude of the maximal prolongation obtained with

TAFIa is directly proportional to its stability. Variants with a

relatively short half-life therefore show only a small maximal

increase, whereas those with a long half-life show the opposite. In

fact, mutagenesis studies have shown that the maximal

prolongation is directly proportional to the half-life of the variant

(61,62). An example is shown in Figure 20-3. This observation had

proven difficult to rationalize, because the expectation is that a

relatively short half-life could be offset with an elevated TAFIa

concentration. Further studies, however, provided an explanation

for this and disclosed a curious property of the fibrinolytic system

and its regulation by TAFIa (65,66). The studies showed that the

TAFIa concentration dependence of lysis prolongation is not best

represented by a saturating function, but rather by relation,

whereby the lysis time increases linearly with respect to the TAFIa

concentration up to some critical value, and then logarithmically

thereafter. Therefore, the lysis timeversus TAFIa concentration

relation does not show a true plateau; it gives only a superficial

impression of a plateau because of the linear-to-logarithmic switch

in the relation. The explanation for this switch is based on the

proposition that so long as TAFIa is present at or above some key

threshold value, fibrin degradation essentially ceases, only to

begin again once TAFIa decays to a level below the threshold

value. The time interval over which the TAFIa level stays above the

threshold is determined by both the initial input concentration of

TAFIa and its half-life for first-order decay. Therefore, although

TAFIa might, in principle, totally suppress fibrinolysis, it does not

appear to do so because it decays. A stable carboxypeptidase

(pancreatic carboxypeptidase B), however, can virtually stop

fibrinolysis if present at a sufficiently high level (66). If TAFIa

were to be generated acutely, and then decay, the effect would be

to delay, but not eliminate, eventual fibrinolysis. If it were to be


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generated chronically, however, such that it were replenished over

time, a situation might exist whereby fibrinolysis would be

eliminated so long as the coagulant stimulus were present.

Whether this can or does occur in vivo is not known, however. This

raises the intriguing possibility, although, that under some

conditions, TAFIa might function as an absolute inhibitor of

fibrinolysis.

FIGURE 20-3. Prolongation of fibrinolysis by variants of

thrombin-activatable fibrinolysis inhibitor (TAFI) with different

half-lives. The time to lyze a clot is prolonged when TAFIa is

included. Pseudosaturation occurs in the relation between the

lysis time and the TAFIa concentration. The maximum

prolongation depends on the half-life of the TAFIa. The data

shown in solid circles was obtained with a TAFIa variant having

threonine at position 325 and a half-life of 10 minutes. The

data shown in solid squares were obtained with a TAFIa variant


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having isoleucine at position 325 and a 20-minute half-life. The

other data were obtained mixtures of the two. They gave

maximal lysis times between the extremes, as indicated in the

insert, where TAFI-TI is the proportion of the sample consisting

of the Ile325 variant. [From Schneider M, Boffa M, Stewart R,

et al. Two naturally occurring variants of TAFI (Thr-325 and

Il-325) differ substantially with respect to thermal stability and

antifibrinolytic activity of the enzyme. J Biol Chem

2002;277(2):10211030.]

The combination of pseudosaturation in the relation between the

time to lyze a clot and the TAFIa concentration, and a tendency for

reversible inhibitors of TAFIa to stabilize it, gives rise to a complex

relation between effects on fibrinolysis and the concentration of

such inhibitors. Therefore, when reversible inhibitors of TAFIa were

examined in vitro for their effects on the time to lyze a clot, they

both prolonged and promoted lysis, depending on the dose

(67,68). Typically, at relatively low concentrations, such inhibitors

actually retard fibrinolysis, sometimes by a considerable margin,

because they stabilize the TAFIa population but do not completely

inhibit all the TAFIa molecules. Only at relatively high

concentrations are they able to sufficiently inhibit the whole

population to overcome the stabilizing effect.

THROMBIN-ACTIVATABLE FIBRINOLYSISINHIBITOR AND THE FACTORXIDEPENDENT PATHWAY OFCOAGULATIONWhen clotting is triggered in whole blood or plasma, a series of

events occur that collectively have been designated, in sequence,

the initiation, propagation, and termination phases (69). In the

initiation phase, events such as platelet activation, factor V and

factor VIII activation, and prothrombin activation at a low level

occur. At the end of the initiation phase, clotting occurs. At this

point, approximately 1% or 2% of the prothrombin has been

activated. This is followed by the propagation phase in which the


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factor XIdependent (intrinsic) pathway is triggered, presumably

through activation of factor XI by thrombin (70), and massive

prothrombin activation occurs within the clot. This is followed by

the termination phase in which the reactions of the coagulation

cascade subside and thrombin is consumed by antithrombin.

Samples of plasma with defects in the factor XIdependent

pathway, such as those with severe hemophilia A or B, display the

initiation phase (69,71), but not the intense thrombin formation of

the propagation phase.

Several investigators noted early on that when clotting and

subsequent fibrinolysis were induced by adding tPA and thrombin

to normal plasmas, or those with defects in the factor

XIdependent pathway, fibrinolysis occurred early in the defective

plasmas (72,73,74). For example, clots made in normal plasma

would lyze under the extant condition in 2 hours, and those made

in the deficient plasmas would lyze in approximately 30 minutes.

The phenomenon was designated premature lysis (72). The

mechanism for this subsequently was shown to be dependent on

the TAFI pathway, in that normal plasma showed premature lysis

when TAFIa was inhibited, and normal lysis could be restored in

hemophilia plasma by promoting TAFI activation (72,74). These

and other studies showed that the massive level of thrombin

transiently formed after clotting in normal plasma is sufficient,

even in the absence of thrombomodulin, to activate enough of the

TAFI pool to subsequently suppress fibrinolysis (71,72,73,74,75).

This suggests the concept that a role of the factor

XIdependent pathway of coagulation is to suppress fibrinolysis

through the activation of TAFI, thereby stabilizing the newly

formed clot. The concept of premature lysis has also led to the

hypothesis that bleeding in hemophiliacs is caused as much by a

failure to trigger the TAFI pathway and therefore suppress

fibrinolysis as it is by the formation of a clot in the first place. This

hypothesis, although very plausible, has yet to be tested in a

systematic way.

ASSAYS FOR THROMBIN-ACTIVATABLEFIBRINOLYSIS INHIBITOR AND ACTIVE


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THROMBIN-ACTIVATABLE FIBRINOLYSISINHIBITORNumerous assays for TAFI and TAFIa have been described, and

some are available commercially. The assays for TAFI are either

immunologic assays based on measurements of antigen or

functional assays based on measurements of the activity of TAFIa

following the complete activation of TAFI by

thrombinthrombomodulin. Some of the immunologic assays are

compromised somewhat because they respond differentially to

various forms of the antigen that can arise because of proteolysis

of it (76,77). In addition, some assays have been shown to

respond very differently to the Ile325/Thr325 isoforms of TAFI

(78). Such assays have been applied to the determination of the

average TAFI concentration and its distribution about the average

in several large populations. Substantial difference in the averages

have been reported by several groups, but all report a fairly broad

concentration distribution (77,79). The individual variations have

been reported to be determined mostly by genetics, as opposed to

by environment (80). The difference in average values reported by

different groups may reflect differences in concentrations assigned

to assay standards rather than real differences between the

populations studied.

Assays for the enzyme TAFIa are based on measuring its

carboxypeptidase Blike function with a variety of substrates.

These assays are complicated by the existence at relatively high

levels of the constitutively active carboxypeptidase Blike enzyme,

known as carboxypeptidase N, in plasma. Its concentration is

approximately 100 nM, which is about 100 times the level at which

TAFIa would have a significant effect on fibrinolysis. Therefore,

detecting TAFIa at levels in the range of 1 nM, for example,

requires a substrate that is highly selective for TAFIa or an

inhibitor that is highly specific for carboxypeptidase N. No

synthetic substances with absolute specificity have been described

to date, but the judicious use of partially selective substrates has

indicated that the endogenous basal level of TAFIa is less than 100

pM (81,82). Another assay has been described for TAFIa that is on

the basis of its ability to downregulate plasminogen activation

(83). In this assay, high-molecular-weight soluble fibrin


18 de 41 24/06/2006 01:13 p.m.

degradation products are incubated for a designated period with

the plasmin sample containing TAFIa. Following this, the residual

cofactor activity of the fibrin degradation products is measured in

cleavage of a fluorescent plasminogen derivative by the vampire

bat plasminogen activator. This assay is very specific for TAFIa, as

opposed to carboxypeptidase N, and it responds to TAFIa in the

sample at levels ranging from 5 to 200 pM. Therefore, it is both

very specific and highly sensitive. The application of it to five

freshly drawn plasma samples from apparently healthy volunteers

showed the basal level of plasma TAFIa to be 11 pM. This is only

approximately 0.01% of the TAFI level in plasma, suggesting very

little systemic activation of the TAFI pathway under basal

conditions.

ACTIVATION OF THROMBIN-ACTIVATABLEFIBRINOLYSIS INHIBITOR IN VITRO ANDIN VIVOIndirect evidence for the activation of TAFI upon clotting in vitro is

provided by the timing of subsequent fibrinolysis when a

plasminogen activator is included in the experiments. The time to

achieve lysis after clotting is considerably reduced when a

carboxypeptidase B inhibitor is included. Quantitatively similar

results are obtained if a monoclonal antibody directed at TAFI that

prevents its activation is included. From such observations, the

conclusion is reached that TAFI activation occurs following clotting

in plasma. Studies to directly measure TAFIa over time, when a

clot is formed through the coagulation cascade and subsequently

lyzed because of included tPA, have shown that TAFIa is formed

shortly after clotting. Its concentration exhibits a transient peak

that decays with a half-life of approximately 10 minutes. The

activator is presumably thrombin, formed after the clot is made.

Some time later, fibrinolysis occurs, and this is accompanied by a

second transient burst of TAFIa activity; in this case, the activator

is presumably plasmin (81). TAFIa generated in the first peak

appears to delay fibrinolysis, but that which occurs in the second

peak does not because it is likely formed too late in the sequence

of events. Evidence for activation of TAFI in spontaneously clotting

whole blood is provided by the transient increase in


19 de 41 24/06/2006 01:13 p.m.

carboxypeptidase Blike activity in serum. This activity is unstable

and therefore appears only transiently (6). Its appearance, in

retrospect, provided the first clue to the existence of TAFI. Studies

in thrombolysis models in animals indirectly indicate that TAFI can

be activated in vivo and that TAFIa can retard fibrinolysis

(76,84,85). A particularly revealing study (76) within an arterial

thrombolysis model in the rabbit showed that a TAFIa inhibitor

included along with tPA could increase the apparent potency of the

activator by approximately threefold. It could also reduce the time

to reperfusion and markedly enhance patency, with no appreciable

increase in bleeding. A similar study in a dog model showed

directly that TAFI is activated during thrombolysis and that this

could be diminished or eliminated with a reversible synthetic

thrombin inhibitor (86). All of these studies together show that

TAFI is activated postclotting in vitro and that it can be activated

in vivo. However, the scope of conditions under which it is

activated in vivo, the extent to which it is activated, and the

duration are not yet known in detail.

PHYSIOLOGIC AND PATHOPHYSIOLOGICROLES OF THE THROMBIN-ACTIVATABLEFIBRINOLYSIS INHIBITOR PATHWAYStudies in vivo and in selected animal models indicate that the

TAFI pathway suppresses the activity of the fibrinolytic cascade

when coagulation is triggered. This observation strongly suggests

that it contributes to the balance between fibrin deposition and

removal. Because the plasma level of TAFI is considerably lower

than the Km value for its activation by thrombinthrombomodulin,

its rate of activation, all other things being equal, would be

expected to be proportional to its plasma concentration. Therefore,

the impact on fibrinolysis could be expected to vary with the

plasma concentration, and this expectation has been confirmed

experimentally (87). Theoretically, therefore, variations in plasma

levels would associate with tendencies to bleed or thrombose.

Whether this occurs has been examined in numerous epidemiologic

studies

(88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107

results of which are summarized in Table 20-1. Among the


20 de 41 24/06/2006 01:13 p.m.

P.387

P.388

P.389

P.390

P.391

findings are the following: (a) elevated levels of plasma TAFI are

found in patients with stroke; (b) men requiring coronary artery

bypass grafting because of stable angina pectoris had a higher

plasma TAFI level than age-matched controls; (c) the restenosis

rate 6 months after percutaneous coronary intervention correlated

with the plasma TAFI level; (d) increased plasma TAFI correlates

with an increased incidence of angina pectoris in France but not in

Northern Ireland; (e) no difference is observed between plasma

levels of TAFI in those who have a myocardial infarction or

coronary death compared to controls, but fewer patients than

controls had a TAFI concentration above the 90th percentile; (f)

oral contraceptives or hormone replacement therapy are variously

associated with changes in plasma TAFI levels; (g) plasma TAFI is

low in patients with liver cirrhosis or dengue hemorrhagic fever;

and (h) in promyleocytic leukemia, plasma TAFI measured as

antigen is normal but measured as activity is low. Studies to date

generally suggest association of the TAFI pathway with various

thrombotic pathologies, but no definitive mechanistic connections

have yet been identified.

TABLE 20-1 OVERVIEW OF STUDIES OF proCPU

(THROMBIN-ACTIVATABLE FIBRINOLYSIS INHIBITOR) AS

A RISK FACTOR FOR CARDIOVASCULAR DISEASE

The TAFI knockout mouse is viable and has no obvious thrombotic

or bleeding phenotype (137). When crossed with a heterozygous

plasminogen knockout, however, a TAFI-deficient phenotype is

clearly evident in models involving both clot lysis and leukocyte

migration in peritoneal inflammation (138). Therefore, in the

context of the partially plasminogen-deficient mouse, the observed

phenotypes with respect to TAFIa deficiency are consistent with

the conclusion that the TAFI pathway modulates fibrinolysis in


21 de 41 24/06/2006 01:13 p.m.

P.392

vivo. The TAFI knockout mouse has been demonstrated to have

readily measured deficiencies in wound healing (139,140). This,

too, could be a consequence of deregulated fibrinolysis, but it also

might be a consequence of other actions of the TAFI pathway.

OTHER POTENTIAL FUNCTIONS OF THEACTIVE THROMBIN-ACTIVATABLEFIBRINOLYSIS INHIBITOR PATHWAYThat the TAFI pathway might have functions other than modulation

of fibrinolysis is plausible because TAFIa is able to target

molecules other than partially degraded fibrin. Therefore, it might

function, like many other members of the carboxypeptidase family

of enzymes, as modulators of other processes.

Targets of TAFIa other than plasmin-modified fibrin have been

identified. It is very active toward bradykinin and some

encephalins, for example (8). It also effectively catalyzes removal

of carboxy-terminal arginine or lysine residues from peptides

associated with inflammation, such as the anaphylatoxins C5a, and

C3a, and thrombin-cleaved osteopontin, which has adhesive and

cell-signaling functions thought to be important in inflammatory

responses (141,142,143). A study by Myles et al. (142) suggested

that the enzyme TAFIa is considerably more efficient than the

constitutively active plasma enzyme carboxypeptidase N in

catalyzing cleavage of peptides with sequences based on

anaphylatoxins, osteopontin, and bradykinin. They also provided

data that indicated in their experimental animal model that TAFIa

was more potent than carboxypeptidase N in preventing a

hypotensive response to bradykinin. They also suggested that

thrombin could upregulate the proinflammatory properties of

osteopontin and that subsequent action of TAFIa could

down-regulate them.

Clinical evidence for a potential role of the TAFI pathway comes

from a recent study by Hovinga et al. on the association between a

functional single-nucleotide dimorphism in the coding region of the

TAFI gene and outcome (survival or death) in meningococcal

disease (144). The dimorphism codes for either threonine or


22 de 41 24/06/2006 01:13 p.m.

isoleucine at amino acid 325 in the TAFI protein. The Ile325

variant of TAFIa is twice as stable and 60% more potent as an

antifibrinolytic than the Thr325 variant. The genotype of survivors

and many of their relatives were determined, as were the

genotypes of relatives of the nonsurvivors. The analysis indicated

that patients whose parents were carriers of the TAFIa Ile325

genotype had a 1.6-fold [confidence interval (CI), 0.7 to 3.7]

higher risk of contracting meningococcal disease and a 3.1-fold

(CI, 1.0 to 9.5) increased risk of dying from the disease compared

with all other genotypes. The mechanistic basis for this can only be

speculated upon at this point, but the observations suggest that

the TAFI pathway might be significant in the response to sepsis.

Two recent reviews have been published on the potential

connections between the TAFI pathway and inflammation

(145,146). In them, evidence suggesting that TAFI participates in

crosstalk between coagulation or fibrinolysis and inflammation is

discussed. However, a definitive understanding of these linkages

remains to be gathered.

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