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

MINIREVIEW Antiplasmin 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. 4852 FEBS Journal 272 (2005) 4852–4857 ª 2005 FEBS associated with post phlebitic symptoms and an increased risk of recurrence [5] while residual pulmon- ary artery emboli lead to pulmonary 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 · 10 7 mol )1 Æs )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 P 1 -P 1 ¢ 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. P. B. Coughlin Antiplasmin – the forgotten serpin? FEBS Journal 272 (2005) 4852–4857 ª 2005 FEBS 4853 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 P 10 and P 11 ) 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 (P 8 ¢) 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 [ 14 C]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. Antiplasmin – the forgotten serpin? P. B. Coughlin 4854 FEBS Journal 272 (2005) 4852–4857 ª 2005 FEBS 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 addition 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 · 10 5 m )1 Æs )1 vs. 2 · 10 7 mol )1 Æs )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 (P 1 Arg-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 P. B. Coughlin Antiplasmin – the forgotten serpin? FEBS Journal 272 (2005) 4852–4857 ª 2005 FEBS 4855 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 mortality, 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. 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FEBS Journal 272 (2005) 4852–4857 ª 2005 FEBS 4857 . 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. Antiplasmin – the forgotten serpin? FEBS Journal 272 (2005) 4852–4857 ª 2005 FEBS 4855 approaches to therapeutic manipulation of AP are to block either the N-