Characterisation of the complex of plasminogen activator inhibitor type 1 with tissue-type plasminogen activator by mass spectrometry and size-exclusion chromatography

Strömqvist, Mats; Karlsson, K.; Björquist, Petter; Andersson, Jan-Olof; Byström, Mona; Hansson, Lennart; Johansson, Tomas; Deinum, Johanna

doi:10.1016/0167-4838(96)00035-0

Cited by 18 publications

(11 citation statements)

References 33 publications

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“…However, the addition of a cocktail of protease inhibitors was unable to prevent the cleavage during incubation with TVASS. The mutant was exclusively cleaved at the P1-P1′ site (Arg346-Met347) as confirmed by the appearance of the terminal 3804 Da moiety [8] and the absence of other fragments. Dissolved crystals of PAI-1-Ala335Glu in complex with TVASS were shown to be similarly cleaved.…”

Section: Enhancement Of the Substrate Properties Of Pai-1-ala335glumentioning

confidence: 83%

“…Mass spectra were recorded using a Finnigan TSQ 700 triple quadrupole mass spectrometer equipped with a Finnigan electrospray ion source [8] or a MALDI spectrometer from PerCeptive Biosystems, Voyager Elite, using a Sinapinic acid matrix. Immediately before recording the spectra, a buffer exchange of the proteins was performed on a NAP-5 column (Pharmacia Biotech) equilibrated in 1% acetic acid/ water (v/v).…”

Section: Mass Spectrometrymentioning

confidence: 99%

“…Active PAI-1 spontaneously converts into latent PAI-1 [2]. Active PAI-1 forms a tight, presumably covalent, complex with tPA in which PAI-1 is cleaved at the P1-P1′ peptide bond [8]. So far the structural features of the PAI-1-tPA complex remain uncertain, but recent results from fluorescence energy transfer experiments [9][10][11] and antibody mapping [12] have provided some insight into the positioning of the two proteins in relation to each other.…”

Section: Introductionmentioning

confidence: 99%

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Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

et al. 1998

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This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between beta strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into beta sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.

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Section: Enhancement Of the Substrate Properties Of Pai-1-ala335glumentioning

confidence: 83%

Section: Mass Spectrometrymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

et al. 1998

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“…3A and 6, corresponding to crmA (both N ϩ C-terminal fragments) ϩ p20, illustrates how, even for a very tightly but non-covalently associated C-terminal fragment, most of the peptide dissociates during the analysis. Equivalent behavior was seen for the covalent acyl intermediate of the serpin plasminogen activator inhibitor-1 (PAI-1) with the serine proteinase tissue-type plasminogen activator (tPA), examined by electrospray mass spectrometry, where a dominant peak for the N-terminal PAI-1 ϩ tPA was seen, reflecting the covalent linkage between the two species, and a smaller peak for N ϩ C fragments of PAI-1 ϩ tPA (33).…”

Section: ϫ6mentioning

confidence: 99%

Cytokine Response Modifier A Inhibition of Initiator Caspases Results in Covalent Complex Formation and Dissociation of the Caspase Tetramer

Dobó

Swanson

Salvesen

et al. 2006

Journal of Biological Chemistry

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Active caspases are generally composed of two catalytic domains, each containing a large (p20) and a small (p10) subunit so that a fully active caspase has the organization (p20-p10) 2 . The cowpox serpin crmA suppresses host apoptosis and inflammation by inhibiting endogenous caspases. We report on the mechanism crmA uses to inhibit caspases 1, 6, and 8. Native PAGE showed formation of tight crmA-caspase complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided evidence for a covalent crmA-p20 thioester linkage. SDS-PAGE of isolated complexes showed near complete loss of the p10 subunit from initiator caspases 1 and 8 but not from the executioner caspase-6. This was confirmed for caspase-1 by sequencing and Western blotting. Size exclusion chromatography indicated a size of ϳ60 kDa for complexes with caspases 1 and 8, consistent with a crmA⅐p20 species, suggesting that the p20-p10 interface and possibly the p10-p10 interface had been disrupted. In contrast, crmA⅐caspase-6 complex behaved as an equilibrium mixture of crmA 2 ⅐(p20-p10) 2 and crmA⅐(p20-p10). Complex deacylation rates were all slow, suggesting effective kinetic trapping of the covalent thioacyl intermediate. These results suggest a novel serpin inhibition mechanism through which crmA down-regulates apoptosis and inflammation. This involves (i) rapid formation of covalent complex with initiator caspases 8 or 1, (ii) very slow deacylation, and (iii) loss of the caspase p10 subunit for initiator but not for executioner caspases, so that any free p20 formed by deacylation of initiator caspases cannot reassociate to active heterotetramer, thus resulting in irreversible inhibition of apoptosis and inflammation.Caspases are intracellular cysteine proteinases involved in inflammation and apoptosis (1). Caspase-1, also known as interleukin-1␤ converting enzyme (ICE), 3 activates the pro-inflammatory cytokine interleukin-1␤ by proteolysis (2), whereas caspase-8, itself activated by dimerization through an extrinsic receptor-mediated pathway (3, 4), acts as an initiator of apoptosis through downstream proteolytic activation of so-called "executioner" caspases, such as caspases -3, -6, and -7 (5). Active caspases are obligate homodimers, with each monomer composed of a large (p20, 17-20 kDa) and a small (p10, 9 -12 kDa) subunit, which are formed after the activation cleavage of the procaspase (6). Structures of several caspases are known, and all show that two p20-p10 heterodimers are associated in an anti-parallel arrangement through the ␤-sheet of the two p10 subunits (7-11). The p20 subunit, however, contains both the catalytic cysteine and histidine (6).Orthopox viruses, such as the cowpox virus, enhance their infectivity through specific inhibition of caspases and consequent abrogation of the inflammatory response and of apoptosis (12). They do this using an inhibitor that is a member of the serpin family (13). In the case of cowpox virus, the serpin is crmA (14). crmA can inhibit a range of caspases, although at ver...

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“…(24, 25) Indeed, decreased levels of PAI-1 activity have been demonstrated in trauma patients with hyperfibrinolysis and PAI-1 is known to be inactivated by interaction with a number of proteolytic enzymes including aPC and neutrophil elastase, which may themselves be upregulated in conditions of injury, inflammation and shock. (26-29) It has therefore become widely accepted that degradation of PAI-1 by aPC with resulting unchecked tPA is chiefly responsible for the pathologically high level of plasmin-mediated fibrinolysis in trauma.…”

Section: Introductionmentioning

confidence: 99%

Overwhelming tPA release, not PAI-1 degradation, is responsible for hyperfibrinolysis in severely injured trauma patients

Chapman

Moore

et al. 2016

Journal of Trauma and Acute Care Surgery

185

202

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Background Trauma induced coagulopathy (TIC) is associated with a four-fold increased risk of mortality. Hyperfibrinolysis is a component of TIC, but its mechanism is poorly understood. PAI-1 degradation by activated protein C has been proposed as mechanism for deregulation of the plasmin system in hemorrhagic shock, but in other settings of ischemia, tPA has been shown to be elevated. We hypothesized that the hyperfibrinolysis in TIC is not the result of PAI-1 degradation, but is driven by an increase in tPA, with resultant loss of PAI-1 activity through complexation with tPA. Methods 86 consecutive trauma activation patients had blood collected at the earliest time after injury, and were screened for hyperfibrinolysis using thrombelastography (TEG). Twenty-five hyperfibrinolytic patients were compared to 14 healthy controls using ELISAs for active tPA, active PAI-1 and PAI-1/tPA complex. Blood was also subjected to TEG with exogenous tPA-challenge as a functional assay for PAI-1 reserve. Results Total levels of PAI-1 (the sum of the active PAI-1 species and its covalent complex with tPA) are not significantly different between hyperfibrinolytic trauma patients and healthy controls: median 104 pM (IQR 48—201 pM) versus 115 pM (IQR 54—202 pM). The ratio of active to complexed PAI-1, however, was two orders of magnitude lower in hyperfibrinolysis than controls. Conversely, total tPA levels (active plus complex) were significantly higher in hyperfibrinolysis than controls: 139 pM (IQR 68—237 pM) versus 32 pM (IQR 16—37 pM). Hyperfibrinolytic trauma patients displayed increased sensitivity to exogenous challenge with tPA: median LY30 of 66.8% compared to 9.6% for controls. Conclusions Depletion of PAI-1 in TIC is driven by an increase in tPA, not PAI-1 degradation. The tPA-challenged TEG, based on this principle, is a functional test for PAI-1 reserves. Exploration of the mechanism of upregulation of tPA is critical to an understanding of hyperfibrinolysis in trauma.

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Characterisation of the complex of plasminogen activator inhibitor type 1 with tissue-type plasminogen activator by mass spectrometry and size-exclusion chromatography

Cited by 18 publications

References 33 publications

Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

Cytokine Response Modifier A Inhibition of Initiator Caspases Results in Covalent Complex Formation and Dissociation of the Caspase Tetramer

Overwhelming tPA release, not PAI-1 degradation, is responsible for hyperfibrinolysis in severely injured trauma patients

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