Tài liệu Báo cáo khoa học: Antiplasmin The forgotten serpin? doc

Although free antiplasmin is an excellent inhibitor of plasmin, the fibrin bound form of the serpin appears to be the major regulator of clot lysis.. Abbreviations AP, antiplasmin; PAI-1,

The forgotten serpin?

Paul B Coughlin

Australian Centre for Blood Diseases, Monash University, Prahran, Australia

Introduction

The fibrinolytic system is clearly important in human

biology and its major components are highly conserved

through vertebrate evolution It is therefore very

sur-prising that it is so hard to find good evidence of

genetic anomalies in the fibrinolytic system commonly

associated with thrombotic or other diseases Patients

deficient in plasminogen suffer from ligneous

conjunc-tivitis but not thrombosis [1,2] while there have been

no convincing reports of tissue plasminogen activator

(tPA) deficiency associated with thrombosis On the

other hand mice rendered deficient in the fibrinolytic

proteases urokinase plasminogen activator (uPA), tPA

and plasminogen demonstrate significant phenotypes,

particularly when challenged with thrombotic or

inflammatory stimuli Regulators of fibrinolytic

pro-teases should be important in thrombolysis and indeed variation in the levels of plasminogen activator inhib-itor-1 (PAI-1) appear to be important in the genesis of atherothrombotic disease (reviewed in [3]) It may be that these effects relate more to the role of PAI-1 in regulating cell growth and migration rather than any direct relationship to fibrinolysis While there is no evi-dence for variation in the level of antiplasmin (AP) playing a part in thrombotic disease, complete defici-ency causes a variable, but often severe, bleeding dis-order [4]

Although at a clinical level it is unclear how import-ant fibrinolytic abnormalities are in pathological clot formation, it is well known that the rate and complete-ness of clot lysis play a role in determining patient outcomes On the venous side of the circulation partic-ularly the persistence of clot burden in leg veins is

Keywords

antiplasmin; fibrinolysis; plasminogen;

serpinF2

Correspondence

P Coughlin, Australian Centre for Blood

Diseases, Monash University, Level 6,

Burnet Tower, Commercial Road, Prahran,

3181, Australia

E-mail: Paul.Coughlin@med.monash.edu.au

(Received 9 May 2005, accepted 25 July

2005)

doi:10.1111/j.1742-4658.2005.04881.x

Much of the basic biochemistry of antiplasmin was described more than

20 years ago and yet it remains an enigmatic member of the serine protease inhibitor (serpin) family It possesses all of the characteristics of other inhibitory serpins but in addition it has unique N- and C-terminal exten-sions which significantly modify its activities The N-terminus serves as a substrate for Factor XIIIa leading to crosslinking and incorporation of antiplasmin into a clot as it is formed Although free antiplasmin is an excellent inhibitor of plasmin, the fibrin bound form of the serpin appears

to be the major regulator of clot lysis The C-terminal portion of anti-plasmin is highly conserved between species and contains several charged amino acids including four lysines with one of these at the C-terminus This portion of the molecule mediates the initial interaction with plasmin and is a key component of antiplasmin’s rapid and efficient inhibitory mechanism Studies of mice with targeted deletion of antiplasmin have con-firmed its importance as a major regulator of fibrinolysis and re-empha-sized its value as a potential therapeutic target

Abbreviations

AP, antiplasmin; PAI-1, plasminogen activator inhibitor-1; PEDF, pigment epithelium derived factor; serpin, serine protease inhibitor; tPA, tissue plasminogen activator; TUG, transverse urea gradient; uPA, urokinase plasminogen activator.

associated with post phlebitic symptoms and an

increased risk of recurrence [5] while residual

pulmon-ary artery emboli lead to pulmonpulmon-ary hypertension,

right heart failure and death

Antiplasmin – the serpin

Like so many other molecular systems in biology,

fibrinolysis is organized on a surface, assembling and

orienting key components in close proximity With

respect to intravascular clot the framework for

assem-bly is provided by fibrin polymers and fibrils (Fig 1)

Both tPA and plasminogen bind fibrin leading to

enhanced rates of activation Plasmin(ogen) is also

protected from inactivation by AP because the lysine

binding domains are occupied by fibrin Conversely,

both PAI-1 and AP bind to fibrin during clot

forma-tion PAI-1 binds via vitronectin which is incorporated

into the clot and AP is crosslinked to fibrin by Factor

XIIIa Thus fibrin facilitates two opposing processes,

namely, the assembly and activation of profibrinolytic

enzymes and the binding of antifibrinolytic serpins

How the balance between these pro- and

antifibrino-lytic factors plays out on the fibrin surface is unclear

If there is an equilibrium between free and

fibrin-bound enzymes (tPA and plasmin) within the clot then

the inhibitors (PAI-1 and antiplasmin) are well placed

to regulate the unbound, unprotected proteases

AP is present in plasma at a concentration of

approximately 70 lgÆmL)1 (1 lm) and is the principal

physiological inhibitor of plasmin which is present in

plasma at a concentration of 2 lm [6] It forms a

con-ventional serpin-enzyme complex with plasmin but its

activity is modified by the presence of N- and

C-ter-minal extensions that are unique among the serpin

family The rate of association of AP with plasmin is

extremely fast at 2· 107mol)1Æs)1 This is comparable

to the rate of association of antithrombin and

throm-bin in the presence of unfractionated heparin AP

interacts with plasmin in a two stage process There is

an initial reversible association between the AP

C-ter-minal extension and the kringle domains of plasmin

followed by the formation of an irreversible serpin–

enzyme complex (reviewed in [7])

AP possesses the conserved core structure of the

serpin family of proteins Its functional importance

is highlighted by the highly conserved amino-acid

sequence between species Human AP is 80% identical

with the bovine protein and is 74% identical with the

murine homolog This sequence conservation includes

the reactive centre loop with the P1-P1¢ Arg-Met

identi-cal and strong conservation in both the N- and

C-ter-minal extensions Phylogenetically AP is most closely

related to the noninhibitory serpin pigment epithelium derived factor (PEDF) and these two are grouped together in the F clade [8] AP and PEDF are located together on chromosome 17p13.3 AP and PEDF share

a common intron-exon structure except at the N-termi-nus AP has 10 exons [9] while PEDF has eight exons The intron–exon structure is conserved between the

A

B

C

Fig 1 Schematic representation of the assembly of fibrinolytic pro-teins (A) In the absence of fibrin clot formation the principal fibrino-lytic proteins are free in plasma (B) Upon activation of coagulation the fibrin clot forms Antiplasmin is crosslinked by its N-terminus to fibrin tPA and plasminogen assemble on fibrin leading to the gen-eration of plasmin tPA can be inhibited by PAI-1 either in solution

or at the fibrin surface Kringle domains on plasmin allow binding to lysine residues on fibrin or alternatively in the C-terminus of anti-plasmin (C) While plasmin remains bound to fibrin it is relatively protected from antiplasmin and fibrinolysis occurs Antiplasmin is crosslinked to the fibrin surface and is well placed to inactivate free plasmin in this microenvironment.

two genes, except at the N-terminus where two

addi-tional exons are inserted between exons 2 and 3 of

PEDF corresponding to the AP N-terminal extension

The intron–exon structure of AP is conserved between

Homo sapiens and Mus musculus in keeping with the

high overall sequence homology

AP deficiency in humans has been well described

although only five variants have been characterized at

the molecular level [4] AP Enschede is caused by an

alanine insertion at position 366 (between P10and P11)

lengthening the proximal hinge of the reactive centre

loop and causing the serpin to behave as a substrate

rather than inhibitor [10–12] Reactive centre loop

length is known to be critical for correct inhibitory

function of serpins and insertions in other members

has resulted in similar disruption of function [13] AP

V384M is a substitution in strand 1C just C-terminal

to the reactive center loop (P8¢) and is analogous to a

mutation in C1-inhibitor (V451M) which leads to

instability and polymerization AP Okinawa (Glu149

deletion) disrupts the beginning of helix E and is

there-fore likely to cause malfolding and deficiency The

other two antiplasmin variants are caused by frame

shift mutations

Heterozygosity does not appear to have a clinical

phenotype but homozygous deficiency leads to a

vari-able but often severe bleeding disorder related to

excessive fibrinolysis [11,14] In contrast, targeted

dele-tion of the AP gene in mice has been performed and

produces a remarkably normal phenotype with no

effect on fertility, growth, development or

post-trau-matic bleeding [15] It was noted that AP–⁄ – mice had

significant residual plasmin neutralizing activity (22%)

presumably related to other plasma protease

inhibi-tors When the AP–⁄ – mice were injected with

pre-formed clot to induce pulmonary embolism the

clearance of emboli from the lungs was markedly

enhanced in AP deficient animals Surprisingly the

acceleration of clot clearance occurred irrespective of

whether the thrombi were derived from wild type or

AP–⁄ – mice In a separate report where pulmonary emboli were induced in situ, AP–⁄ –mice had a reduced mortality compared to wild type (42% vs 69%) [16] The same experiment in plasminogen deficient mice was universally fatal When purified human AP was infused into AP–⁄ – mice the mortality was identical to wild type

N-terminal extension There are differences in the literature with respect to the amino-acid numbering of the AP protein although there is general agreement that the signal peptide cleavage occurs at the Asp-Met bond 27 resi-dues from the translation start site giving rise to a mature N-terminus of MEPL- (Fig 2) In keeping with the convention with other secreted serpins this review will use numbering from the mature N-ter-minal methionine However, 70% of circulating AP

is truncated at the N-terminus by a further 12 amino acids giving rise to the Asn form [17] Even with this additional 12 amino-acid deletion AP possesses

a 42 residue N-terminal extension before the start of the first conserved secondary structural element of the serpin core (the A-helix) During clot formation the N-terminus of AP is crosslinked to fibrin by Factor XIIIa Gln14 appears to be the main target for trans-glutamination as it is labelled preferentially using the small molecule substrate [14C]methylamine

by Factor XIIIa and mutant serpin lacking Gln14 is poorly crosslinked to fibrin [18] The functional importance of crosslinking to AP was illustrated by Aoki et al [19] in experiments using plasma from patients deficient in AP It was shown that clot lysis was accelerated and when AP was added to plasma the rate of lysis was related to the amount of inhib-itor crosslinked to the clot and was relatively insen-sitive to free AP

Fig 2 Homology of human, bovine and murine antiplasmin at the N- and C-terminus Homology diagram showing conservation of the first 54 residues of the antiplasmin N-terminus and final 55 residues at the C-terminus Signal peptide cleavage gives rise to the mature N-terminus (MEPL-) Further cleavage at position 12 (.) gives rise to the Asn form of the protein The preferred target for trans-glutamination is Gln14 (m) The conserved Cys43 situated 12 residues before the A-helix is shown (n) Conserved residues are shown with a grey background.

As noted above, AP exists in two forms at the

N-terminus and this affects susceptibility for

crosslink-ing to fibrin Lee et al [20] showed that the Asn form

of AP was crosslinked to fibrin 13 times faster than

the native Met form They were also able to

demon-strate a protease in plasma capable of cleaving AP at

position 12 to generate the Asn form although the

reaction appeared to be slow It remains to be seen

whether this represents the physiological mechanism

for modifying the N-terminus

Some attention has been paid to the potential for

disulfide bond formation in AP The serpin has four

cysteine residues at positions 43, 76, 116 and 125 By

comparison bovine AP has three cysteines (lacking 76)

and the mouse has only two (lacking 76 and 125) It

was originally reported that human AP contained two

disulfide bonds [21] but Christensen et al [22]

demon-strated that the protein contains free thiols and

identi-fied a single bond between residues 43 and 116 This

result is much more consistent with the

nonconserva-tion of residues 76 and 125 From a structural point of

view Cys43 lies approximately 12 amino acids

N-ter-minal to the A-helix, the first conserved secondary

structural element of the serpin (Fig 2) Cys116 is

located in the C-D interhelical region although it is

dif-ficult to be certain of the precise structural location as

this is an area of marked variability between serpins

In particular there is very little similarity between AP

and its closest relation PEDF, for which there is a

crystal structure, in this region Cysteine residues in

the C-D interhelical region are relatively uncommon

with the exceptions being the ovalbumin serpins PAI-2,

bomapin, PI-8, ovalbumin and gene-Y [8] It has

been proposed that Cys79 in the PAI-2 C-D

interheli-cal loop can form a disulfide bond with Cys161 at the

bottom of the F helix and that this bond leads to

opening of the A b-sheet and consequent

polymeriza-tion [23,24] In addipolymeriza-tion angiotensinogen possesses a

highly conserved cysteine residue in the C-D

inter-helical region Interestingly this cysteine has also been

shown to be disulfide bonded to a cysteine in the

N-terminal region suggesting that this is a mechanism

for imposing structural constraint on the N-terminus

of serpins with possible functional significance [25]

Christensen et al [22] compared native AP to the

reduced and alkylated form of the protein and

exam-ined structural stability by transverse urea gradient

(TUG) gels and the association rates with trypsin No

difference was found but the sensitivity of TUG gels

would be inadequate to detect the relatively small

effect expected from an additional disulfide bond not

involving core serpin secondary structural elements

The disulfide bond from Cys43 to Cys116 is most

likely to position the N-terminus at the ‘lower’ end of the protein so that after crosslinking to fibrin the react-ive centre loop at the ‘top’ end of the molecule would

be optimally exposed for interaction with plasmin

C-terminal extension

AP possesses a C-terminal extension of 55 amino acids beyond the conserved proline at the end of strand 5 of b-sheet B This is strongly conserved between species with 67% identity between human and bovine and 61% identity between human and mouse (Fig 2) It does not show any similarity to other proteins but con-tains a number of conserved charged amino acids including a C-terminal lysine which is believed to asso-ciate with the lysine-binding domain of plasmin The interaction between AP and microplasmin (lacking kringles with lysine binding domains) is 30–60 times slower than with plasmin (6.5· 105m)1Æs)1 vs 2· 107 mol)1Æs)1) [26] Frank et al [27] studied the interaction

of recombinant AP C-terminal fragment (Asn398-Lys452) and demonstrated high affinity binding to recombinant plasmin kringles 1 and 4 If the C-ter-minal lysine was deleted then the affinity decreased five fold indicating that, while Lys452 was important, other residues contribute significant binding capacity They proposed a sequential zipper-like model for the inter-action of the AP C-terminus with the plasmin kringles Surprisingly, approximately 40% of circulating AP binds plasmin slowly [28] Sasaki et al [29] made the same observation and demonstrated that the slow form was truncated at the C-terminus by at least the final 26 amino acids When this observation is taken together with the fact that 70% of antiplasmin is in the Asn12 cleaved form which is the best substrate for crosslink-ing to fibrin, it implies that only 40% of total circula-ting protein is optimal for both incorporation into clot and rapid inhibition of plasmin

AP as a therapeutic target

AP is clearly a key regulator of the principal clot lys-ing enzyme plasmin and is therefore a rational thera-peutic target The domains within AP that are accessible to manipulation are the N- and C-terminal extensions and the reactive centre loop Lee et al dem-onstrated that the addition to plasma of mutant AP (P1Arg-Ala), which has no plasmin inhibitory activity, accelerated clot lysis by competitively crosslinking to fibrin [30] Similarly, when a monoclonal antibody which bound and blocked the AP reactive centre loop was added to plasma it dramatically enhanced the effectiveness of tPA induced clot lysis [31] Other

approaches to therapeutic manipulation of AP are to

block either the N- or C-terminal extensions which are

required for crosslinking to fibrinogen and interaction

with plasmin, respectively Kimura et al [32] showed

that when plasma was clotted in the presence of

12-mer synthetic N-terminal AP peptide that there

was a marked enhancement of spontaneous and

tPA-induced clot lysis This corresponded to inhibition

of AP incorporation into the clot and was not seen in

plasma deficient in either AP or Factor XIII

The C-terminal portion of AP inhibits fibrinolysis in

two ways; it competes with lysine residues in fibrin for

plasminogen binding and it binds directly to plasmin

kringle domains thereby dramatically enhancing the rate

of AP inhibition Lysine analogues such as EACA and

tranexamic acid compete for binding at the

plas-min(ogen) kringles impairing interaction of plasmin to

both AP and fibrin In vivo the dominant effect of these

analogues is antifibrinolytic presumably because the

ant-agonism of interaction with fibrin is more important By

contrast when a synthetic AP C-terminal 26-mer peptide

was added to plasma there was a twofold increase in the

rate of fibrinolysis probably as a result of a direct

inter-action with plasminogen causing conformational change

and accelerated activation to plasmin [33] This is

con-sistent with the observation that it is clot bound AP

which modulates fibrinolysis and that interaction of free

plasmin and antiplasmin is probably more important in

preventing a systemic lytic state

Whether antiplasmin will be useful as a therapeutic

target remains to be seen There is, however, unmet

clinical need in the area of fibrinolysis An agent that

increased the efficiency of endogenous fibrinolysis

would be useful to assist in the clearance of venous

thrombi Furthermore, calf vein thrombosis is a

com-mon complication of surgery which often becomes

clinically manifest upon extension into proximal veins

Antiplasmin inhibitors which biased in favour of clot

lysis may well be useful either post-operatively or in

long-term secondary prophylaxis for patients with

thrombotic disorders Other situations in which this

approach may be useful is in diseases where fibrin

deposition is a key component of disease progression

such as glomerulonephritis

Despite the fact that arterial and venous thrombosis

are common problems, responsible for major

morbid-ity and mortalmorbid-ity, there are relatively few

antithrom-botic drugs available to the clinician So far the

pharmaceutical industry has mainly focused on the

development of plasminogen activators for use in acute

arterial occlusion Attention is beginning to shift to

PAI-1 as a possible target in view of its role in

athero-thrombotic disease There is however, ample scope for

the investigation of therapeutic approaches using new targets like antiplasmin to broaden the range of agents available to clinicians and their patients

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