Decrease in matrix metalloproteinase­3 activity in systemic sclerosis fibroblasts causes α2­antiplasmin and extracellular matrix deposition, and contributes to fibrosis development.

Systemic sclerosis (SSc) is a connective tissue disease of autoimmune origin characterized by fibrosis of the skin and visceral organs, and peripheral circulatory disturbance. α2­antiplasmin (α2AP) is the major circulating inhibitor of plasmin and is a key regulator of fibrinolysis. It has been demonstrated that the expression of α2AP is elevated in dermal fibroblasts obtained from patients with SSc patients. It has also been determined that α2AP is associated with the development and progression of fibrosis in SSc. The present study assessed the relationship between α2AP and matrix metalloproteinase­3 (MMP­3), an extracellular matrix (ECM)­degrading enzyme. Serum levels of α2AP and MMP­3 were measured in healthy controls and patients with SSc using ELISA. No significant differences were determined between these two groups. α2AP, MMP­3 and tissue inhibitor of metalloproteinase­1 (TIMP­1) expression was subsequently evaluated in normal and SSc fibroblasts via western blotting. The results revealed that α2AP expression increased in SSc dermal fibroblasts, while the ratio of MMP­3/TIMP­1 decreased. Additionally, incubation of recombinant α2AP with MMP­3 caused α2AP degradation. The mixture of recombinant α2AP with MMP­3 was subsequently added to normal fibroblasts prior to western blotting. The results revealed decreased α­smooth muscle actin (α­SMA; a marker of the myofibroblast phenotype) and type I collagen expression. The stimulation of SSc fibroblasts with MMP­3 decreased protein levels of α2AP, α­SMA and type I collagen, thus reversing the pro­fibrotic phenotype of SSc fibroblasts. SSc fibroblast transfection with microRNA­29a resulted in a decreased TIMP­1 expression, but also decreased the protein expression of α2AP. The results indicated that MMP­3 attenuated fibrosis progression by degrading α2AP and ECM, and might therefore contribute to a novel therapeutic approach for SSc treatment.

Identification of glycated and acetylated lysine residues in human α2-antiplasmin.

BACKGROUND: The post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues. AIM: We sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin. METHODS: We analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of ß-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM ß-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer. RESULTS: Eleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with ß-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with ß-d-glucose or ASA. Incubation with concomitant ASA and ß-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM ß-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM ß-d-glucose was associated the absence of acetylation at that site. CONCLUSION: Human α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII.

UNLABELLED: Essentials Factor XIIIa inhibits fibrinolysis by forming fibrin-fibrin and fibrin-inhibitor cross-links. Conflicting studies about magnitude and mechanisms of inhibition have been reported. Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots. Cross-links of α2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot. SUMMARY: Background Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links α2 -antiplasmin to fibrin, the topic remains a matter of debate. Objective To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of α2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of α2 -antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of α2 -antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction.

Natural heterogeneity of α2-antiplasmin: functional and clinical consequences.

Human α2-antiplasmin (α2AP, also called α2-plasmin inhibitor) is the main physiological inhibitor of the fibrinolytic enzyme plasmin. α2AP inhibits plasmin on the fibrin clot or in the circulation by forming plasmin-antiplasmin complexes. Severely reduced α2AP levels in hereditary α2AP deficiency may lead to bleeding symptoms, whereas increased α2AP levels have been associated with increased thrombotic risk. α2AP is a very heterogeneous protein. In the circulation, α2AP undergoes both amino terminal (N-terminal) and carboxyl terminal (C-terminal) proteolytic modifications that significantly modify its activities. About 70% of α2AP is cleaved at the N terminus by antiplasmin-cleaving enzyme (or soluble fibroblast activation protein), resulting in a 12-amino-acid residue shorter form. The glutamine residue that serves as a substrate for activated factor XIII becomes more efficient after removal of the N terminus, leading to faster crosslinking of α2AP to fibrin and consequently prolonged clot lysis. In approximately 35% of circulating α2AP, the C terminus is absent. This C terminus contains the binding site for plasmin(ogen), the key component necessary for the rapid and efficient inhibitory mechanism of α2AP. Without its C terminus, α2AP can no longer bind to the lysine binding sites of plasmin(ogen) and is only a kinetically slow plasmin inhibitor. Thus, proteolytic modifications of the N and C termini of α2AP constitute major regulatory mechanisms for the inhibitory function of the protein and may therefore have clinical consequences. This review presents recent findings regarding the main aspects of the natural heterogeneity of α2AP with particular focus on the functional and possible clinical implications.

Effect of fibrin degradation products on fibrinolytic process.

Fibrin clot lysis by plasminogen/plasmin system results in fibrin degradation products formation with subsequent release into bloodstream. The fragments contain specific binding sites for fibrinolytic system components and can interact with them. In this study, we investigated the way in which fibrin fragments effect fibrinolytic process. We have shown that high molecular weight products of fibrin degradation and fibrin fragments of DDE-complex and DD, but not end product Е3, stimulate plasmin formation. Additionally, components of DDE-complex mixture of fragments Е1 and Е2 have potentiation ability. The intermediate fibrin fragments hmFDPs and DDE attenuate clot lysis by plasmin and hmFDPs protect plasmin from α2-antiplasmin inhibition but under further fragmentation to endpoint fibrin fragments loose this ability. The plasma inhibitors reduce fibrinolytic system activity generated by the degradation products. Thus, fibrin fragments formed during the clot lysis can bind and move out fibrinolytic system components from clot volume and in this way result in clot resistance to hydrolysis.

Functional factor XIII-A is exposed on the stimulated platelet surface.

Factor XIII (FXIII) stabilizes thrombi against fibrinolysis by cross-linking α2-antiplasmin (α2AP) to fibrin. Cellular FXIII (FXIII-A) is abundant in platelets, but the extracellular functions of this pool are unclear because it is not released by classical secretion mechanisms. We examined the function of platelet FXIII-A using Chandler model thrombi formed from FXIII-depleted plasma. Platelets stabilized FXIII-depleted thrombi in a transglutaminase-dependent manner. FXIII-A activity on activated platelets was unstable and was rapidly lost over 1 hour. Inhibiting platelet activation abrogated the ability of platelets to stabilize thrombi. Incorporating a neutralizing antibody to α2AP into FXIII-depleted thrombi revealed that the stabilizing effect of platelet FXIII-A on lysis was α2AP dependent. Platelet FXIII-A activity and antigen were associated with the cytoplasm and membrane fraction of unstimulated platelets, and these fractions were functional in stabilizing FXIII-depleted thrombi against lysis. Fluorescence confocal microscopy and flow cytometry revealed exposure of FXIII-A on activated membranes, with maximal signal detected with thrombin and collagen stimulation. FXIII-A was evident in protruding caps on the surface of phosphatidylserine-positive platelets. Our data show a functional role for platelet FXIII-A through exposure on the activated platelet membrane where it exerts antifibrinolytic function by cross-linking α2AP to fibrin.

Increased N-terminal cleavage of alpha-2-antiplasmin in patients with liver cirrhosis.

BACKGROUND: The activity of alpha-2-antiplasmin (α2AP), the main fibrinolytic inhibitor, is modified by N- and C-terminal proteolytic cleavages. C-terminal cleavage converts plasminogen-binding α2AP (PB-α2AP) into a non-plasminogen-binding derivative. N-terminal cleavage by antiplasmin-cleaving enzyme (APCE), a soluble, circulating derivative of fibroblast activation protein (FAP), turns native Met-α2AP into Asn-α2AP, which is more quickly crosslinked into fibrin. OBJECTIVES: We developed two novel enzyme-linked immunosorbent assays (ELISAs) to determine the N-terminal variation of α2AP to test the hypothesis that liver cirrhosis, characterized by increased expression of FAP/APCE, results in increased N-terminal cleavage of α2AP. PATIENTS/METHODS: α2AP and FAP/APCE antigen levels were measured in the plasma samples of 75 patients with cirrhosis with different severities and 30 healthy control individuals. The percentage of N-terminal cleavage of α2AP was calculated. RESULTS: Compared with levels (median [interquartile range]) in control individuals, total PB-α2AP levels and Met-PB-α2AP levels were reduced in cirrhosis patients (27.3 [21.4-41.3] µg mL(-1) vs. 56.2 [49.6-62.8] µg mL(-1) , P < 0.001, and 2.7 [1.7-5.5] µg mL(-1) vs. 12.1 [11.0-15.3] µg mL(-1) , P < 0.001, respectively). Interestingly, the percentage of N-terminal cleavage was increased in the patients (87.8 [85.0-91.6]% vs. 77.2 [72.2-79.8]% in controls, P < 0.001), as well as the plasma FAP/APCE levels (166 [60-550] ng mL(-1) in patients vs. 107 [67-157] ng mL(-1) in controls, P < 0.001). Additionally, all variables significantly correlated with the severity of disease. CONCLUSIONS: Using our novel ELISAs we found increased N-terminal cleavage of α2AP in liver cirrhosis patients, which correlated with the severity of disease and is likely to have reflected the increased FAP/APCE levels in these patients.

In vivo near-infrared imaging of fibrin deposition in thromboembolic stroke in mice.

OBJECTIVES: Thrombus and secondary thrombosis plays a key role in stroke. Recent molecular imaging provides in vivo imaging of activated factor XIII (FXIIIa), an important mediator of thrombosis or fibrinolytic resistance. The present study was to investigate the fibrin deposition in a thromboembolic stroke mice model by FXIIIa-targeted near-infrared fluorescence (NIRF) imaging. MATERIALS AND METHODS: The experimental protocol was approved by our institutional animal use committee. Seventy-six C57B/6J mice were subjected to thromboembolic middle cerebral artery occlusion or sham operation. Mice were either intravenously injected with the FXIIIa-targeted probe or control probe. In vivo and ex vivo NIRF imaging were performed thereafter. Probe distribution was assessed with fluorescence microscopy by spectral imaging and quantification system. MR scans were performed to measure lesion volumes in vivo, which were correlated with histology after animal euthanasia. RESULTS: In vivo significant higher fluorescence intensity over the ischemia-affected hemisphere, compared to the contralateral side, was detected in mice that received FXIIIa-targeted probe, but not in the controlled mice. Significantly NIRF signals showed time-dependent processes from 8 to 96 hours after injection of FXIIIa-targeted probes. Ex vivo NIRF image showed an intense fluorescence within the ischemic territory only in mice injected with FXIIIa-targeted probe. The fluorescence microscopy demonstrated distribution of FXIIIa-targeted probe in the ischemic region and nearby micro-vessels, and FXIIIa-targeted probe signals showed good overlap with immune-fluorescent fibrin staining images. There was a significant correlation between total targeted signal from in vivo or ex vivo NIRF images and lesion volume. CONCLUSION: Non-invasive detection of fibrin deposition in ischemic mouse brain using NIRF imaging is feasible and this technique may provide an in vivo experimental tool in studying the role of fibrin in stroke.

Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition.

α(2)-Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of α(2)-antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of α(2)-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α(2)-antiplasmin and plasmin. When the C terminus of α(2)-antiplasmin was removed, the binding affinity for active site-blocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.

Proteolytic and genetic variation of the alpha-2-antiplasmin C-terminus in myocardial infarction.

Alpha-2-antiplasmin (α2AP) undergoes both N- and C-terminal cleavages, which significantly modify its activities. Compared with other Ser protease inhibitors (serpins), α2AP contains an ~50-residue-extended C-terminus, which binds plasmin(ogen). We developed 2 new ELISAs to measure the antigen levels of free total α2AP and free C-terminally intact α2AP to investigate whether α2AP antigen levels or α2AP C-terminal cleavage were associated with myocardial infarction (MI) in 320 male MI survivors and 169 age-matched controls. Patients had 15.2% reduced total α2AP antigen levels compared with controls (93.8 vs 110.6 U/dL, P < .001), with a 10.1-fold (95% confidence interval [CI]: 5.5-18.9) increased MI risk for levels in the 1st quartile compared with the 4th quartile. The percentage of C-terminal cleavage did not differ between patients and controls (38.7% and 38.1%, respectively, P = .44). In addition, all individuals were genotyped for the polymorphism Arg407Lys, which is located near the start of the extended C-terminus. Arg407Lys was not associated with α2AP C-terminal cleavage, total α2AP antigen levels, or MI risk (odds ratios compared with Arg/Arg: Arg/Lys 0.74, 95% CI: 0.50-1.10; Lys/Lys 0.77, 95% CI: 0.31-1.92). Our data show that levels of free full-length α2AP were decreased in MI, that the percentage of C-terminally cleaved α2AP was unaltered, and that Arg407Lys did not influence α2AP levels or MI risk.

The antifibrinolytic function of factor XIII is exclusively expressed through α2-antiplasmin cross-linking.

Factor XIII (FXIII) generates fibrin-fibrin and fibrin-inhibitor cross-links. Our flow model, which is sensitive to cross-linking, was used to assess the effects of FXIII and the fibrinolytic inhibitor, α2-antiplasmin (α2AP) on fibrinolysis. Plasma model thrombi formed from FXIII or α2AP depleted plasma lysed at strikingly similar rates, 9-fold faster than pooled normal plasma (PNP). In contrast, no change was observed on depletion of PAI-1 or thrombin activatable fibrinolysis inhibitor (TAFI). Inhibition of FXIII did not further enhance lysis of α2AP depleted thrombi. Addition of PNP to FXIII or α2AP depleted plasmas normalized lysis. Lysis rate was strongly inversely correlated with total cross-linked α2AP in plasma thrombi. Reconstitution of FXIII into depleted plasma stabilized plasma thrombi and normalized γ-dimers and α-polymers formation. However, the presence of a neutralizing antibody to α2AP abolished this stabilization. Our data show that the antifibrinolytic function of FXIII is independent of fibrin-fibrin cross-linking and is expressed exclusively through α2AP.

Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2-antiplasmin.

BACKGROUND AND OBJECTIVE: Resistance of thrombi to plasmin digestion depends primarily on the amount of α(2)-antiplasmin (α(2)AP) incorporated within fibrin. Circulating prolyl-specific serine proteinase, antiplasmin-cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met-α(2)AP between -Pro12-Asn13- to yield Asn-α(2)AP, which is crosslinked to fibrin approximately 13× more rapidly than Met-α(2)AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met-α(2)AP to Asn-α(2)AP and thereby enhance endogenous fibrinolysis. METHODS AND RESULTS: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl-Arg-(8-amino-3,6-dioxaoctanoic acid)-D-Ala-L-boroPro selectively inhibited APCE vs. DPPIV, with an apparent K(i) of 5.7 nm vs. 6.1 µm, indicating that an approximately 1000-fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K(i) of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose-dependent decrease of APCE-mediated Met-α(2)AP cleavage, which ultimately shortened plasminogen activator-induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC(50) value in plasma remaining comparable to that in phosphate buffer. CONCLUSION: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.

Noncovalent interaction of alpha(2)-antiplasmin with fibrin(ogen): localization of alpha(2)-antiplasmin-binding sites.

Covalent incorporation (cross-linking) of plasmin inhibitor alpha(2)-antiplasmin (alpha(2)-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that alpha(2)-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of alpha(2)-AP to fibrin(ogen) and its fragments by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance. The experiments revealed that alpha(2)-AP binds to polymeric fibrin and surface-adsorbed fibrin(ogen), while no binding was observed with fibrinogen in solution. To localize the alpha(2)-AP-binding sites, we studied the interaction of alpha(2)-AP with the fibrin(ogen)-derived D(1), D-D, and E(3) fragments, and the recombinant alphaC region and its constituents, alphaC connector and alphaC domain and its subdomains, which together encompass practically the whole fibrin(ogen) molecule. In the ELISA, alpha(2)-AP bound to immobilized D(1), D-D, alphaC region, alphaC domain, and its C-terminal subdomain. The binding was Lys-independent and was not inhibited by plasminogen or tPA. Furthermore, the affinity of alpha(2)-AP for D-D was significantly increased in the presence of plasminogen, while that to the alphaC domain remained unaffected. Altogether, these results indicate that the fibrin(ogen) D region and the C-terminal subdomain of the alphaC domain contain high-affinity alpha(2)-AP-binding sites that are cryptic in fibrinogen and exposed in fibrin or adsorbed fibrinogen, and the presence of plasminogen facilitates interaction of alpha(2)-AP with the D regions. The discovered noncovalent interaction of alpha(2)-AP with fibrin may contribute to regulation of the initial stage of fibrinolysis and provide proper orientation of the cross-linking sites to facilitate covalent cross-linking of alpha(2)-AP to the fibrin clot.

The human alpha(2)-plasmin inhibitor: functional characterization of the unique plasmin(ogen)-binding region.

The human alpha(2)-plasmin inhibitor (A2PI) possesses unique N- and C-terminal extensions that significantly influence its biological activities. The C-terminal segment, A2PIC (Asn(398)-Lys(452)), contains six lysines thought to be involved in the binding to lysine-binding sites in the kringle domains of human plasminogen, of which four (Lys(422), Lys(429), Lys(436), Lys(452)) are completely and two (Lys(406), Lys(415)) are partially conserved. Multiple Lys to Ala mutants of A2PIC were expressed in Escherichia coli and used in intrinsic fluorescence titrations with kringle domains K1, K4, K4 + 5, and K1 + 2 + 3 of human plasminogen. We were able to identify the C-terminal Lys(452) as the main binding partner in recombinant A2PIC (rA2PIC) constructs with isolated kringles. We could show a cooperative, zipper-like enhancement of the interaction between C-terminal Lys(452) and internal Lys(436) of rA2PIC and isolated K1 + 2 + 3, whereas the other internal lysine residues contribute only to a minor extent to the binding process. Sulfated Tyr(445) in the unique C-terminal segment revealed no influence on the binding affinity to kringle domains.

Antiplasmin activity of natural occurring polyphenols.

The equilibrium between proteolytic enzymes and their cognate inhibitors is crucial in a number of physiological as well as pathological processes, including cancer, inflammatory processes and thrombosis. Therefore, both synthetic and natural small molecule inhibitors are object of extensive studies as drugs in the treatment of these pathologies. Two natural occurring polyphenolic compounds, representative of glycosylated and unglycosylated flavonoid structures, namely quercetin and rutin, were thereby tested as potential ligands of plasmin(ogen), a serine (pro)protease, whose role in tumor cell invasion and migration has been reported. Quercetin showed a ten folds higher affinity with plasmin with respect to rutin in terms of equilibrium dissociation constant, both compounds acting as in vitro moderate reversible inhibitors; additionally, quercetin and rutin prevented plasmin-incubated BB1 cells from releasing E-cadherin fragment to a different extent, respectively. Furthermore, a feasible mechanism of interaction was analyzed and discussed using a molecular modeling approach.

X-ray crystal structure of the fibrinolysis inhibitor alpha2-antiplasmin.

The serpin alpha(2)-antiplasmin (SERPINF2) is the principal inhibitor of plasmin and inhibits fibrinolysis. Accordingly, alpha(2)-antiplasmin deficiency in humans results in uncontrolled fibrinolysis and a bleeding disorder. alpha(2)-antiplasmin is an unusual serpin, in that it contains extensive N- and C-terminal sequences flanking the serpin domain. The N-terminal sequence is crosslinked to fibrin by factor XIIIa, whereas the C-terminal region mediates the initial interaction with plasmin. To understand how this may happen, we have determined the 2.65A X-ray crystal structure of an N-terminal truncated murine alpha(2)-antiplasmin. The structure reveals that part of the C-terminal sequence is tightly associated with the body of the serpin. This would be anticipated to position the flexible plasmin-binding portion of the C-terminus in close proximity to the serpin Reactive Center Loop where it may act as a template to accelerate serpin/protease interactions.

Reversible inactivation of serpins at acidic pH.

The inhibitory activity of the serpins alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, alpha(2)-antiplasmin, antithrombin and C(1)-esterase inactivator is rapidly lost at pH 3 but slowly recovers at pH 7.4 with variable first-order rates (t(1/2)=1.4-19.2 min). All except alpha(1)-antichymotrypsin undergo a variation in intrinsic fluorescence intensity upon acidification (midpoint ca. 4.5) with a slow bi-exponential return to the initial intensity at pH 7.4 (mean t(1/2)=2.3-23 min). No correlation was found between the time of fluorescence recovery and that of reactivation. The acid-treated serpins are proteolyzed at neutral pH by their target proteinases. alpha(1)-Proteinase inhibitor was studied in more detail. Its acidification at pH 3 has a mild effect on its secondary structure, strongly disorders its tertiary structure, changes the microenvironment of Cys(232) and causes a very fast change in ellipticity at 225 nm (t(1/2)=1.6s). Neutralization of the acid-treated alpha(1)-proteinase inhibitor is an exothermic phenomenon. It leads to a much faster recovery of activity (t(1/2)=4+/-1 min) than of fluorescence intensity (t(1/2)=23+/-19 min), ellipticity (t(1/2)=32+/-4 min) and change in total energy, indicating that the inhibitory activity of alpha(1)-proteinase inhibitor does not require a fully native structure.

Characterization of three distinct catalytic forms of human tryptase-beta: their interrelationships and relevance.

Human tryptase-beta (HTbeta) is a serine protease that is isolated as a tetramer of four identical, catalytically active subunits (HTbeta-AT). Tetramer activity is not affected by protein-based physiological inhibitors but instead may be regulated by an autoinactivation process we have called spontaneous inactivation. Unless stabilized by heparin or high salt, the active tetramer converts to an inactive state consisting of an inactive-destabilized tetramer that reversibly dissociates to inactive monomers upon dilution. We refer to this mixture of inactive species as siHTbeta and show in this study that previous reports of monomeric catalytic forms are derived from this mixture. siHTbeta itself did not hydrolyze model substrates but unlike the tetramer did react slowly with the serpin alpha2-antiplasmin (alpha2-AP), suggesting a highly limited catalytic potential. In the presence of heparin (or other highly charged polysaccharides), we demonstrate that siHTbeta formed a well-defined complex with the heparin (siHTbeta-HC) that reacted 70-fold faster with alpha2-AP than siHTbeta and also hydrolyzed model substrates and fibrinogen. Formation of siHTbeta-HC was limited to dilute subunit solutions since high subunit concentrations resulted in the reformation of the active tetramer. By compensating for changes in the strength of heparin binding, siHTbeta-HC could be formed over the pH range of 6.0-8.5. The activity dependence on pH was bell-shaped with highest activity between pH 6.8 and pH 7.5. In contrast, HTbeta-AT activity showed no dependence upon heparin, increased over the pH range of 6.0-8.5, and was much higher than that of siHTbeta-HC especially above pH 6.8. HTbeta-AT incubated with excess heparin of different size (3-15 kDa) was functionally stable at 25 degrees C but lost activity regardless of heparin size at 37 degrees C above pH 6.8. The change in stability, which is likely due to weakened heparin binding, did not result in the formation of a stable catalytic monomer. These results confirm that siHTbeta is for the most part an inactive species and that any active monomer is a consequence of heparin binding to siHTbeta under dilute conditions where unfavorable thermodynamics and/or kinetics restrict formation of active tetramer. Heparin binding under these conditions drives a limited reorganization of the active site to a conformation that is catalytic but not the equivalent of a subunit within the active tetramer.

[Regulation with alpha-2-antiplasmin of Glu-plasminogen activation by tissue activator on fibrin].

Interaction of tissue plasminogen activator with alpha-2-antiplasmin and its influence on tissue activator binding to fibrin was studied. Alpha-2-Antiplasmin decreases the binding of tissue activator to fibrin by 20%. The inhibitor formed a complex with tissue plasminogen activator (Kd 78.2 nM) and had no effect on amidolytic activity of the activator. The tissue activator binding to alpha-2-antiplasmin decreases by 20-35% in the presence of 6-aminohexanoic acid. It indicates that not only kringle 2 of the tissue activator molecule takes part in complex formation with alpha-2-antiplasmin, but also other activator domains. Two models were proposed to explain the alpha-2-antiplasmin effect on the Glu-plasminogen activation by tissue activator on fibrin. In the first place, the inhibitor binds to fibrin in the site where the activator complex is localized. It can create steric hindrances for the proenzyme interaction with its activator on fibrin. In the second place, alpha-2-antiplasmin in a complex with tissue plasminogen activator can bring to a change in the activator conformation and a decrease of its functional activity.